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Challenges to implementation of alternative methods to animal testing for drug safety assessment in North America

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Challenges to implementation of alternative methods to animal testing for drug safety assessment in North America

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CHALLENGES TO IMPLEMENTATION OF ALTERNATIVE METHODS TO ANIMAL
TESTING FOR DRUG SAFETY ASSESSMENT IN NORTH AMERICA

by

Sunita Singh Babbar

A Dissertation Presented to the
FACULTY OF THE USC SCHOOL OF PHARMACY
UNIVERSITY OF SOUTHERN CALIFORNIA
In Fulfillment of the
Requirements for the Degree
DOCTOR OF REGULATORY SCIENCE

December 2021

Copyright 2021 Sunita S. Babbar

ii
Dedication
It is with pride, I dedicate this dissertation to parents: my father Prof. Jaswant Singh and
my late mother Asha Singh. My father inspired me to believe in myself, continue my academic
pursuits to highest level possible and never give up. My mother taught me the value of
unconditional love and nurturing.
My father gave one advice to me as a child – “no matter what goal you pursue in life, be
the best at it.” He believed I could achieve anything I wanted to. That has truly shaped my life
and I would not be the person I am today without that belief in me!

iii
Acknowledgements
The completion of this degree would not have been possible without the support and
encouragement of several very special people. I would like to take this opportunity to express
my gratitude to those who helped me along this incredible journey.
First, I extend my thanks and appreciation to the survey and focus group participants for
their valuable insights in making this research study possible.
My heartfelt thanks to my advisor Dr. Frances J Richmond who has guided me with her
expert knowledge and experience. I shall forever be grateful to her for enabling my life-long
desire and passion to complete this monumental academic endeavor after my first incomplete
attempt to finish my doctoral degree almost three decades ago. Of course, this would not be
possible without her vision and strength with which she created this unique Regulatory Science
program at USC.
My sincere gratitude to Dr. Keith Bley who has been a friend, manager and mentor
throughout my personal and professional career. He not only provided his invaluable mentorship
when I was a young scientist and a mother of two little children but continued his support and
encouragement in completing this research and doctoral degree many years later.
Many thanks to Dr. Mary Ellen Cosenza in volunteering her precious time in the review
of this thesis and for her scientific advice and knowledge and many insightful suggestions.
I also want to thank the members of my PhD committee - Dr. Daryl L Davies and Dr.
Terry David Church, for their time, helpful advice and valuable suggestions.
Thanks to my friends who provided the much-needed encouragement to apply to this
program and pursue this degree when it almost seemed impossible and those who were a
constant source of support. Finally, I want to acknowledge my two best friends, my son Salil

iv
and my daughter Anisha, who have been a source of motivation and strength during moments of
despair.
Without you all none of this would have been possible and I cannot thank you all
enough…

v
TABLE OF CONTENTS
Acknowledgements ........................................................................................................................ iii
List of Tables ............................................................................................................................... viii
List of Figures ..................................................................................................................................x
Abstract ......................................................................................................................................... xii
Chapter 1. Overview ........................................................................................................................1
1.1 Introduction ................................................................................................................. 1
1.2 Statement of the Problem ............................................................................................ 8
1.3 Purpose of the Study ................................................................................................... 9
1.4 Importance of the Study .............................................................................................. 9
1.5 Limitation, Delimitations, and Assumptions ............................................................ 10
1.6 Organization of Thesis .............................................................................................. 12
1.7 Definitions ................................................................................................................. 12
Chapter 2. Literature Review .........................................................................................................15
2.1 Introduction ............................................................................................................... 15
2.2 History of Animal Contributions to Medical Research ............................................. 16
2.3 The Evolution of Toxicological Oversight by US Regulatory Systems ................... 21
2.4 Introduction of the “3Rs” .......................................................................................... 26
2.5 Alternative methods .................................................................................................. 27
2.5.1 Are In Vitro Assays Alternative or Complementary to Animal Tests? ....... 28
2.5.2 Specific Issues related to Cell-Based Tests ................................................. 30
2.6 Challenges to Animal Use and Paradigm Shift ......................................................... 32
2.6.1 Scientific and Technological Drivers .......................................................... 32
2.6.2 Economic/ Business Drivers ........................................................................ 34
2.6.3 Ethical or Societal Drivers ........................................................................... 36
2.6.4 Political Drivers ........................................................................................... 38
2.6.5 Regulatory Drivers ...................................................................................... 41
2.6.5.1 FDA’s Critical Path Initiative ......................................................... 42
2.6.5.2 FDA’s Predictive Toxicology Roadmap......................................... 43
2.7 Research Direction .................................................................................................... 44
Chapter 3. Methodology ................................................................................................................49
3.1 Introduction ............................................................................................................... 49
3.2 Identification of Survey Participants ......................................................................... 49
3.3 Development of Initial Survey .................................................................................. 50
3.4 Survey Deployment and Analysis ............................................................................. 52
3.5 Data Analysis ............................................................................................................ 53
Chapter 4. Results ..........................................................................................................................54
4.1 Survey Participation .................................................................................................. 54

vi
4.2 Demographic Profile of Respondents ....................................................................... 54
4.3 Current Status and General Outlook Regarding Animal Use in Safety Testing ....... 59
4.4 Barriers and Drivers of Implementation of Alternatives ........................................... 62
4.5 Exploration and Installation Stages ........................................................................... 69
4.5.1 In Silico/Computer-Based Alternatives ....................................................... 69
4.5.2 Tissue/Cell Culture Technologies ................................................................ 76
4.5.3 Lower Vertebrates/Invertebrates-Based Alternatives .................................. 83
4.5.4 Molecular Imaging/Other Novel Methods ................................................... 90
4.6 Potential Policy Recommendations .......................................................................... 97
Chapter 5. Discussion ..................................................................................................................103
5.1 Summary ................................................................................................................. 103
5.2 Methodological Considerations .............................................................................. 103
5.2.1 Limitations ................................................................................................. 103
5.2.2 Delimitations ............................................................................................. 107
5.3 Considerations of the Results .................................................................................. 110
5.3.1 Exploration and Installation ....................................................................... 110
5.3.1.1 How commonly are different alternatives explored? .................... 111
5.3.1.2 What resources were useful during those explorations? ............... 114
5.3.1.3 How often did explored alternatives advance to the installation
phase? ........................................................................................... 115
5.3.1.4 How often do sponsors seek regulatory guidance from the FDA
during the exploration and installation stages? ............................. 116
5.3.1.5 What were the main challenges encountered during
installation? ................................................................................... 117
5.3.2 Implementation .......................................................................................... 119
5.3.2.1 Why is validation such a large concern? ...................................... 119
5.3.2.2 How does regulatory acceptance influence alternative model
development? ................................................................................ 120
5.3.2.3 What ways are available to increase acceptance for safety
testing? .......................................................................................... 121
5.4 Current Status and Outlook ..................................................................................... 121
5.4.1 Will alternative methods reduce animal studies in the near future? .......... 121
5.4.2 How much impact have alternative tests really had? ................................. 122
5.5 Areas of Policy Focus ............................................................................................. 124

vii
References ....................................................................................................................................127
Appendix A. .................................................................................................................................139
Appendix B. .................................................................................................................................179
Appendix C. .................................................................................................................................180
Appendix D. .................................................................................................................................182
Appendix E. .................................................................................................................................184
Appendix F...................................................................................................................................185
Appendix G. .................................................................................................................................186
Appendix H. .................................................................................................................................191

viii
List of Tables
Table 1: Animal Research in the US in 2018 .............................................................2
Table 2: Questionnaire Instrument: Breakdown of Areas of Inquiry ......................50
Table 3: List of Participants in the Focus Group .....................................................51
Table 4: Focus Group Agenda .................................................................................52
Table 5: Survey Response Data ...............................................................................54
Table 6: Other-Locations .........................................................................................57
Table 7: Other-Therapy ............................................................................................59
Table 8: Other-Barriers (Main Themes) ..................................................................64
Table 9: Other-Useful Sources .................................................................................71
Table 10: Factors that Drove the Decision to not Pursue In Silico Methods During
Exploration .................................................................................................73
Table 11: Factors that led to Abandonment of In Silico Methods after Initial
Implementation ..........................................................................................73
Table 12: Other-Challenges to Implementation of In Silico Alternatives .................75
Table 13: Description of In Silico Methods Implemented (Main Themes) ...............76
Table 14: Other-Information Sources for Tissue/Cell Culture Methods ...................78
Table 15: Factors Causing Abandonment of Tissue/Cell Culture Technologies
After Initial Implementation ......................................................................80
Table 16: Other-Challenges .......................................................................................81
Table 17: Description of Implemented Tissue/Cell Culture Technologies (Main
Themes)......................................................................................................83
Table 18: Factors Driving the Decision to not Pursue Using Lower Vertebrates/
Invertebrates Alternatives During Exploration ..........................................86
Table 19: Factors Causing Abandonment of Lower Vertebrates/Invertebrates-
Based Methods After Initial Implementation ............................................87
Table 20: Other-Challenges .......................................................................................88
Table 21: Description of Alternatives Using Lower Vertebrates/ Invertebrates
(Main Themes) ...........................................................................................90
Table 22: Other-Useful Information Sources .............................................................92
Table 23: Factors that Drove the Decision to not Pursue Molecular Imaging/ Other
Novel Methods ...........................................................................................93
Table 24: Factors Causing Abandonment of Molecular Imaging/Other Novel
Methods After Initial Implementation .......................................................94
Table 25: Description of Molecular Imaging/Other Novel Methods Implemented ...97
Table 26: Other-Recommendations ...........................................................................99

ix
Table 27: Addressing Studies When Non-Animal Alternative Do Not Exist (Main
Themes)....................................................................................................102
Table 28: Exploration and Installation of Various Categories of Alternatives ........111

x
List of Figures
Figure 1: Number of Animals Used in Research in the US in 2018 ...........................3
Figure 2: General Nonclinical Testing Paradigm ......................................................25
Figure 3: The Cost of Developing a New Drug Since the 1970s ..............................36
Figure 4: Pew Survey Results on Animal Use ..........................................................38
Figure 5: Organizational Affiliations ........................................................................55
Figure 6: Size of Organizations .................................................................................56
Figure 7: Age of Organizations .................................................................................56
Figure 8: Geographical Location of Organizations ...................................................57
Figure 9: Years of Personal Experience in Safety Assessment .................................58
Figure 10: Types of Products ......................................................................................58
Figure 11: Appropriateness of Animal Numbers Used for Safety
Assessments ...............................................................................................59
Figure 12: Impact of Alternatives on Numbers of Animals Used ...............................60
Figure 13: Expectation from ICH Guidances in the Near Future ................................60
Figure 14: Alternatives: Add-ons or Reduced Use of Animals? .................................61
Figure 15: Possibility of Animal-Free Safety Assessment ..........................................61
Figure 16: Views on FDA’s success ...........................................................................62
Figure 17: Implementation Barriers ............................................................................63
Figure 18: Implementation Drivers .............................................................................65
Figure 19: Lack of Validation .....................................................................................66
Figure 20: Cost of Validation ......................................................................................68
Figure 21: Strategies for Acceptance of Validated Alternatives .................................69
Figure 22: Exploration of In Silico/Computer-Based Alternatives .............................70
Figure 23: Usefulness of Information Sources During Exploration of In
Silico Methods ...........................................................................................71
Figure 24: Outcome of Exploration of In Silico Methods ...........................................72
Figure 25: Challenges to In Silico Alternative Development .....................................74
Figure 26: Regulatory Agency Feedback Regarding In Silico Methods .....................75
Figure 27: Impact of In Silico Method Implementation on Animal Use ....................76
Figure 28: Exploration of Tissue/Cell Culture Technologies as Alternatives .............77
Figure 29: Usefulness of Information Sources During Exploration of
Tissue/Cell Culture Technologies ..............................................................78
Figure 30: Outcome of Exploration of Tissue/Cell Culture Technologies ..................79

xi
Figure 31: Challenges to Tissue/Cell Culture-Based Alternative
Development ..............................................................................................81
Figure 32: Regulatory Agency Feedback During Implementation of
Tissue/Cell Culture Methods .....................................................................82
Figure 33: Impact of Tissue/Cell Culture Method Implementation on Animal
Use .............................................................................................................82
Figure 34: Exploration of Lower Vertebrates/Invertebrates-Based
Alternatives ................................................................................................84
Figure 35: Usefulness of Information Sources Regarding Lower Vertebrate/
Invertebrate Alternatives ............................................................................85
Figure 36: Outcome of Exploration of Lower Vertebrates/Invertebrates-
Based Methods ...........................................................................................86
Figure 37: Challenges to Development of Alternatives Using Lower
Vertebrates/Invertebrates ...........................................................................88
Figure 38: Regulatory Agency Feedback During Implementation of Lower
Vertebrates/Invertebrates-Based Alternatives ...........................................89
Figure 39: Impact of Lower Vertebrates/Invertebrates-Based Alternative
Implementation on Animal Use .................................................................89
Figure 40: Exploration of Molecular Imaging/Other Novel Methods .........................90
Figure 41: Usefulness of Information Sources During Exploration of
Molecular Imaging/Other Novel Methods .................................................92
Figure 42: Outcome of Exploration of Molecular Imaging/Other Novel
Methods......................................................................................................93
Figure 43: Challenges to Development of Alternatives Based on Molecular
Imaging/Other Novel Methods ..................................................................95
Figure 44: Regulatory Agency Feedback During Implementation of
Molecular Imaging/Other Novel Methods .................................................96
Figure 45: Impact of Implementation of Molecular Imaging/ Other Novel
Methods on Animal Use ............................................................................96
Figure 46: Efforts to Accelerate Validation of Alternatives .......................................98
Figure 47: Recommendations to Make Implementation More Efficient .....................99
Figure 48: Arguments for Failing to Develop Alternatives.......................................101

xii
Abstract
Animal studies have been an essential part of drug development to evaluate the efficacy
and safety of biopharmaceutical products. Notably, they are a prerequisite for the
conduct of human clinical trials and marketing authorization for pharmaceuticals, but
their use adds significant cost to the drug development process, cannot always predict
effects in humans and can raise significant ethical concerns. Thus, efforts to replace
animal studies with “alternative methods” have been ongoing for several decades. The
objective of this study was to explore the current state of alternative methods and identify
barriers to their adoption and acceptance for the safety assessment of pharmaceuticals.
The research was conducted using a survey tool and explored the status of the “3Rs”
(Replacement, Reduction, and Refinement) with a focus on the implementation of
alternative methods by two key stakeholders – pharmaceutical industry and contract
research organizations in North America. A total of 170 respondents with a background
in toxicology and animal studies completed the survey. The results suggest a field in
transition. The implementation of alternative methods has grown steadily in the early
screening stage of drug development but the field of safety assessment still mostly
depends on animal studies. However, significant regulatory barriers exist, including
those related to the validation of the alternative tests and to requirements that the tests are
as good or better than comparable animal experience. Further, uncertain costs and
outcomes make companies wary of pursuing alternative methods. Thus, most respondents
do not expect that the implementation of alternative methods will greatly change the
nature of drug safety testing in the next decade.

1
Chapter 1. Overview
1.1 Introduction
Animal research has played a crucial role in supporting countless scientific and
medical advancements of the past century. It has not only helped to extend and improve
the quality of life for millions of people but has also facilitated the development of
important treatments for cats, dogs, farm animals, wildlife, and endangered species.
Animal research has advanced the treatment of infections, provided insights into
immunization, improved cancer treatments, and had a major impact on managing heart
disease, brain disorders, arthritis and organ transplantation. Because animals and humans
share many physiological similarities, animal studies have continued to provide valuable
information that cannot yet be completely replaced by computer modelling, in vitro
studies or non-invasive human experimentation.
Nonetheless, animal research is only one tool- and sometimes an imperfect tool-
to study medical products. Differences between animals and humans can restrict the
usefulness of animals to replicate certain disease states in humans. Further, the value and
appropriateness of animal use in research has been questioned from an ethical viewpoint
for many decades. It was a driving force in the 1950s to promote the development of
Laboratory Animal Science (LAS) as a distinct multi-disciplinary branch of science
(Baumans, 2005), intended to promote responsible use of animals in research by
contributing both to the quality of animal experiments and to the welfare of laboratory
animals. One of the most important outcomes of this field of study was the critical and

2
influential work by Russell and Burch (1959) that led to the framework known today as
the 3Rs – the replacement, reduction, and refinement of the use of animals in research.
A decrease in animal use for research was observed in the early 1980s as the
public became more uncomfortable with animal use and stricter legislation was put in
place. More recently, however, the use of animals has increased, mainly due to the
development of genetically modified animals, particularly mice. In 2018, United States
Department of Agriculture (USDA) statistics put the number of laboratory animals used
in research under the Animal Welfare Act at 780,070 (USDA, 2018) (Figure 1) (Table 1).
However, these statistics greatly underestimate true numbers because they do not include
rats, mice, birds or fish- species not covered by the Animal Welfare Act, although they
are still protected under other regulations.
Table 1: Animal Research in the US in 2018
Reproduced with permission, www.speakingofresearch.com
Animal Research in the US in 2018
Species
Number of
Animals
% of Total
% Change
from 2017
Guinea Pigs 171,406 22% -6%
Rabbits 133,634 17.1% 11%
Hamsters 80,539 10.3% -17%
Non-Human Primates 70,797 9.1% 12%
Dogs 59,401 7.6% 4%
Pigs 50,094 6.4% 4%
Cats 18,619 2.4% 4%
Sheep 13,000 1.7% -1%
Other Covered Species 182,580 23.4% 31%
Total 780,070 100% 5.7%

3
Figure 1: Number of Animals Used in Research in the US in 2018
Reproduced with permission, www.speakingofresearch.com

In parallel with the social and legislative trends has been a revolutionary change
in toxicological research. Today, the principles of replacing, reducing and refining the
use of animals in scientific research sits at the core of modern toxicology. Development
of non-animal methods such as mathematical and computer models, advancements in
tissue and cell cultures and radiological imaging technologies have already contributed to
a significant reduction in the number of animals used in medical research. Reducing that
number much further, however, may be unrealistic in some fields of scientific research in
the immediate future because the biggest challenge is still the difficulty in reproducing
the complex biological systems of living organisms.
One area of drug development that still relies heavily on animal models is that of
safety assessment, in which toxicity studies must be carried out to evaluate safety of the
drug product being developed. Nonclinical safety testing of new drug candidates entails a

4
comprehensive series of in silico, in vitro and in vivo tests to characterize potential
toxicities to ensure human safety, aid in identifying a starting clinical dose level for a
First-In-Human (FIH) clinical study and identify potential target organs of toxicity along
with any applicable safety biomarkers, if possible. That large body of toxicological data
obtained from these studies is evaluated by nonclinical safety scientists and clinical
investigators to determine the potential risks of the new drug candidate and is used by
regulators to judge the acceptability of subsequent testing in humans.
At present, in vivo testing in experimental animals is an essential regulatory
requirement before a drug candidate can be tested in clinical trials. However, in the last
two decades, many in vitro assays have been developed and validated, some of which are
now commonly employed in pre-clinical evaluations. Most are used for early-stage
screening, with the goal of identifying and eliminating molecules with a higher potential
for toxicity (Sewell et al., 2017). However, in a few cases, they are used to replace or
reduce the use of certain in vivo tests. The pharmaceutical industry has shown a strong
commitment in developing new in vitro alternatives to replace, reduce and refine the use
of animals in nonclinical safety testing. Most notable examples include the skin irritancy
test and Draize eye irritancy test that have been replaced with in vitro cell cultures
(Vinardell and Mitjans, 2008).
Regulators and scientific organizations, too, have expressed commitment to
encourage the replacement of animals with alternative forms of testing. In 2007, the
National Research Council (NRC) published the landmark report, “Toxicity Testing in
the 21st Century: A Vision and a Strategy”, that outlined new approaches requiring fewer
animals and focused on in vitro methods for assessing risks that chemicals may pose to

5
biological systems (National Research Council, 2007). Shortly thereafter, in 2008, a
collaboration was formed involving the National Toxicology Program (NTP), the
National Institute of Health Chemical Genomics Center (NCGC), and the Environmental
Protection Agency (EPA) to establish the Toxicity Testing in the 21st Century (Tox21)
program. Its aim was to develop and promote toxicity tests on human cells or cell lines in
vitro to show that key molecular or cellular processes had changed in a way that were
predictive of adverse effects (Collins, Gray and Bucher., 2008). However, this is easier
said than done. It is even more difficult given the expectation that alternative methods
should only be considered acceptable if they are better or at least as effective at
predicting toxicity as the animal tests that they would replace (Burden, Sewell and
Chapman, 2015). Hence, it is imperative to evaluate the clinical relevance and reliability
of these test methods in validation studies.
Governmental initiatives play a key role in promoting development of the new
alternative methods and in validating those already available. These activities are
occurring not only in the US but also across many other countries, and some of these
initiatives have resulted in harmonization activities. In 1993, the European Center for the
Validation of Alternative Methods (ECVAM) was established (Marafante, Smyrniotis
and Balls, 1994) and eventually has become a global leader in this field. In 1997, US
government agencies formed the Interagency Coordinating Center for the Validation of
Alternative Methods (ICCVAM). The ICCVAM consists of 15 research and regulatory
agencies, including the EPA, the Food and Drug Administration (FDA), and the Agency
for Toxic Substances and Disease Registry (ATSDR). The ICCVAM Committee,
through these federal agencies, seeks to coordinate discussions on the development,

6
validation, and approval, in addition to national and international standardization, of
toxicological tests (ICCVAM, 2003). In 2005, the Japanese government created the
JaCVAM (Japanese Center for the Validation of Alternative Methods), and in 2011 the
BraCVAM (Brazilian Center for the Validation of Alternative Methods) was founded in
Brazil.
However, the validation of alternative tests can take years and the processes can
be tedious, complicated, cumbersome, confusing and costly. Often, in vitro assays
developed for one industry frequently cannot be transferred seamlessly to another. In the
European Union, separate legislation for different industries (e.g., chemicals, food
additives, cosmetics, pharmaceuticals and detergents) can exist in parallel with little
communication or collaboration between those industries. Thus, tests and assays may be
different in different industries; to use an accepted test from one industry, that test may
have to be submitted to a different agency to be accepted as a qualified method in that
new sector. At the same time, however, some promising steps were being taken. For
example, Directive 2010/63/EU that aims to protect animals for research purposes across
all industries in all European Union countries has established one of the most stringent
ethical and welfare standards worldwide. It requires that the 3Rs and welfare standards
for animals be integrated in all aspects of drug development. Its ultimate goal is to
replace animals in research with non-animal methods (Cozigou et al., 2015).
If a new method is to be used as a basis for safety assessment, it must first be
validated. Validation criteria for new toxicological test methods are driven by
three organizations: The Organization for Economic Cooperation and Development
(OECD), ECVAM, and ICCVAM. Several alternative methods, such as certain

7
genotoxicity and local cutaneous toxicity tests, have already successfully undergone full
validation (Kandarova and Letasiova, 2011). However, achieving validation does not
automatically ensure regulatory acceptance or international recognition of these test
methods. It is a complex process involving not only scientific considerations, but also
policy issues, such as cost-benefit evaluations, traditional practices and requirements that
may be unique to each country for international acceptance (Burden, Sewell and
Chapman, 2015; Schiffelers et al., 2014). Thus, implementation can be challenging and
slow. Increased communication and collaboration between these organizations and the
industry are important activities that would enable improvements in the validation
processes and their eventual adoption worldwide.
The European Coalition to End Animal Experiments (ECEAE) expressed concern
in a meeting held by the In Vitro Testing Industrial Platform (IVTIP) in May 2013 that
the implementation of alternative methods is not made available after validation,
preventing a widespread uptake by companies. The alliance suggested that “post-
validation stages need to be considered to speed and smooth the implementation of new
tests and summarized the validation and post-validation stages as assessment, decision,
acceptance, policing and transparency (ADAPT)”. Participants at that forum argued that
“the regulatory requirements and processes must be simplified, and the frequency and
effectiveness of collaborations and communication must increase transparency” (Ashton
et al., 2014, p.360). One major hurdle is the need for international acceptance which can
substantially delay implementation. Thus, key regulatory agencies internationally will
need to agree on the mutual acceptance of alternative methods that have been
successfully validated using principles of validation established by OECD, ECVAM and

8
ICCVAM. Further, it may be important to organize the full array of available methods,
including in silico, in vitro, and in vivo methods, in a central repository to enable more
universal access and application of the methods if they are to be implemented effectively
in future.
1.2 Statement of the Problem
Most stakeholders in drug development see advantages in the further development
of testing methods that reduce animal use. However, despite the introduction of new
technologies in the field of safety assessment, companies tend to follow familiar and
established methods. Thus, new alternatives may be used as add-on methods rather than
stand-alone methods to help improve our understanding with respect to the safety or
efficacy of the drug. Further, anecdotal evidence suggests that new test methods, even
after having received regulatory acceptance, do not ‘automatically’ find widespread
application. We do not know much about the magnitude and types of efforts that the
industry has made to adopt the in vitro alternatives despite extensive encouragement and
investment by the FDA. We also do not know whether the industry is seeing this
commitment as a positive step or a step that will be costly and confusing for different
aspects of the preclinical drug development program. Obstacles such as cost, expertise,
resources, time and inexperience have been suggested to hinder the implementation of
alternative methods, but we do not know the relative degree to which these impediments
contribute to slow implementation.

9
1.3 Purpose of the Study
The goal of this exploratory study was to understand the challenges in
development and implementation of alternative methods to animal testing in the field of
safety assessment in North America through the eyes of one group of key stake holders –
the US pharmaceutical industry and the contract research organizations (CROs) working
with them to conduct preclinical testing. Using an implementation framework developed
by Fixsen (Fixsen et al., 2005) to base a survey, this study explored the use of non-animal
alternatives in pharmaceutical safety assessment with the goal of understanding where the
greatest impediments to implementation exist and to gather the potential
recommendations of the stake holders.
1.4 Importance of the Study
Today, most studies to assess the safety of drugs seeking marketing approval are
still based on extensive animal research as evident from the guidance outlined in
International Council for Harmonization (ICH M3 (R2)) guidelines (ICH, 2009). The
results of this study provide industry stakeholders with a better understanding of the
current state of alternative methods in safety assessment. These results may also play an
important role for future policy decisions and tactical approaches to facilitate better
implementation. The study identified some of the challenges serving as barriers to the
adoption and implementation of alternative methods. The findings of this study may be
important to the regulators as they assess the effectiveness of the current regulatory
strategies to foster the development and implementation of alternative strategies. Results
highlight issues and gaps that need to be addressed specifically by the FDA. This may be

10
also be useful in improving the process for promoting the use of qualified in vitro
methods for regulatory safety testing.
Cost, ethical issues, and quality of scientific data are areas of importance and the
results of this study will help policy makers from both government and the private sector
to understand if more efforts need to be undertaken for effective implementation of 3 Rs.
1.5 Limitation, Delimitations, and Assumptions
The research described here was conducted using a survey and therefore has
certain limitations. The validity of survey methods can be affected if many potential
respondents fail to participate, either because of time constraints or lack of interest. To
reduce attrition, it was important that the survey be kept as short as possible. However, a
short survey may miss important areas of concern. If the participants are not evenly
distributed across companies of different sizes and product lines, the results may not be
fully representative of the larger group that the sampled respondents are purported to
represent. Some respondents may not share the opinions of the organization for which
they work and may be hesitant to provide candid feedback.
The research has also been delimited in ways that could affect its external
validity. Although the use of animal alternatives is of interest in several sectors, such as
chemical, cosmetic and food sectors, the primary focus of this dissertation is the use of
alternative models in the pharmaceutical and biologics industries. The majority of survey
participants were drawn from scientific groups, including those engaged in research,
development and toxicology. Thus, it may not reflect the experience of individuals in
other areas such as translational science, pharmacology or basic research, even though

11
those individuals may have experiences and opinions about animal research. It is further
directed at scientists working for biopharmaceutical companies in the United States.
Thus, the data will be US-centric; EU-based companies may have slightly different views
on the subject. Notably, academic laboratories in the US represent a large sector involved
in animal research but are not captured in this study. However, this latter fact does not
detract from the importance of this survey tool.
Data in this study represents a snapshot in time for the years close to 2021. There
has been a significant increase during this time in the numbers of small
biopharmaceutical companies. Individuals working in these companies may have less
experience about historical trends in their companies given that those organizations have
not been in existence for any length of time. However, they should be able to speak to
current company practices and to their views on future trends. An attempt was made to
survey companies across the country. However, a large majority of the companies are
primarily located on the west coast and east coast. Data would be assumed to represent
the current state of affairs for all biopharmaceutical companies in the US.
The 21
st
century has seen the robust emergence of biopharmaceuticals, which
often are human-specific and are mainly evaluated in non-human primates (NHPs).
Since these are a relatively new class of drugs, insufficient work has been done to
evaluate alternatives methods for their safety assessment. This may artificially skew the
views and results. Nevertheless, the data collected is assumed to be generally
representative of the current trends in drug development.

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1.6 Organization of Thesis
This dissertation is organized into five chapters. Chapter 1 provides an
introduction to the research study, statement of the problem, the purpose and importance
of the study being undertaken, and the limitations, delimitations, and assumptions that
should be taken into consideration in evaluating the research. Chapter 2 presents a
literature review that synthesizes the current state of knowledge. Chapter 3 describes the
research methodology devised to investigate and analyze the issue for the study. Chapter
4 reports the results obtained from the survey. Chapter 5 discusses the results, author
opinions based on the results, and recommendations for additional steps, if any.
1.7 Definitions
3 R’s Replacement, Reduction, and Refinement
ABPI Association of the British Pharmaceutical Industry
ACT American College of Toxicology
AD Anno Domini (after death)
ADAPT Assessment, decision, acceptance, policing and transparency
APHIS Animal and Plant Health Inspection Service
AOPs Adverse outcome pathways
ATSDR Agency for Toxic Substances and Disease Registry
AWA The Animal Welfare Act
AZT Azidothymidine
BraCVAM Brazilian Center for the Validation of Alternative Methods
CAAT Center for Alternatives to Animal Testing
CAGR Compound Annual Growth Rate
CDER Center of Drug Evaluation and Research
CPI Critical Path Initiative
CPSC Consumer Product Safety Commission
C-PATH Critical Path Institute

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CSDD Tufts Center for the Study of Drug Development
CRO Contract Research Organization
CT Computed Tomography
CULABBR Committee on Use of Laboratory Animals in Biomedical and
Behavioral Research
CYP Cytochrome P450
DARPA Defense Advanced Research Projects Agency
DNA Deoxyribonucleic Acid
ECEAE European Coalition to End Animal Experiments
ECI European Citizens' Initiative
ECVAM European Center for the Validation of Alternative Methods
EMA European Medicines Agency
EPA Environmental Protection Agency
EU European Union
EURL European Union Reference Laboratory for Alternatives to Animal
Testing
FDA Food and Drug Administration
FDCA Food, Drug, and Cosmetic Act
FIH First-In-Human
FNIH Foundation for the National Institutes of Health
HIV Human Immunodeficiency Virus
IACUC Institutional Animal Care and Use Committee
ICCVAM Interagency Coordinating Center for the Validation of Alternative
Methods
ICH International Conference on Harmonisation of Technical
Requirements for Registration of Pharmaceuticals for Human Use
IMI Innovative Medicines Initiative
IVTIP In Vitro Testing Industrial Platform
JaCVAM Japanese Center for the Validation of Alternative Methods
JRC Joint Research Centre
KOL Key Opinion Leader
LAS Laboratory Animal Science

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LD50 Lethal Dose, 50%, or median lethal dose
MDR Multi-Drug Resistance
MRI Magnetic Resonance Imaging
NaBDEN Nonclinical and Biological Discovery Expert Network
NAS National Academy of Sciences
NASA National Aeronautics and Space Administration
NCGC National Institute of Health Chemical Genomics Center
NHP Non-human primate
NIH National Institutes of Health
NIRN National Implementation Research Network
NME New Molecular Entity
NRC National Research Council
NSMR National Society for Medical Research
NTP National Toxicology Program
OECD Organization for Economic Cooperation and Development
PET Positron Emission Tomography
REACH Registration, Evaluation, Authorization and restriction of Chemicals
R&D Research and Development
SEURAT Safety Evaluation Ultimately Replacing Animal Testing
SME Subject Matter Expert
SOT Society of Toxicology
TOX21 Toxicity Testing in the 21st Century
TOXCAST Toxicity Forecaster
TSCA Toxic Substances Control Act
UFAW Universities Federation for Animal Welfare
UK United Kingdom
US United States
USC University of Southern California
USDA United States Department of Agriculture
VA Veterans Administration
WoE Weight of Evidence

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Chapter 2. Literature Review
2.1 Introduction
The use of animals in biomedical research and drug development has been viewed
historically as a scientifically crucial step in predicting the effects that a medical product
will have on humans. The structural and physiological similarities between humans and
other animal species have led to their use in demonstrating proof-of-concept studies for
experimental medicines or procedures, elucidating pathophysiological mechanisms, and
evaluating the safety of novel candidate therapies before applying them to humans.
Animals have also served as surrogates to understand effects of medical procedures and
surgical interventions prior to their use in human patients. Animal models of infectious
diseases have been essential to evaluate antimicrobial agents and to develop vaccines.
Animals have even been used as biological factories to source and manufacture biological
products. To accomplish all of these goals, a wide range of animals- mice, rats, hamsters,
rabbits, guinea pigs, cats, dogs, and primates, as well as various species of birds, fish and
amphibians – have had specific uses as animal models (National Research Council and
Institute of Medicine, 1988).
As stated by a Royal Society report in 2006 (The Royal Society, 2006, p.1):
We have all benefited immensely from scientific research involving
animals. From antibiotics and insulin to blood transfusions and
treatments for cancer or HIV, virtually every medical achievement in the
past century has depended directly or indirectly on research on animals.

Animal research has played an important role in biomedical research as 70% of
the Nobel prizes are awarded in categories of physiology or medicine (Foundation for

16
Biomedical Research, 2019b) were based on animal experiments. As an example, the
Nobel laureate Albert Sabin once said in an interview in 1993, “there could have been no
oral polio vaccine without the use of innumerable animals” (Speaking of Research, 2011,
para. 20). Animals are still needed to test every new batch of polio vaccine produced
today.
Nonetheless, the continued reliance on animals has had many critics. Animal
research is now often viewed as anachronistic. Advances in science continue to provide
new tools to reduce or in some cases replace animal research altogether. In the present
study, we attempted to trace the use of animal research, examine its current state, and
describe the extent of current literature related to the views of industry-employed
toxicological experts regarding the need for animal use as part of pharmaceutical
development.
2.2 History of Animal Contributions to Medical Research
Animals have been used by humans for thousands of years for food, transport,
labor, protection and companionship. It is thus not surprising that their use in medicine
also has a long history. According to Baumans,
the use of animals in experimental research parallels the development of
medicine, which had its roots in ancient Greece where Aristotle and
Hippocrates laid down their knowledge on structure and function of the
human body based on their dissection of animals (Baumans, 2004, p.S64).
Galen (130–201 AD), physician of the Roman Emperor Marcus Aurelius,
dissected animals and conducted physiological experiments on pigs, monkeys and dogs to
learn how the body functions. His experiments formed the basis for medical practices for
many centuries thereafter (Baumans, 2005). Reports on the historical use of animals in

17
the quest to gain insight into human anatomy and physiology are captured in several
reviews throughout the last millennium, a history well-reviewed elsewhere (Franco,
2013) that will only be abstracted here.
In 1859, Darwin’s publication, “On the Origins of Species”, emphasized the
biological similarities between man and animal, and thus contributed to the justification
of animal experimentation. In 1865, Claude Bernard published his book, “Introduction à
l’Etude de la Médecine Expérimentale” in which he introduced basic principles of
scientific research for designing experiments. Over time, the creation of other biomedical
sciences such as microbiology, biochemistry, pharmacology, toxicology, cell biology,
immunology and neuroscience eventually led to a sharp increase in the use of animals for
medical research into and throughout the 20th century.
It is not possible to enumerate the thousands of ways in which animals have
contributed to the development of important medical advances and scientific revolution.
An excellent review of these contributions can be found in a comprehensive review, titled
“Medical advances and animal research” (Research Defense Society, 2007). Even a few
examples described below underline the value of that research. Blood transfusion was
developed when citrated blood was shown to be safe for transfusion in dogs in 1914; this
technique has saved the lives of countless people and animals. Banting and Best won the
1923 Nobel Prize for the discovery of insulin in dogs; this discovery has saved millions
of lives. Florey and Chain first tested the effects of penicillin in mice in 1940 and by
1941, penicillin was being used to treat dying soldiers; the researchers won the Nobel
Prize for this work in 1945. The discovery of heparin, an anticoagulant occurring
naturally in mammals, became a crucial drug after purified extracts were shown in 1937

18
to be a safe and effective anticoagulant in dogs, rabbits, guinea pigs, mice, and
subsequently, human patients. A hundred years ago, tuberculosis was one of the most
common causes of death. Nobel Prize-winning research on guinea pigs in the 1940s led
to the antibiotic, streptomycin, the first successful treatment for tuberculosis.
Experiments on guinea pigs, rabbits, dogs and monkeys formed the basis for peritoneal
dialysis and paved the way for the first successful treatment of a patient with acute
kidney failure by Willem Kolff, in 1945. About 40 years of research using monkeys, rats
and mice led directly to the introduction of the Salk and Sabin polio vaccines in the
1950s. In 1987, AZT (azidothymidine), the first drug treatment for human
immunodeficiency virus (HIV), became available through research on mice, rats, dogs
and primates. In 1992, vaccines for meningitis were tested in mice and have resulted in a
huge decrease in the incidence of the disease.
The beginning of the 21st century saw the first steps toward understanding and
manipulating the genetics of humans and other animals. In 1996, Dolly the sheep became
the first mammal to be cloned from an adult cell and demonstrated the enormous power
of DNA to create an entire organism (Wilmut et al., 1997). In 2002, the initial sequencing
and analysis of the mouse genome was published by the Mouse Genome Sequencing
Consortium (Waterston et al., 2002 ). This accomplishment was a major milestone
because it provided the experimental key to the human genome, as 99% of mouse genes
have human counterparts (Dutta and Sengupta, 2016). It also opened the door to
“humanizing” mice so that they could accept human proteins and tissues without
responding immunologically to them (Devoy et al., 2011). Breast cancer, once a deadly
disease, can now be treated successfully with tamoxifen, one of the most effective

19
treatments that was first tested in animals. In the last decade, the use of stem cells for
spinal cord repair, angiogenesis inhibitors for cancer, and gene therapy for muscular
dystrophy, cystic fibrosis, retinal degeneration and spinal muscular atrophy are just a few
of numerous medical advances in progress due to genetic research in animals.
Although animal research is conducted in many countries, it is not possible to
estimate accurately the total number of vertebrates used in research because numbers are
estimated in different ways. Some countries count the number of animal experiments or
procedures while others count the actual number of animals used; furthermore, no
country maintains a count of invertebrates used in research. The EU counts all vertebrates
as well as cephalopods (octopuses, squid etc.). However, in the US, only warm-blooded
animals are counted, and these numbers exclude rats, mice, and birds. In the United
Kingdom, 97% of procedures were conducted on animals such as rats, mice, birds or fish
and across the EU, 93% of research was conducted on the same categories of animals.
Similarly, most of the millions of animals used in research in the US have a similar
distribution across species. However, their numbers, suggested to be 11-23 million in the
US (Speaking of Research, 2019), are difficult to estimate because they are not counted
under the US Animal Welfare Act. The Animal Welfare Act was the first federal law in
the US regulating animals in research and applies to animal carriers, handlers, dealers,
breeders, and exhibitors in addition to research laboratories. It sets minimum standards
of care that must be provided for animals- including housing, handling, sanitation, food,
water, veterinary care and protection from weather extremes. (USDA, 2012). According
to government statistics, the use of non-rodent animals has been declining over the past
two decades, as genetically modified mice become the species of choice for most

20
experiments. Non-rodent mammals account for 1% or less of the animals used in
research every year. The statistics suggest a significant decrease in the number of
procedures on dogs and primates compared to ten years ago (Understanding Animal
Research, 2019). To put this in perspective, fewer animals covered by the Act are used in
research than ducks eaten per year in the US (Speaking of Research, 2019).
Mice and rats are the most frequently used research animal species for a few
reasons. They have sufficient anatomical, physiological and genetic similarities to
humans to serve as good models in many types of research (Foundation for Biomedical
Research, 2019a). Additionally, due to their small size they require little space or
resources to maintain them, they reproduce quickly but produce large numbers of
offspring, have rapid development to adulthood and relatively short life spans. Using
genetic engineering, scientists can “knock in”, “knock out” or “knock-down” disease-
related traits in mice and rats to make them more “human-like” (Yang, Wang and
Jaenisch, 2014). Research with genetically modified transgenic mice and rats has
provided new opportunities to treat difficult conditions including those based on genetic
abnormalities.
At the same time, the patterns of animal usage are also shifting as the types of
medicinal products under development are changing. Today, fewer small molecules than
larger biologics are entering the market than was typical even a decade ago (Torre and
Albericio, 2017). The types of safety testing required for large molecules- often with
potentially allergenic components- differ from those typically associated with small
molecules. The safety testing of small molecules most commonly follows a ‘standard’
nonclinical approach based on toxicological studies in rodent and canine or minipigs.

21
However, most biopharmaceuticals must be tested in NHPs, because the greater
immunological homology of NHP with humans can improve the predictability of immune
responses and efficacy in humans. Hence, a majority of safety assessment studies are
conducted only in NHPs such as cynomolgus or rhesus monkeys. As biologics continue
to become a larger part of the pharmaceutical compendium, the number of NHPs used for
testing is also projected to grow. Currently, non-human primates represent a very small
proportion of experimental animals, estimated to be fewer than 1 in 1,000 in the EU and
approximately 3 in 1,000 in the US. According to the 2017 animal-research statistics
report from USDA/APHIS, non-human primates are one of the few species whose usage
has risen, from an average of 54,000 animals per year from 1977-2006, to 67,000 in
2007-2016 (Speaking of Research, 2017). Nevertheless, much of the primate testing is
done overseas so this number still may exceed 100,000 worldwide each year (Chatfield
and Morton, 2018).
2.3 The Evolution of Toxicological Oversight by US Regulatory Systems
Until the 1930s, the US had few laws to prevent the sale of unsafe food or drugs.
This situation changed in 1937, when the Massengill Company, a reputable
pharmaceutical manufacturer, created a new liquid version of sulfanilamide, a drug used
to treat streptococcal infections, by dissolving the drug in diethylene glycol - better
known today as antifreeze. The new formulation, marketed without safety testing in
animals, was later discovered to cause severe organ toxicities that caused more than 100
deaths, mostly in children (Ballentine, 1981). The subsequent difficulties of holding
Massengill liable for these deaths made it clear that the existing laws were insufficient to

22
protect the public from unsafe drugs. In response, Congress passed the Food, Drug, and
Cosmetic Act (FDCA) of 1938, which required manufacturers to provide evidence of
drug safety from animal toxicity studies before a new drug could be approved for
marketing (FDA Review, 2019).
For the next few decades, many companies added some type of testing to their
development strategies, but that testing was often less than systematic. In the 1950s,
another tragedy, associated with a newly introduced drug called thalidomide, drew
attention to the relatively underdeveloped methods that were being used to test food and
drug products. Thalidomide was a new sedative placed on the European market by the
West German pharmaceutical manufacturer, Grünenthal, in 1957. The drug was widely
prescribed to alleviate the symptoms of morning sickness in women during the first
trimester of pregnancy. By 1962, evidence was mounting that the drug caused birth
defects including an unusual syndrome called phocomelia (Greek for “seal limb”) in
which babies were born with truncated limbs that resembled flippers. By the time that
the birth defects could be associated with thalidomide, the drug had been sold in forty-six
countries and had injured thousands of newborn babies (Fintel, Samaras and Carias,
2009). The tragedy became headline news throughout the world.
The US was less affected than many other countries by the thalidomide crisis.
Concerns about the adequacy of safety testing had led the FDA reviewer, Frances Kelsey,
to delay the approval of the drug, thus preventing its use in the US before the drug’s
teratogenic effects became known (Seidman and Warren, 2002). Nevertheless, the fears
that drugs could enter the market without a good understanding of their adverse effects
prompted a major amendment to the FDCA known as the Kefauver-Harris Amendment,

23
in 1962 (FDA, 2012). This amendment strengthened the original law to require stronger
proof not only of drug safety but also of drug efficacy. To satisfy this requirement,
animal models were quickly incorporated into drug development.
A further requirement for safety testing arose in 1958, when The Food Additive
Amendment of 1958 (FDCA) was added in response to concerns about the safety of new
food additives. It required manufacturers to demonstrate the safety of food additives
before they could be introduced into the food supply (Frankos and Rodricks, 2001). The
FDA was given authority to develop toxicity studies for assessing food additives. To that
end, it engaged toxicologists in FDA, academia, and industry during the 1950s and 1960s
in developing protocols and standards, most of which are still used today (FDA, 1959).
In the late 1960s, the development of toxicological testing strategies was also
affected by the concerns of environmental groups. Rachel Carson published “Silent
Spring” in 1962 documenting the adverse environmental effects caused by the
widespread overuse of pesticides (Carson, 1962). In 1970, the U.S. EPA was established
with the mission “to protect human health and the environment” (EPA, 2018). The EPA
then also became involved in developing toxicity testing to evaluate pesticides and
industrial chemicals that could eventually appear as food residues or environmental
contaminants.
The FDA’s drug- and food-additive testing programs and EPA’s pesticide testing
strategies included safety evaluations of chemicals that had specific uses in medical,
food, or other commercial products. Additional testing was also developed in response to
other specific regulatory concerns (Krewski et al., 2010). For example, potentially
hazardous agents in the environment which may be of public-health concern are

24
subjected to testing through the National Toxicology Program, an interagency program
established in 1978 (National Toxicology Program, 2019).
The NTP developed the rodent cancer bioassay, the current gold standard for
carcinogenicity testing, that involves exposing specified groups of standard-bred male
and female rats or mice to the test agent for 2 years, a period encompassing their normal
life span. NTP has also initiated the development of medium- and high-throughput non-
animal alternative tests to evaluate newly introduced chemicals and existing chemicals.
These tests are formally reviewed by ICCVAM established in 2000, to ensure that they
have value in regulatory decision making (Krewski et al., 2010).
Another organization that has had significant influence on regulatory toxicology
is the Organization for Economic Cooperation and Development (OECD). In 1982,
OECD became the lead organization to develop harmonized international guidelines for
the testing of chemicals (Walker, 1984). The goal of the harmonization program is to
eliminate unnecessary duplication of toxicity tests conducted by member countries. As
the regulatory requirements for developing new drugs grew to be more expensive and
time-consuming, regulators and industry in the EU, US and Japan began to recognize the
value of working together in order to further harmonize the technical requirements for
new drugs. A significant further step towards harmonization occurred with the launch of
the International Conference on Harmonization of Technical Requirements for
Registration of Pharmaceuticals for Human Use (ICH) in 1990. The purpose of the ICH
was to
…make recommendations on ways to achieve greater harmonization on
the interpretation and application of technical guidelines and
requirements for product registration in order to reduce or obviate the

25
need to duplicate the testing carried out during the research and
development of new medicines (Ohno, 2002, p.S95).
According to the International Council for Harmonization (ICH M3 (R2))
guidelines (ICH, 2009), the nonclinical safety assessment for marketing approval of a
pharmaceutical would include a rather specific grouping of pharmacology studies, safety
pharmacology studies, general toxicity and toxicokinetic studies, metabolism and
nonclinical pharmacokinetic studies, reproduction toxicity studies, and genotoxicity
studies. For drugs that have special cause for concern or are intended for long durations
of use, an assessment of carcinogenic potential is also required. Other nonclinical studies
to assess phototoxicity, immunotoxicity, juvenile animal toxicity and abuse liability are
conducted on a case-by-case basis (Figure 2). Because of harmonization of these
guidelines for safety testing, a significant reduction in animals was achieved simply by
accepting a common nonclinical strategy across different regions.
Figure 2: General Nonclinical Testing Paradigm

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2.4 Introduction of the “3Rs”
All of the activities identified above to formalize animal testing strategies have
been carried out against a backdrop of societal advocacy regarding animal welfare that
became apparent by the middle of the twentieth century. At that time, scientists, ethicists,
and the general public were already expressing concerns about the need for so many
animal experiments of different types. This prompted the Universities Federation for
Animal Welfare (UFAW), an independent charity founded in England that promotes
animal welfare, to undertake large scale scientific studies of humane experimental
techniques that might be employed in animal experiments. That project led to a
pioneering book, The Principles of Humane Experimental Technique (Russell and Burch,
1959) in which Russell and Burch proposed the concept of “3Rs” as a framework to
guard the welfare of animals used in science.
The acronym “3Rs”, are a short form that stands for the “Replacement,
Reduction, and Refinement” of animals using scientific ingenuity without compromising
scientific rigor. Each of these areas of activity has a specific set of initiatives.
“Replacement” referred to ways in which investigators could avoid using whole, sentient
animals, by substituting: (i) non-animal approaches such as in vitro methods,
microorganisms, ethical human studies, and computer simulation, (ii) experiments using
invertebrates, or early-stage vertebrate embryos, and (iii) anesthetized vertebrates with
the goal of minimizing pain and distress. Over time, use of anesthetized vertebrates has
come to be viewed as a refinement rather than replacement.
“Reduction” referred to any strategy involving better design and analysis of
animal experiments that would reduce the use of animals. Russell and Birch argued that

27
the spirit of reduction was not served when experiments were poorly designed or
analyzed. They also called for the increased use of genetically uniform animals or the
offspring of crosses between two different in-bred lines, as a means of controlling inter-
individual variation and thus reducing the variability that would require the use of more
animals to assure a statistically demonstrable difference between groups (Stephens et al.,
2014).
“Refinement” referred to modifications of experimental procedures to minimize
pain, suffering, or distress to animals. Russell and Burch considered a wide range of
approaches toward this goal, including the effective use of anesthesia and analgesia; the
management of euthanasia; the selection of injection sites; the use of less sentient
species; and the adoption of less intense experimental procedures to induce stress. The
scope of refinement eventually expanded to include animal welfare enhancement through
the implementation of social housing and enrichment of the caged animals (Weary,
2011).
Today, the 3Rs are the guiding principles underpinning all scientific research
involving animals not only in the US but also in most countries throughout the world.
2.5 Alternative methods
It seems obvious that alternative methods could offer many benefits with respect
to economy and efficiency. These data may be used to increase the efficiency of whole-
animal studies and decrease the number of animals for testing. Alternative methods can
be used as partial or full replacements of animals depending on the objective of the study.
They can be generally grouped into certain subtypes of methodologies: (a) in

28
vitro methods such as primary cultures, finite lifespan cell lines, continuous cell lines,
and reconstructed 3D tissues; (b) ex vivo methods such as the use of isolated animal
tissues and organs; and (c) in silico methods such as computer simulations and
mathematical models (Kandarova and Letasiova, 2011).
There are many advantages to studies that do not rely on living animals. It is
easier to control the testing conditions and thus assure a higher level of standardization
and reduced variability between experiments. Toxicological effects in a particular tissue
or substrate can be isolated from systemic effects. Such tests are also typically
inexpensive and fast. They may rely on a much smaller amount of test material and
produce a much smaller amount of toxic or biological waste. Finally, it is possible to use
human cells and tissues or transgenic cells carrying human genes. However, these
methods also have serious limitations. They do not permit the evaluation of systemic or
chronic effects. They do not permit multi-generational studies and provide little insight
into pharmacokinetics and other interactions between tissues and organs (Kandarova and
Letasiova, 2011).
2.5.1 Are In Vitro Assays Alternative or Complementary to Animal Tests?
Modern technology has enabled scientists to cultivate in vitro preparations of
almost every kind of cell from every animal species, including humans. However, it is
essential to determine objectively whether a particular in vitro test is truly an alternative
and can therefore replace an in vivo experiment, where the complexities of absorption
and metabolism defy easy replacement. Drugs are easy to deliver directly to cells in vitro.
However, the delivery is nothing like that encountered when a drug is administered, for
example, by mouth. In such a case, the drug will face several absorptive barriers, such as

29
the blood-brain and the intestinal barriers that are hard to reproduce in vitro. Further,
during in vivo absorption a drug will interact with the microbiome, affect intestinal
motility, be adsorbed onto fibrous matter in food, be metabolized by cytochrome P450
(CYP) enzymes in the intestine, or be transported or excreted after absorption by the
multi-drug resistance (MDR) complex (Bodo et al., 2003).
Once a drug is absorbed by the intestine, it may bind to circulating proteins and
distribute to various organs. The drugs may experience “first pass metabolism” in the
liver which can cause drugs to be metabolized to form several metabolites with similar or
different activities. In some cases, those metabolites may even be toxic or neutralize the
action of the parent drug. Therefore, the effects of a drug in vivo, in contrast to that in
vitro, may be related not just to a single chemical species but also to a mixture of multiple
chemical forms. It is very difficult to replicate such metabolic effects in vitro, even when
CYP enzyme mixtures responsible for the degradation of some drugs are added to mimic
the in vivo process. Further, in vivo activity is more dynamic than that seen under in
vitro conditions because absorption, distribution, metabolism and excretion change with
age, time and physiological conditions. Cells or tissue cultures by themselves cannot
mimic the complexity of a living organism whose organs are affected by nervous,
hormonal, immunological and circulatory systems. Interactions between drugs and
functional conditions such as blood pressure, sleep or cognitive activities cannot at
present be studied in vitro. Animal species employed for pre-clinical tests have many
features similar to humans; they have similar organs, similar circulatory, hormonal,
neural and immunological functions. The genomic organization, too, is similar although
not fully homologous. Animal proteins are in some cases very similar or identical and

30
metabolic processes are similar. All of these considerations suggest that while cell or
tissue culture methods are useful for studying some of the effects of drugs, they still may
be considered as complementary, and not an alternative, to in vivo studies.
2.5.2 Specific Issues related to Cell-Based Tests
Cell lines are popular because of their low cost and multifaceted nature. However,
they have shortcomings that can affect their usefulness. Cell lines are frequently
misidentified or contaminated, and this can be a particular problem in certain fields, such
as cancer research, where drugs are initially tested using a cell line derived from the type
of tumor that the patient is considered to have (Weinstein, 2012). Testing of a drug on an
incorrect or degraded cell line can lead to misleading results that will delay or defeat the
development of effective treatments. Since the 1960s, more than 400 cell lines used
worldwide have been shown to have been misidentified (Capes-Davis et al., 2010). In
some cases, cells thought to have been derived from one tissue type were found to belong
to a different tissue; sometimes even the cell species had been identified incorrectly. A
2011 study of 122 different head and neck cancer cell lines revealed that 37 (30%) were
misidentified (Zhao et al., 2011). An analysis conducted on various tissue cultures and
cells from labs in the US, Europe, and Asia suggested that at least 15% of cell lines are
misidentified or contaminated (MacLeod and Drexler, 2006). Misidentified cell lines can
compromise medical research at many levels. For example, experimental results based
on several contaminated cell lines have been identified to result in inappropriate clinical
trials. They are implicated in at least one incorrect US patent, 100+ misleading scientific
publications and three undeserved research grants funded by the National Institutes of
Health (NIH) (Boonstra et al., 2010).

31
Further, cell lines often contain many chromosomal anomalies as they replicate
from one generation to the next. Thus, cells from the same line in two laboratories may
not be identical biologically. It is thus critical that cell-line identity be validated and
reported accurately. This step is not widely recognized by researchers and regulatory
agencies. Greater efforts are required to develop technology to determine the extent to
which genetic drift may affect the reproducibility of results on such cell lines. The time
and cost required for quality control in terms of authentication, contamination and
stability of cell lines has also been a barrier to its widespread adoption (Lorsch, Collins
and Lippincott-Schwartzl, 2014).
Tissue culture conditions are inherently unable to replicate the interactions
between different cell types that are found in most tissues. Furthermore, such test
systems cannot be used for studies that require tests over several generations
(teratogenicity, endocrine disrupting activity). Therefore, it seems unlikely that cell and
tissue cultures can sufficiently replace animal research in the near future.
In the absence of realistic alternatives to replace the animals used in research, the
emphasis often shifts toward reduction and refinement. As the knowledge of human
biology continues to grow, some of the limitations of current in vitro methods may be
overcome as innovative technologies, for example, in the field of tissue engineering, are
used to develop more effective and efficient interventions. Organs or Tissues-on-Chips
are some of those innovative, alternative tools that can be used for drug screening and
safety testing to provide early readouts of efficacy and toxicity. This technology is
expected to “merge advantages of animal models and cell culture by combining high

32
physiological relevance with high throughput capacities” (Probst, Schneider and Loskil,
2018, p.1). It is hoped that these devices will be able eventually to replace animal testing.
2.6 Challenges to Animal Use and Paradigm Shift
In the years since the initial promulgation of the 3 Rs, scientists, regulators and
the public in general have agreed that the practice of toxicology needs to change from the
present traditional paradigm of conducting most tests in laboratory animals. Several
forces appear responsible for driving this shift. For purposes of discussion, they can be
grouped broadly into five areas of concern: (a) scientific and technological, (b) economic/
business, (c) ethical/societal, (d) political, and (e) regulatory.
2.6.1 Scientific and Technological Drivers
To date, drug development has relied heavily on animal models that may
recapitulate only selected aspects of human physiology or disease (Zerhouni, 2014;
DiMasi, Grabowski and Hansen, 2016). The failure of animal studies to predict drug
efficacy and toxicity in humans can have several causes, including flaws in experimental
design or investigator bias, but species variations also contribute to erroneous predictions
(Bracken, 2009). Toxicity predictions can depend on many factors including the species,
the study duration and the specifics of the test (Greaves, Williams and Eve, 2004). The
concordance between animal and human toxicity also varies greatly according to the
organ under study. Furthermore, the customary practice to expose animals to much higher
doses of drug than would be used in humans is viewed as flawed by many scientists
(Sewell et al., 2017). Thus, scientific bodies are concerned that more needs to be done to
assure that toxicological data is more human- relevant (EU, 2016; National Research

33
Council, 2007). A number of scientific organizations, including the National Institutes of
Health, the US Environmental Protection Agency, the US Department of Defense, the
Defense Advanced Research Projects Agency (DARPA), now advocate and work toward
better methods based on the growing understanding of human physiology (Zerhouni,
2017). Advances in computer science enabling data storage and mining have also been
explored to determine if they can provide additional tools to revise toxicological
methods.
The biggest barrier or driver for achieving widespread acceptance is the
standardization and validation of the alternative methods. Alternative tests must
demonstrate that they are relevant, robust, reproducible, and fit for the purpose intended.
The regulatory agency can then have a basis for deciding if the performance standard of a
new test method makes the new method “fit for purpose” in terms of its applicability for a
particular regulatory requirement, and whether it has limitations with respect to such
features as reliability, reproducibility, and sensitivity. For this reason, an assay may be
“qualified” for a narrower application, called a “context of use”, defined as “a conclusion
that the results of an assessment using the model or assay can be relied on to have a
specific interpretation and application in product development and regulatory decision-
making” (FDA, 2017, p.7). Once a new test method has been qualified, it can be then
employed with confidence for the qualified purpose.
Formal mechanisms for validation and qualification have now been established by
different organizations. One important US organization is the ICCVAM, whose
report, Guidelines for the Nomination and Submission of New, Revised, and Alternative
Test Methods serves as a good textbook on how to qualify a method (ICCVAM, 2003).

34
In the EU, European Union Reference Laboratory for Alternatives to Animal Testing
(EURL-ECVAM) plays a similar role. However, the processes needed to qualify a
method are rigorous and involve significant investments of money and time. Thus, many
feel that government agencies should provide incentives to encourage greater
participation in methods validation by academic laboratories and companies. Further,
given the complexity of qualification, they feel that validation methods would profit from
a more well-defined and streamlined approach (Burden, Sewell and Chapman, 2015). For
example, the current process of validation involves benchmarking results against the
current animal models, which are still considered the gold standard. This practice may
need to change, and the regulators should focus on addressing the specificity and
sensitivity of human-relevant tests (Burden, Sewell and Chapman, 2015).
2.6.2 Economic/ Business Drivers
Current animal-testing programs are lengthy, expensive and resource intensive.
Their costs contribute significantly to the 15-20 percent of revenues that industry invests
each year on research and development activities (International Trade Administration,
2016). According to Tufts Center for the Study of Drug Development (CSDD), the
average pre-approval cost of developing a prescription drug is estimated to be $2.6
billion. (DiMasi, Grabowski and Hansen, 2016). This represents a 145% increase over the
estimate made in 2003 (Mullin, 2014) (Figure 3). It is therefore not surprising that the
yield of new drugs approved per billion US dollars spent has been declining (Scannell et
al., 2012). Part of the high costs can be attributed to the fact that fewer than 10% of the
compounds entering clinical trials gain regulatory approval, mainly because of
insufficient efficacy and/or unacceptable toxicity (Khanna, 2012). Thus, pharmaceutical

35
companies have strong incentives to put into place a better system to reduce the costs of
the preclinical trial programs if only to eliminate unsafe drugs at an earlier point in
development. For this reason, industry has been engaged in a variety of public-private
partnerships aimed to develop alternative strategies. One such example is the Innovative
Medicines Initiative (IMI) which is the world’s largest such partnership between the
European Union and the European pharmaceutical industry established in 2009 (Stevens
et al., 2015). The IMI was set up to boost the competitiveness of Europe in the
biopharmaceutical field by bringing different stakeholders (pharmaceutical companies,
small- and medium-sized enterprises, universities, public research laboratories, patient
organizations, and healthcare regulators) together.
As a more directed example, the FDA recently formed a partnership with Emulate
Inc., to evaluate and qualify the use of their Organs-on-Chips technology as a platform
for toxicology testing to meet regulatory evaluation criteria for products including foods,
dietary supplements and cosmetics. The system is designed to provide a more predictive
and precise model of human response to diseases, medicines, chemicals, and foods.

36
Figure 3: The Cost of Developing a New Drug Since the 1970s
Reproduced with permission, Tuft CSDD

Source: Tufts Center for the Study of Drug Development
2.6.3 Ethical or Societal Drivers
Animal testing has always been a deeply divided issue from an ethical
perspective. At one extreme are those who argue that animal experimentation should be
abolished regardless of its value in creating new knowledge. They believe that animals
should have the same rights and moral status as human beings. At the other extreme are
proponents of animal research, who argue that animal experimentation is essential and
reasonable if conducted within a suitable ethical framework (Foëx, 2007).
Views of the public on the usefulness and acceptability of animal research has
changed over time. In 1948, the first national poll found a public that supported animal

37
research - 84 percent of respondents approved and only 8 percent disapproved (National
Opinion Research Center, 1949). That view has changed significantly over the
subsequent 70 years. In 2018, the U.S. public appears to be split quite evenly; 47%
approve, while 52% disapprove of animal research (Strauss, 2018) (Figure 4). The
majority of individuals with higher levels of science education were open to animal use in
scientific research (63%). However, only 44% of those with a medium level of science
knowledge level and 37% of those with a low level of science knowledge were
supportive. Views also vary depending on the type of animal and the field of research
being conducted. Experiments on mice and rats are more acceptable than experiments on
dogs or primates; in a 1985 poll, 88 percent accepted the use of rats but only 55 percent
accepted the use of dogs. In the same poll, experiments perceived to provide important
health benefits received much greater support; only 12 percent opposed the use of
animals for cancer or diabetes research, but 27 percent opposed the use of animals for
allergy testing (Rowan and Loew, 2001). Much less support was given to animal
research if that is perceived to cause pain, suffering or some level of harm. Results from
several studies indicated that the people are less likely to support animal research if the
words “pain” or “death” are used (Hagelin, Carlsson and Hau, 2003) or if the research
resulted in harm to animals (Henry and Pulcino, 2009). A majority support strengthening
of federal legislation or regulations along with development and promotion of
alternatives (Rowan and Loew, 2001). Such sentiments and emotions are driving the
regulatory authorities to adopt successfully validated techniques quickly into mainstream
drug development.

38
Figure 4: Pew Survey Results on Animal Use

2.6.4 Political Drivers
Cascading from societal concerns have been political drivers. In 1975, Peter
Singer published the powerful and influential book, “Animal Liberation”, which focused
on protection of animals in research. The book was a central text for the then-nascent
animal rights movement. Animal rights groups criticized how animals were used in
research and advocated “alternatives” in their animal protection campaigns. These more
organized efforts were often coupled with militant activities such as threatening
prominent scientists engaged in animal research, accosting corporate employees of
animal testing facilities, and vandalizing research laboratories. Such tactics played a
significant role in gaining the attention of federal and state legislators, who then were
challenged to respond.
*Respondents who did not give an answer are not shown.
Source: PEW Research Center

39
Europe has perhaps been the most proactive, driven particularly by legislative
changes in the cosmetic and chemical industries. In 2013, the EU banned the sale of
finished cosmetic products and ingredients that were tested on animals (European
Commission, 2013). In 2015, the European Citizens' Initiative (ECI), “Stop Vivisection”,
made a legislative proposal to the European Commission after having gathered the
required one million signatures of support to phase-out the use of all animals in research
completely (Menache, 2016).
In the chemical industry, the introduction of REACH (Registration, Evaluation,
Authorization and restriction of Chemicals) legislation in 2006 mandated the use of
validated alternatives and allowed animal research only when no alternative existed (EU,
2006). Chemical manufacturers and importers were required to assess information about
the hazardous properties of those products. In the absence of adequate data, toxicological
tests of some form still would be required. Another chemical law in the EU called the
“Classification, Labelling and packaging of substances and mixtures Regulation”
requires, or strongly encourages, the replacement of animal testing (European Chemicals
Agency, 2011).
When animal research is carried out in the EU, it is regulated under Directive
2010/63/EU, which replaced the 1986 Directive 86/609/EEC “on the protection of
animals used for scientific purposes”. The goal of the Directive is to protect animals in
scientific research, with the ultimate goal of replacing all animal research with non-
animal alternatives (EU, 2010). The Directive took full effect in the EU on 1 January
2013. This led to the establishment of the European Union Reference Laboratory for
Alternatives to Animal Testing (EURL ECVAM) whose mission includes multiple

40
initiatives to advance the 3Rs of animal procedures. Another large collaborative program
called Safety Evaluation Ultimately Replacing Animal Testing (SEURAT) was launched
in 2011 (www.seurat-1.eu/). Their vision is to introduce fundamental changes in the way
safety of chemicals is assessed by superseding traditional animal experiments with a
predictive toxicology that is based on a comprehensive understanding of how chemicals
can cause adverse effects in humans.
In the US, the first federal law regulating animal research was the “Laboratory
Animal Welfare Act” passed by Congress in 1966 to protect all warm-blooded animals
except rats, mice, and birds bred for research. The Act has been amended several times.
The amendment of 1985, called "The Improved Standards for Laboratory Animals Act",
had two significant outcomes. First, it established an Animal Welfare Information Center
(www.nal.usda.gov/awic) to promote the humane care and use of animals by providing
information on alternatives. Second, it required research facilities to register with the
USDA and establish an Institutional Animal Care and Use Committee (IACUC). The
new IACUC committees would review all experimental protocols involving live, warm-
blooded animals with the aim of assuring the promotion by the researchers of the 3Rs.
At the same time, attention was also paid to the chemical industry, much as it had
been in the EU. Estimates even today suggest that few of the approximately 50,000
chemicals used in U.S. consumer products and industrial processes have been tested fully
(Fischetti, 2010). “The Frank R Lautenberg Chemical Safety for the 21st Century Act”
(EPA, 2016), which amends the “Toxic Substances Control Act” (TSCA) created a
“mandatory requirement to evaluate existing chemicals with clear and enforceable
deadlines”. However, this has proven to be easier said than done. The number of

41
chemicals to be tested greatly exceeds the capacity of contract testing laboratories. The
need has been a major driver to develop efficient, inexpensive and higher-throughput
methods. As a result, the EPA has devoted significant resources to develop new
technologies as a part of the ToxCast (Toxicity Forecaster) system (EPA, 2017) designed
to generate data and predictive models on thousands of chemicals of interest to the EPA.
ToxCast uses cost-effective, state-of-the-art high-throughput screening methods and
computational toxicology approaches to predict potential toxicity in humans. That data is
used to rank and prioritize chemicals for further attention, based on potential human
health risks.
2.6.5 Regulatory Drivers
A number of issues must still be clarified with respect to the use of alternative
testing models, especially if those are to be used for regulatory purposes. Companies
need to feel confident that the data derived using alternative models will be accepted by
the regulatory agencies worldwide. The routine use of non-animal alternative
methodologies for safety assessment will need to be coded into regulatory guidance to
encourage widespread uptake by the industry and other stakeholders. This will also
require consideration of how to integrate and prioritize the multiplicity of approaches,
including in silico, in vitro, and in vivo methods, that will over time become available
(Worth et al., 2014; Rovida et al., 2015; Rouquie et al., 2015). Ten years after the 2007
NRC’s report “Toxicity Testing in the 21st Century: A Vision and a Strategy),
the National Academy of Sciences has issued a 2017 report titled “Using 21st Century
Science to Improve Risk-Related Evaluations” (National Academies of Sciences, 2017),

42
that provides recommendations for integrating new scientific methodologies for assessing
risk.
The FDA has long been involved in efforts to regulate nonclinical testing and give
guidance on best practices. Some of these activities, described below, have been carried
out by the agency itself but more often they are multilateral, and often involve public-
private consultations or even partnerships.
2.6.5.1 FDA’s Critical Path Initiative
Perhaps the flagship for FDA’s involvement in the remodeling of animal testing
regulations has been its support for the “Critical Path Initiative” (CPI). In 2004, the FDA
launched this initiative as a broad effort to reverse what was seen as a steep decline in the
number of innovative medical products submitted for approval. The landmark report,
“Innovation/Stagnation: Challenge and Opportunity on the Critical Path to New Medical
Products”, was a starting point for CPI (FDA, 2004). The report recognized that currently
employed methods were resource-intensive, and sometimes inaccurate in their
predictions. This could lead industry to conduct unwarranted and expensive clinical trials
that ultimately would fail. FDA acknowledged that most of the tools used for toxicology
safety testing were decades old.
The CPI identified several projects that focused on identification of translational
biomarkers (e.g., nephrotoxicity, hepatotoxicity, cardiotoxicity, vascular injury and
muscle injury) to be used as predictive tools for assessing safety (Woodcock and
Woosley, 2008). Some of these projects were then taken up by the Critical Path Institute
(C-Path), an industry-government partnership formed in 2005 under the auspices of the
CPI program. The goal of C-Path is:

43
to accelerate the pace and reduce the costs of medical product
development through the creation of new data standards, measurement
standards, and methods standards that aid in the scientific evaluation of
the efficacy and safety of new therapies (Critical Path Institute, 2005).
C-Path put into place the biomarker qualification process and had the first
successful biomarker qualification with the FDA, EMA, and PMDA. In 2018, the FDA
approved the first ever qualification of a clinical safety biomarker based on data
submitted jointly by the Foundation for the National Institutes of Health (FNIH)
Biomarkers Consortium and the Predictive Safety Testing Consortium of C-Path (PSTC,
2018).
2.6.5.2 FDA’s Predictive Toxicology Roadmap
In 2017, the FDA launched a comprehensive plan for integrating multiple
predictive toxicology methods into safety and risk assessment. This six-part initiative is a
product of the FDA Toxicology Working Group. It relied on recommendations in the
NRC’s reports, Tox21 and the 2017 follow-on report to develop the new roadmap. This
predictive roadmap is intended “to foster the development and evaluation of emerging
toxicological methods and new technologies and incorporate these methods and
technologies into regulatory review, as applicable” (FDA, 2017, p.6).
This initiative identified some of the areas of highest priority for promoting
greater FDA engagement. In September 2018, the FDA held a public hearing to solicit
comments from stakeholders on the Agency’s proposed Predictive Toxicology Roadmap.
Its goal was to gain insights on how to foster the development and evaluation of
innovative technologies and their integration in the regulatory process.

44
One of the difficulties with national programs, however well-intentioned, is the
challenge of ensuring harmonization. Companies are wary of using methods that might
be acceptable by one regulatory authority but not by another. Thus, creating an
infrastructure for harmonization and standardization across countries is critical step for
sharing of information. An important development in this direction is the release of
biocompatibility standard, ISO 10993-1 for evaluation of medical devices in August 2018
involving further reduction of animal testing. For many endpoints, in vitro alternative
method tests are recommended as “first choice” methods (FDA, 2016). Success in this
area would help to eliminate multiple concurrent tests as companies try to satisfy the
regulatory agencies in different countries. Although the OECD member countries do
have mutual acceptance agreements, these must be enforced to see any advantage.
2.7 Research Direction
Significant progress has been made in reducing laboratory animal use and in
improving the welfare of animals for drug development, but more progress should be
possible. It is, however, difficult to understand where gains can be made without a sound
understanding of the underlying factors that impact the choice of testing models at the
preclinical toxicology-testing stage. Submissions for drug approval either at the clinical
investigational stage or at the time of market approval have typically been treated as
confidential, so public information on testing strategies is not readily available. It is
therefore difficult to know what alternatives are being used to replace or supplement
animal testing. Some of these tests might be developed “in house” in testing laboratories

45
or companies for proprietary use on a single product. Others might be tests already
developed and even qualified but not in general use yet.
Delays in the adoption of new practices are not uncommon. However, little
information appears to be available on the implementation of alternative methods in order
to replace the animal studies in the US (including Canada), particularly in the area of
safety assessment of medicinal products. One study in the UK that might give some
insight was sponsored by the Nonclinical and Biological Discovery Expert Network
(NaBDEN). That study previously examined the use of in vitro and in silico techniques
in preclinical safety testing by the pharmaceutical industry, by using a survey sent to the
Association of the British Pharmaceutical Industry (ABPI) member companies. Only four
pharmaceutical companies and 3 contract research organizations (CROs) participated in
the survey (Goh et al., 2015). The survey examined the number of compounds screened
to assess the trends in the uptake of in vitro methods and confirmed an increase in the
uptake of alternative methods. However, the impact on animal usage was not evaluated.
Another study supported by the Dutch Ministry of Economic Affairs sought to
understand the opinions concerning inclusion of alternative methods to characterize
food contaminants, additives and food-contact materials. Key stakeholders actively
working in the field of food safety evaluations (UK, Switzerland and the Netherlands)
were surveyed for their opinions on what they consider the most relevant barriers and
drivers in the acceptance and use of 3R methods for safety evaluations of food chemicals.
It found that the major barriers included (i) uncertain predictability of 3R methods/lack of
validation, (ii) insufficient guidance from regulators and industry and (ii) insufficient
harmonization of legislation (Punt et al., 2018). No assessment of animal use was made.

46
Given that so little systematic information appears to be available on the adoption
and use of non-animal alternatives by the pharmaceutical industry, especially in the U.S.,
further research would appear to be important. In this study, I aimed to understand the
extent to which new methods are being developed, and if these alternatives are being
deployed as “add-on” methods instead of “substitute” methods to replace an in vivo
animal study. Further, I was interested in learning whether programs to change testing
methods have been initiated and then abandoned, and, if so, for what reasons. A good
understanding of the challenges experienced as new methods are developed and
implemented would help both industry and regulators to understand where impediments
exist and whether policy or other measure could be used to reduce those hurdles.
This study used survey methods, using an adoption and implementation
framework to help systematize the data collection. A variety of models and frameworks
have been proposed to study or guide the adoption and implementation of new
interventions or innovations in the health care industry (Tabak et al., 2012). One useful
methodology well-suited to this study was published in 2005 by the National
Implementation Research Network (NIRN), which described how to apply
implementation science systematically in diverse settings (Fixsen et al., 2005). There,
implementation is defined as “a process of carefully considered organizational
adjustments” that take place over a few years (Bertram, Blase and Fixsen, 2015, p. 470).
It consists of 4 stages:
A. The exploration and adoption phase: This phase involves the growth in
awareness of an issue or problem or in recognizing that a better method can be used to
address an existing challenge. This phase can reflect an organization’s readiness and

47
receptiveness for change. At this stage, the organization will examine their options and
the corresponding activities and resources that will be needed to proceed, including
barriers and drivers of the implementation process under consideration.
B. The installation phase: During this phase, organizations prepare for
implementation after the decision has been made to implement a particular change or
refine a particular practice. This will involve assessing staff competency, purchasing
equipment, and assuring readiness of the organization and the leadership, all of which are
considered “implementation drivers”. These efforts will require additional financial
resources and training of staff.
C. The Initial implementation phase: The new processes/changes will be put
into practice during this phase and those changes will need to be managed across the
organization. The support of company leadership is important during this phase as new
challenges develop, and personnel resist the change. This phase may include
improvement cycles that use data to assess the implementation, address barriers by
identifying problems and solve those problems systematically.
D. Full implementation phase: This phase occurs once the new processes
become integrated into the organization’s regular practices. During this phase the
organization begins to use the new or refined model repeatedly and with good fidelity.
In this study, the implementation framework was used as an organizing skeleton
to structure a novel survey tool to explore the barriers and enablers to the implementation
of non-animal alternatives in safety assessment during the drug discovery and
development process. Information was gathered from a principal stakeholder in
development - the industry and its allied contract research organizations (CROs) in US

48
and Canada. The study gathered data concerning the drivers and barriers of
implementing new in vitro alternative methods as replacement to in vivo methods.

49
Chapter 3. Methodology
3.1 Introduction
This research study explored current trends in the use of animal studies and
implementation status of non-animal alternative in vitro and in silico methods by the
pharmaceutical industry and Contract Research Organizations (CROs) in North America.
An interactive focus group guided the development of a self-administered online survey
to capture the perspectives of toxicologists, pharmacologists and other research
professionals involved in safety assessment of small molecules and biologics.
3.2 Identification of Survey Participants
All participants were selected based on predefined criteria: they were employed in
the pharmaceutical industry, CROs or regulatory agencies as employees or consultants
with extensive experience in the field of drug safety evaluation. The majority of such
toxicologists are members of two large professional and scholarly toxicology
associations:(a) Society of Toxicology (SOT) and (b) American College of Toxicology
(ACT). Using Google search, I compiled an extensive list of companies across all
geographical regions of the US and Canada that were engaged in drugs/biologics
development. As a member of these associations, I was able to search the member
directory using the names of the companies on my list. Then I browsed the individual
member’s profile and targeted mid to senior level board-certified toxicologists. A
proactive effort was made to identify participants across every region of the US. I also
reached out to the leadership of regional chapters and special interest groups who helped
send duplicate invites directly through their offices. About 500 participants eventually

50
received the survey and I personally emailed 250 participants requesting their time to
complete the survey.
3.3 Development of Initial Survey
A self-administered online survey tool was developed using the web-based survey
platform, Qualtrics (https://www.qualtrics.com/). It included approximately 60 questions
covering 5 topic areas as described in the following table. The questions were arranged
so that most respondents were not presented with all of the questions. Instead, some
questions were skipped if respondents indicated that they had insufficient experience with
one or more types of approaches under evaluation. The final survey is provided in
Appendix A.
Table 2: Questionnaire Instrument: Breakdown of Areas of Inquiry
No Areas of Inquiry
1 Demography and professional profiles of participants
2 Current status, trends and outlook
3 Exploration and installation of different category of alternatives
4 Barriers and drivers of alternative implementation
5 Potential policy solutions for transition to non-animal alternatives

Prior to administering the survey, a focus group comprised by 7 participants was
created to seek input on the adequacy, clarity and the structure of the survey. The panel
members were selected on the basis of their extensive knowledge and diverse
experiences, but they all possessed a core knowledge in the field of animal testing and/or
survey methods in regulatory research. The panel included faculty members of the
university and senior regulatory toxicologists from the industry.

51
The list of participants in the focus group is presented in Table 3.
Table 3: List of Participants in the Focus Group
No. Name of Participants
Description of Participant
Professional Background
1 Frances J. Richmond, PhD
Director, D K Kim International Center
for Regulatory Science, University of
Southern California
2
Mary Ellen Cosenza, PhD,
DABT, ATS, ERT, RAC
Regulatory Toxicology Consultant;
Adjunct Professor, USC School of
Pharmacy
3
Terry David Church, DRSc, MA,
MS
Assistant Professor of Regulatory and
Quality Sciences
4 John C Kapeghian, PhD, DABT Toxicology Consultant
5
Chandrashekhar Korgaonkar,
PhD, DABT
Toxicologist, Industry
6 Assad Aslam, BS
Toxicologist/
Business Development, CRO
7 Keith Bley, PhD Nonclinical Development, Industry
8 Chad Oh, MD Clinical Development Consultant

At least 2 weeks before the meeting, all participants were provided an electronic
copy of the Abstract and Chapter 1 used in support of the Qualifying Exam, a copy of the
sample online survey instrument, and a set of logistical instructions. They were provided
with instructions to attend the meeting via video conferencing, and the proceedings were
recorded with the consent of participants.
The focus group was convened on Sept 24, 2020. I served as the moderator for the
meeting. I provided a brief overview describing the expectations and agenda to be
covered in approximately 90 minutes as described in Table 4. Each question in the
survey questionnaire was then reviewed sequentially and debated as needed. I then

52
summarized the meeting and requested any additional feedback on the format and content
of the survey instrument. The survey was revised and updated based on this feedback.
The final survey prior to its launch was validated by Dr. Terry Church and Dr. Eunjoo
Pacifici to ensure that the skip logic worked as expected. These data were not included in
the final survey results.
Table 4: Focus Group Agenda
Item No. Agenda Item Time Allotted
1 Introduction of participants Participant/Moderator
2 Brief overview on research topic Participant/Moderator
3 Review of survey instrument/ feedback Participant/Moderator
4 Summary of feedback and follow-up action Moderator

3.4 Survey Deployment and Analysis
The final survey was deployed on November 17, 2020 to approximately 500
target participants. An email link along with a cover letter describing the purpose of the
survey was sent to each participant directly through Qualtrics. Additionally, an
anonymous link was provided in the cover letter in case the participant wished to share or
forward the survey. Participants were assured that their affiliations with their companies
and organizations would be kept confidential. The survey was kept open for just under 2
months. Survey reminders were sent twice to those who had not responded after 3 and 6
weeks. The survey closed on January 12, 2021.

53
3.5 Data Analysis
All results were collected anonymously and saved electronically within Qualtrics.
Results were summarized using descriptive statistics. Tables and figures were generated
to display the quantitative data. All open text and comment questions/fields were
examined thoroughly and carefully to identify particular trends or common elements
which were captured in the tables.

54
Chapter 4. Results
4.1 Survey Participation
The link to the survey was emailed to 496 individuals directly through the
Qualtrics platform between November 17, 2020 through January 12, 2021. In response to
my personal request, many individuals on the original distribution panel forwarded the
anonymous link to one or more colleagues. Table 5 shows that 170 combined responses
were received and 90% of those were completed. The survey response rate was 26%
(127/496) and 23% (114/496) for those starting and completing the survey, respectively,
for those in the distribution panel to whom direct links were sent. It was not possible to
estimate the completion rate for those to whom an anonymous survey link was sent.
Table 5: Survey Response Data
Distribution
Channel
Audience
Size
Survey
Started
Responses
Completion
Rate
Invite Over Email 496 127 114 90%
Anonymous Link NA NA 56 NA

4.2 Demographic Profile of Respondents
The first block of questions collected demographic information about the
individuals and the organizations with which they were associated. About half of the
respondents worked in pharmaceutical or biotechnology companies (54%, 90/166), a
quarter in CROs/testing facilities (24%, 39/166), and 15% in consultancies (25/166).
Additionally, 4% (7/166) were employed at the regulatory agencies (FDA/Health

55
Canada) and 3% (5/166) in “Other” organizations that were not identified by those
participants (Figure 5).
Figure 5: Organizational Affiliations
Please describe your organization

Respondents were employed by companies/organizations across a full range of
sizes. About a third (33%, 54/165) worked at small companies with less than 100
employees, 21% (35/165) at mid-size organizations with 100-1000 employees, and 10%
(16/165) at large companies with 1001-10,000 employees. Another one-third (36%,
60/165) were employed at very large organizations with more than 10,000 employees
(Figure 6).

56
Figure 6: Size of Organizations
What is the size of your company based on the number of employees? If you are a
consultant, please select the main organization for which you provide service and
estimate its size.

About two-thirds of respondents (66%, 110/166) worked for organizations more
than 20 years old, 22% (37/166) for organizations less than 10 years old and 11%
(19/166) for organizations between 10 and 20 years old (Figure 7).
Figure 7: Age of Organizations
Please describe the approximate age of your organization.

Most commonly, respondents were from companies located on the East Coast
(44%, 73/166) followed by the West Coast (33%, 54/166), and the Mid-West (16%,
27/166). Seven percent (27/166) worked at companies located in “other” locations
(Figure 8, Table 6).

57
Figure 8: Geographical Location of Organizations
Please indicate the geographical location of your company's North American office in the
US or Canada.

Table 6: Other-Locations
UK all over US and Canada
East and West Coast All of the above
Canada Europe, West Coast and East Coast
Worldwide Sites all over NA
I work for a Global Company, the R&D
office I work at is in the east coast
headquarter in San Diego, satellite
offices in Boston and Zug, Switzerland
Multi-national headquartered in Germany
with East coast locations
Outside US, Ethiopia

Most respondents had extensive professional experience in the field of drug safety
assessment. More than half had more than 20 years of experience (54%, 89/164),
one-third (50/164) had 11-20 years of experience, 10% (16/164) had 5-10 years of
experience and 6% (9/164) had less than 5 years of experience (Figure 9).

58
Figure 9: Years of Personal Experience in Safety Assessment
How many years of personal experience do you have in nonclinical safety testing?

Respondents were asked to select the range of products with which they were
involved when developing drugs and biologics (Figure 10). A majority (77%; 128/166)
worked on small molecules, 53% (88/166) on large molecules (therapeutic proteins,
monoclonal antibodies), 33% (55/166) on gene therapy, cell therapy, and vaccines, and
10% (16/166) on “other” products as described in Table 7.
Figure 10: Types of Products
What kind of therapy are you developing for your current organization? If you are
working or consulting for more than one product type, please characterize the area in
which you have had most experience in the last 5 years.

59
Table 7: Other-Therapy
Nucleic acids Agrochemicals Product testing
Multiplex detection
reagents
Therapeutic RNAs,
synthetic (non-biologic)
proteins
pharmaceuticals, consumer and
medical devices, combination
products
Medical Device (n=2) All of the above (n=2) Oligonucleotides (n=3)

4.3 Current Status and General Outlook Regarding Animal Use in Safety Testing
Respondents were asked for their professional opinions on the current use of
animals in safety assessment, and the impact of alternatives in the near future. A large
majority of respondents indicated that the number of animals currently used are
reasonable (82%, 134/166) compared to a small number who felt them to be excessive
(15%, 25/166). Only 2% (3/166) suggested that the numbers are insufficient and 1%
(1/166) did not use animals in their work (Figure 11).
Figure 11: Appropriateness of Animal Numbers Used for Safety Assessments
In your professional opinion, are the number of animals used in nonclinical safety
assessment…

When asked whether alternatives had reduced animal usage, most considered it to
be reduced (yes: 36%, 60/166; somewhat: 46%, 77/166) but 15% (25/166) disagreed and
2% (4/166) did not know (Figure 12).

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Figure 12: Impact of Alternatives on Numbers of Animals Used
Do you think that efforts to implement alternatives to animal testing have helped to
reduce the number of animals used in safety assessment?

The respondents were asked if they expected any changes in the next decade to
the ICH guidances that currently require extensive animal testing. More than half
indicated that the changes would be unlikely (56%, 92/164), one-third (34%, 56/164) that
they were somewhat likely and 8% (13/164) that they were very likely. Three
respondents (2%) were not sure (Figure 13).
Figure 13: Expectation from ICH Guidances in the Near Future
In the US and the EU, ICH guidance requires extensive animal testing of drugs/biologics
before human exposure. Do you see that changing in the next decade or so?

Half of the respondents (51%, 84/166) felt that the in vitro alternatives currently
being employed are mostly add-on tests to assist safety determinations, 38% (63/166) that

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they have mixed purposes as add-ons and methods to reduce animal use, and 7% (12/166)
that they mostly reduce animal use. Four percent (7/166) were not sure (Figure 14).
Figure 14: Alternatives: Add-ons or Reduced Use of Animals?
Research suggests an upward trend in the implementation of in vitro tests for safety
assessment. In your view, are the new alternatives being implemented as “add-on”
methods (to further assist in safety determination) or do they result in reduction in animal
usage?

Most respondents (70%, 116/166) did not believe that drug safety assessment can
be animal-free, but one-fourth (41/166) felt that it might. Only 2% (4/166) believed that it
could be animal-free and 3% (5/166) did not know (Figure 15).
Figure 15: Possibility of Animal-Free Safety Assessment
Do you believe that drug safety assessment can be animal-free?

No respondent expressed the view that the FDA has been “very successful” in the
implementation of alternatives to replace/reduce animal testing. Instead, they were almost
evenly split between those who viewed the FDA as “modestly successful” (46%,

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175/165) and those who thought that it had “not been successful” (47%, 77/165). Eight
percent (13/165) of respondents did not know (Figure 16).
Figure 16: Views on FDA’s success
Do you believe that the FDA has been successful in implementing alternative methods to
replace/reduce animal testing in the field of safety assessment?

4.4 Barriers and Drivers of Implementation of Alternatives
The respondents were asked to rate the barriers that have hindered the
implementation of new alternative methods (Figure 17). On a weighted scale of 1 to 3
(1=very significant [VS], 2=significant [S] and 3=not significant [NS]), weighted means
(WMs) were calculated and the data were rank ordered based on this calculation. On
average, three barriers appeared most troublesome, with WM of 1.5-1.6: lack of
validation of existing alternatives (VS: 57%, 93/164; S: 34%, 55/164; NS: 8%, 13/164),
concerns about regulatory acceptance (VS: 52%, 86/165; S: 42%, 70/165; NS: 5%,
9/165), and lack of confidence in scientific adequacy (VS: 49%, 80/162; S: 39%, 63/162;
NS: 10%, 16/162).
The other offered choices generated mixed responses. A risk averse climate
(WM=2.0) was viewed by most as significant and very significant (S: 43%, 71/164; VS:
25%, 41/164); 28% (46/164) viewed it as not significant. An absence of leadership

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(WM=2.4) was seen by less than half of respondents as very significant or significant
(VS: 12%, 19/159; S:33%, 52/259); a similar number, 48% (76/159), rated it as not
significant. Similarly, the high cost of development (WM=2.5) was rated as very
significant or significant by 40% (VS:11%,18/160; S: 29%, 46/160) but not significant by
about half (53%, 84/160). For each answer, the rate of selecting “not sure” was 2-8%.
“Other” barriers were identified by respondents. The main themes are summarized in
Table 8 (see Appendix B for all data).
Figure 17: Implementation Barriers
What do you believe are the barriers that have impeded the implementation of new
alternative methods?

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Table 8: Other-Barriers (Main Themes)
Cost
Concerns about transability and relevance to
humans
Time Concerns about regulatory acceptance
Lack of Infrastructure Risk aversion
Lack of commitment Resistance to change
Historical failures Lack of Champion and leadership

Respondents were also presented with factors that have helped to promote the
development of alternatives to animal testing (Figure 18). All choices were rated as
important by 40-52% of respondents. They were rank ordered by calculating a WM
using a 4-point scale from “most important” to “not important”. The factor with the
lowest WM of 1.5, reflecting greatest importance, was an improved regulatory
environment (most important (most important: 50%, 82/164; important: 46% 76/164;
marginally important, 4%, 6/164). Two factors with a WM of 1.8, were “ethical
considerations” (most important: 37%, 60/164; important: 52%, 86/164; marginally
important: 10%, 16/164; not important: 1%, 2/164) and “better scientific results” (most
important: 44%, 70/160; important 40%, 64/160; marginally important: 11%, 17/160; not
important: 6%, 9/160). Societal sensitivities (most important: 14% 23/163; important:
52%, 84/163; marginally important: 31%, 50/163; not important: 4%, 6/163) and the
ability to decrease cost (most important:10% 16/163; important: 50%, 82/163; marginally
important: 33%, 53/163; not important: 7% 12/163) were ranked lowest in the list but still
had WM of 2.2 and 2.4, respectively.

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Figure 18: Implementation Drivers
Please rate the following factors that may have played a role in promoting development
of alternatives to animal use in toxicity testing.

When asked to rate the importance of some of suggested barriers that impede the
validation process (Figure 19), respondents rated all the offered options as important or
very important. A WM of 1 to 3 (1=very important [VI), 2=Important [I] and 3=not
important [NI]) was used to order responses by perceived importance.
Highest ranked barriers, with WM of 1.6, included lack of market due to limited
regulatory acceptance (VI: 49%, 81/164; I: 33%, 54/164; NI: 15%, 24/164) and
uncertainty in achieving a successful outcome (VI: 46%, 76/164; I: 42% 68/164; NI:
11%, 18/164). Two additional factors had closely aligned WM of 1.8; highly time-
consuming process (VI: 31%, 51/163; I: 54%, 88/163; NI: 11%, 18/163), and need for
large investment (VI: 32%, 53/163; I: 53%, 87/163; NI: 13%, 18/163). Between 1-4% of
the respondents were not sure about the importance of these factors.

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The remaining options received more mixed responses so had WM between 2.0-
2.3. Half (50%, 82/163) saw the option, “validation too complex technically”, as an
important barrier and a further 21% as very important (34/163), but some also rated it as
not important (22%, 36/163). “Concerns about opinion of peer/scientific community” was
rated as important or very important (VI: 15%, 25/164; I: 43%, 71/164) by most but not
important by more than a third (37%, 61/164). “Alternatives are not animal free” had a
similar distribution of ratings (VI: 9%, 15/162; I: 41%, 66/162; NI: 37%, 60/162 as not
important). Between 1-7% of respondents chose “not sure” for all but one option,
“alternatives are not animal free”, chosen by 13%.
Figure 19: Lack of Validation
Lack of validation of a new alternative method is considered as a major hurdle in
adoption of alternative methods. What are the barriers that hold up the validation of
alternatives?

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Respondents were asked which organizations should bear the cost of validation on
a weighted scale of 1 to 3 (1=primarily responsible [PR], 2=somewhat responsible [SR]
and 3=not responsible [NR] (Figure 20). Most respondents viewed all of the listed
organizations as somewhat responsible. However, a large majority viewed the industry to
be primarily responsible (49%, 81/164) or somewhat responsible (46%, 76/164
(WM=1.5). Public-private consortia and government agencies had lower WMs of 1.8,
because only 28% saw them as primarily responsible (Public-private consortia - PR:
28%, 46/164; SW: 60%, 98/164; NR: 10%, 16/164, and government agencies - PR: 28%,
46/163; SR: 60%, 97/163; NR: 11% 18/163).
International organizations and contract research organizations were ranked
lowest (WM=2.1). Most commonly they were regarded as somewhat responsible but
about a quarter regarded them as not responsible (International organizations - PR: 17%
28/163; SR: 56%, 91/163, NR: 23% 37/163 and contract research organization -
PR: 18%, 30/163; SR: 50%, 82/163; NR: 25% 40/163). Between 1-7 % of respondents
chose “not sure” for all the options provided.

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Figure 20: Cost of Validation
The cost associated with the validation process of new alternatives is considered a
barrier to the development of an alternative method. Who should be responsible for
supporting the cost to validate the new methods for regulatory acceptance?

Respondents were asked to rank the strategies that should be employed to ensure
that a validated alternative method would be accepted, with WMs of 1 to 3 (1=most
effective [ME], 2=effective [E] and 3=not effective [NE]) (Figure 21). All four options
were ranked as most effective or effective approaches by greater than 95% respondents;
less than 5% considered any of the four choices as not effective. Using “weight of
evidence” was ranked overall as most effective (WM=1.4) (ME: 58%, 95/165; E: 39%,
64/165; NE: 2%, 3/165) followed closely by “dissemination through publication”
(WM=1.5) (ME: 49%, 81/165; E: 48%, 80/165; NE: 2%, 3/165). The remaining two
approaches had a WM of 1.6): “dissemination through workshops” (ME: 46%, 76/166; E:
49%, 81/166; NE: 5%, 8/166), and “use of subject matter expert” (ME: 42%, 69/165; E:

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53%, 88/165; NE: 5%, 8/165). Between 1-2 % of respondents chose “not sure” for these
options.
Figure 21: Strategies for Acceptance of Validated Alternatives
Once an alternative method is validated, what strategy should be employed by
stakeholders to work with regulatory agencies to ensure its acceptance?

4.5 Exploration and Installation Stages
Respondents were presented with four categories of non-animal alternatives and
asked for their views during the exploration and installation of such methodologies.
4.5.1 In Silico/Computer-Based Alternatives
When asked about “in silico/computer-based models” as alternatives (Figure 22),
more than half (58%, 96/166) stated that they had explored these alternatives. They were
presented with additional questions related to that exploration. One-third (34%, 56/166)
had not explored alternatives related to such models and 8% (14/166) did not know; these

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respondents skipped to the next category of alternative methods related to tissue/cell
culture technologies, described in section 4.5.2.
Figure 22: Exploration of In Silico/Computer-Based Alternatives
Has your organization explored alternative methods using “in silico/computer-based
models” (including organ/tissue chips) in lieu of or to reduce the size or number of
animal studies?

Respondents who had explored in silico methods were asked to provide their
opinion on the usefulness of various information sources; WMs were calculated on a
scale of 1 to 3 (1=very useful [VU], 2=modestly useful [MU] and 3=not useful [NU
(Figure 23).
Scientific publications (WM=1.4) were viewed as very useful by more than two-
thirds of respondents; almost all found them to be at least modestly useful (VU: 65%,
62/95; MU: 30%, 28/95; NU: 2%, 2/95). “Meetings/workshops/conferences” (WM=1.5)
were also viewed by most as very or modestly useful (VU: 54%, 62/95; MU: 41%, 39/95;
NU: 3%, 3/95). “Information from testing labs” had a somewhat lower WM of 1.8; only
29% saw them as very useful, although most found them modestly useful (VU: 29%,
27/94; MU: 55%, 52/94; NU: 11%, 10/94). Regulatory agency guidance was considered
to be very or modestly useful by 70% of respondents but not useful by nearly a quarter
(WM=1.9, VU: 27%, 26/95; MU: 43%, 41/95; NU: 22% 21/95). Input from consultants

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had a similar distribution (WM=2.0, VU: 22%, 20/93; MU: 44%, 41/93; NU: 23%
21/93). Between 2-7% of respondents chose “not sure” for all but one option, “input from
consultants”, chosen by 12%. Additional sources provided for “other” are presented in
Table 9.
Figure 23: Usefulness of Information Sources During Exploration of In Silico
Methods
As you tried to learn about the use of in silico/computer-based models as an alternative
to animal testing, how useful were the following information sources?

Table 9: Other-Useful Sources
Collaborative efforts between companies Own experience
Informal feedback from colleagues who
tried models
Vendors/companies offering in silico
approaches
This is unfortunately trial and error based on
the training datasets
Internal validation and method development
We developed an internal discovery and investigative tox group that implements the
alternative models in the screening or problem solving phases.

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The respondents were asked about the eventual outcome of their exploration
activities. Thirty six percent (34/95) implemented these methods but did not use them
routinely, 33% (31/95) used them routinely, 28% (27/95) were still exploring, 2% (2/95)
abandoned them after initial implementation, and 1% (1/95) decided to not go forward
with the methods (Figure 24). Those who chose “still exploring” skipped the remaining
questions in this category and were directed to the set of questions described in section
4.5.2.
Figure 24: Outcome of Exploration of In Silico Methods
What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to replace or reduce animal testing?

The single respondent who indicated that the company decided not to go forward
with in silico methods was asked about the factors driving that decision. This respondent
regarded all of the given choices as unimportant, and instead gave “lack of clear validity
and regulatory acceptance” as an important factor associated with “other” (Table 10).

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Table 10: Factors that Drove the Decision to not Pursue In Silico Methods
During Exploration
What factors drove the decision not to go forward with this type of method during the
evaluation/exploration process?
Factors Very
Important
Somewhat
Important
Not
Important
Not
Sure
Development would take too long 0 0 1 0
Replacement too expensive 0 0 1 0
Lack of Necessary infrastructure 0 0 1 0
Team lacked required know how 0 0 1 0
Other 1 0 0 0

The two respondents reporting that that they abandoned the in silico method after
initial implementation were asked to rate the factors contributing to that decision
(Table 11). Both selected “disappointing results from initial runs” and “insufficient
infrastructure”. Only one regarded the “lack of systematic plan and “resistance to
change” as critical.
Table 11: Factors that led to Abandonment of In Silico Methods after Initial
Implementation
What factors played a role in your decision to abandon in silico/computer-based methods
after the initial implementation?
Factors
Very
Critical
Critical Not Critical Not Sure
Lack of systematic training plan 0 1 1 0
Greater comfort with status
quo/resistance to change
0 1 1 0
Disappointing initial runs 0 2 0 0
Insufficient infrastructure 0 2 0 0
Other 0 0 0 0

All respondents were presented with factors that might be challenging when
developing in silico alternatives. WMs were calculated from a scale of 1 to 3 (1=strongly

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agree [SA], 2=agree [A], and 3=disagree [DA]) (Figure 25). Most highly ranked
(WM=1.9) were “lack of experts able to guide development” (SA: 31%, 21/67; A: 43%,
29/67; DA: 25%, 17/67). “Lack of continuous funding” (SA: 20%, 13/66; A: 39%, 26/67;
DA: 29%, 19/67) and “lack of leadership” (SA: 22%, 15/67; A: 37%, 25/67; DA: 36%,
24/67) had WMs of 2.1. Ranked lowest (WM=2.3) were “lack of staff to manage
changes” (SA: 11%, 7/66; A: 47%, 31/66; DA: 33%, 22/66) and “advocates not following
through” (SA: 11%, 7/66; A: 44%, 29/66; DA: 33%, 22/66). For most options, 5-12% of
respondents selected “not sure” but no respondents denied knowing about the “lack of
experts capable of guiding development”. A few “other” challenges were noted and
described in Table 12.
Figure 25: Challenges to In Silico Alternative Development
The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences.

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Table 12: Other-Challenges to Implementation of In Silico Alternatives
To date, only a few of these assays have successfully replaced in vivo studies.
Over-promising of method and misrepresentation of performance value by some vendors
Unrealistic expectations for time needed to develop and validate

Respondents were asked about their interactions with the FDA during the
implementation of in silico alternatives (Figure 26). Forty percent (27/67) did not interact
with the FDA, 30% (20/67) received encouraging feedback, and 27% (18/67) neither
encouraging or discouraging feedback. Only 3% felt that feedback was discouraging.
Figure 26: Regulatory Agency Feedback Regarding In Silico Methods
During this process of evaluation/implementation of new in silico/computer-based
methods, the feedback from the regulatory agencies was:

Almost half (48%, 32/66) thought that these methods reduced animal use, 29%
(19/66) did not and about a quarter (23%, 15/66) did not know (Figure 27).

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Figure 27: Impact of In Silico Method Implementation on Animal Use
In retrospect, do you think that these efforts reduced the use of animals?

Respondents who implemented in silico methods were asked to describe the
method(s). Main themes are summarized in Table 13 from data presented in Appendix C.
Table 13: Description of In Silico Methods Implemented (Main Themes)
If you implemented new in silico/computer-based method, please identify/describe the
nature of that method:
Computer based quantitative structure-activity (QSAR) predictive toxicology software:
Derek-Nexus, Sarah-Nexus, LeadScope, ToxTree, OECD QSAR toolbox
iPSC models, cell cultures for early screening, or determining if toxicity is on or off target
In silico models for cardiac arrythmias, assessment of general toxicity, mutagenicity, organ-
specific toxicity (e.g., kidney, cardiovascular)
Methods to predict key organ system functions, physical chemical properties ADME
Toxicogenomics and adverse outcome pathway software
(Q)SAR evaluation for impurity assessment, leachable
Machine learning method to identify new prodrugs
Integrated systems using organ imaging
Population PKPD
Off-target receptor binding
Lung, organ, cardiac muscle on a chip

4.5.2 Tissue/Cell Culture Technologies
When asked about “tissue/cell culture technologies” as alternatives (Figure 28),
almost half (49%, 80/163) stated that they had explored these alternatives; these
respondents were questioned further about that exploration. Over one-third (37%, 60/163)
had not explored these alternatives and 14% (23/163) did not know; these respondents

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were directed immediately to the next category of alternative methods related to
vertebrates/ invertebrates-based methodologies, described in section 4.5.3.
Figure 28: Exploration of Tissue/Cell Culture Technologies as Alternatives
Has your organization explored alternative methods using “tissue/cell culture
technologies” in lieu of or to reduce the size or number of animal studies?

Respondents who had explored tissue/cell culture technologies were asked to
provide their opinion on the usefulness of various information sources. WMs calculated
from a scale of 1 to 3 (1=very useful [VU], 2=modestly useful [MU] and 3=not useful
[NU]), were used to order the relative usefulness of these sources (Figure 29).
Scientific publications (WM=1.3) were viewed as very useful by more than two-
thirds of respondents; almost all found them to be at least modestly useful and no one
viewed them as not useful (VU: 63%, 49/78; MU: 28%, 22/78). “Meetings/
workshops/conferences” (WM=1.5) were also all viewed by a large majority as very or
modestly useful (VU: 44%, 34/78/; MU: 46%, 36/78; NU: 3%, 2/78). “Information from
testing labs” (WM=1.7) was found to be very or modestly useful by 85% and not useful
by 8% (VU: 38%, 30/79; MU: 47%, 37/79; NU: 8%, 6/79). “Regulatory agency
guidance” (WM=1.9) was seen as very or modestly useful by 66% of respondents but
also seen as not useful by nearly a quarter (VU: 29%, 23/79; MU: 37%, 29/79; NU: 24%
19/79). “Input from consultants “(WM=2.2) was seen as very useful by only a small

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minority (VU: 16%, 12/77). Most were evenly split between considering it as modestly
useful or not useful (MU: 35%, 27/77; NU: 34% 26/77).
Between 8-16% of respondents chose “not sure” for all options. Additional
sources were identified as “other” and presented in Table 14.
Figure 29: Usefulness of Information Sources During Exploration of Tissue/Cell
Culture Technologies
As you tried to learn about the use of tissue/cell culture technologies as an alternative to
animal testing, how useful were the following information sources?

Table 14: Other-Information Sources for Tissue/Cell Culture Methods
Input from other sponsors that the testing works and has been accepted by regulatory agencies
Working with companies developing organs on a chip
Internal investigative efforts
Dermal and corneal assays are well recognized and especially useful
The respondents were asked about the eventual outcome of their exploration
activities. One-third (34%, 27/80) implemented these methods for routine use, one-third

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(33%, 26/80) implemented them but do not use them routinely, and another third (31%,
25/80) were still exploring. Only 3% (2/80) abandoned them after initial implementation.
None decided to not go forward with these methods (Figure 30). Those who chose “still
exploring” were allowed to skip the remaining questions in this category and were
directed to the next set of question described in section 4.5.3.
Figure 30: Outcome of Exploration of Tissue/Cell Culture Technologies
What was the outcome of your evaluation/exploration of tissue/cell culture technologies
to replace or reduce animal testing?

As no respondent (0/80) chose the option “our organization decided not to go
forward with these types of technologies” the question about rating the factors that drove
the decisions to not go forward with tissue/cell culture methodologies during exploration
did not generate any response.
The two respondents who indicated that that they abandoned the tissue/cell
culture methods after the initial implementation were asked to rate the factors
contributing to that decision. All four options were rated as “critical” by at least one
respondent. Lack of systematic plan for training, disappointing results from initial runs,

80
and insufficient infrastructure were rated as very critical or critical. “Greater comfort with
status quo” was perceived as critical or not critical by these two respondents (Table 15).
Table 15: Factors Causing Abandonment of Tissue/Cell Culture Technologies
After Initial Implementation
What factors played a role in your decision to abandon tissue/cell culture technologies
after the initial implementation?
Factors Very
Critical
Critical Not Critical Not Sure
Lack of systematic plan for training 1 1 0
0
Greater comfort with status
quo/resistance to change 0 1 1
0
Disappointing results from initial runs 1 1 0
0
Insufficient infrastructure 1 1 0
0
Other 0 0 0
0

All respondents were presented with challenges that might affect the development
of new tissue/cell culture alternatives. WMs were calculated from a scale of 1 to 3
(1=strongly agree [SA], 2=agree [A], and 3=disagree [DA]) (Figure 31).
Highest ranked (WM= 2.0) was “lack of continuous funding” (SA: 28%, 15/54;
A: 30%, 16/54; DA: 30%, 16/54). “Lack of experts to guide implementation” (WM=2.1)
was second (SA: 22%, 12/54; A: 39%, 21/54; DA: 35%, 19/54). “Lack of leadership”
(SA: 24%, 13/54; A: 33%, 18/54; DA: 39%, 21/54;) and “advocates not following
through” (SA: 17%, 9/54; A: 35% 19/54; DA: 37%, 20/54) had the same WM of 2.2.
“Lack of staff to manage changes” (WM=2.3) was ranked lowest in the list (SA: 15%,
8/54; A: 39%,21/54; DA: 41%, 22/54). For all options, respondents selecting “not sure”
ranged from 4-13%. A few responses were identified as “other” challenges some of
which were specified in Table 16.

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Figure 31: Challenges to Tissue/Cell Culture-Based Alternative Development
The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences.

Table 16: Other-Challenges
Regulatory acceptance
Models didn't capture enough of the biology to replace animal studies
Low translational value of non-animal alternatives

Respondents were asked about their interactions with FDA when implementing
new tissue/cell alternatives (Figure 32). Forty-two percent (22/53) did not interact with
FDA, 26% (14/53) received encouraging feedback, and 25% (13/53) viewed it as neither
encouraging nor discouraging. Only 8% felt that feedback was discouraging.

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Figure 32: Regulatory Agency Feedback During Implementation of Tissue/Cell
Culture Methods
During this process of evaluation/implementation of new tissue/cell culture technologies,
the feedback from the regulatory agencies was:

When asked if the implementation of tissue/cell-based methods resulted in
reduced use of animals, more than half (59%, 32/54) reported a reduction, about one-third
(28% 115/54) no reduction, and 13% (7/54) did not know (Figure 33).
Figure 33: Impact of Tissue/Cell Culture Method Implementation on Animal Use
In retrospect, do you think that the use of alternative cell culture-based technologies
reduced the use of animals?

Respondents who implemented tissue/cell culture methods were asked to describe
them. The main themes, summarized in Table 17, were extracted from data presented in
Appendix D.

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Table 17: Description of Implemented Tissue/Cell Culture Technologies (Main
Themes)
If you implemented new tissue/cell culture-based methods, please identify/describe the
nature of that method:
Organ on a chip technology; tissue in a chip, canine/rat liver: kidney coupled chips; liver on a
chip connected to a kidney on a chip
Use of skin membrane to replace dermal testing; reconstructed skin, MPS
3-dimensional organoids and fluid-based systems CNS in vitro assays
Hepatocyte model Tissue reactivity studies
Multi parameter high content screening Embryonic stem cells
Toxicity Testing using R blood cells Receptor binding assays
Cell cultures using human tissues and tissue organoids Cell-based toxicity assays
Screening of tumor cells and tumor organoids Cardiac assays
Bovine eyes to replace eye testing in rabbits THLE in vitro toxicity assay
Single cell and microphysiologic models Tissue/cell-based CIVM
Engineered T cell 3D liver microspheres

4.5.3 Lower Vertebrates/Invertebrates-Based Alternatives
When asked about alternatives based on lower vertebrates or invertebrates (Figure
34), only 22% (35/162) had explored these alternatives. Two-thirds (62%, 100/162) had
not explored them and 17% (27/162) did not know; these respondents were directed to
the next set of question related to imaging/other novel methods in section 4.5.4.

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Figure 34: Exploration of Lower Vertebrates/Invertebrates-Based Alternatives
Has your organization explored alternative methods using "lower
vertebrates/invertebrates" in lieu of or to reduce the size or number of animal studies?

Respondents who had explored lower vertebrate/invertebrate-based methods were
asked their views on the usefulness of certain information sources. WMs, calculated from
a weighted scale of 1 to 3 (1=very useful [VU], 2=modestly useful [MU] and 3=not
useful [NU]), suggested the relative usefulness of these sources (Figure 35).
Scientific publications (WM=1.3) were viewed as very useful by a majority (68%)
of respondents; (VU: 68%, 23/34; MU: 27%, 9/34; NU: 3%, 1/34).
Meetings/workshops/conferences (VU: 59%, 20/34; MU: 35%, 12/34; NU: 6%, 2/34) and
information from testing labs (VU: 57%, 20/35; MU: 35%, 12/35; NU: 9%, 3/35) had
similar WMs of 1.5, and were viewed by more than half as very useful. Regulatory
agency guidance (WM=1.8) was found to be very useful by only one-third (VU: 32%,
11/34; MU: 38%, 13/34; NU: 21% 7/34). Input from consultants was ranked lowest
(WM=2.1); a higher percent found them to be not useful than very useful (VU: 27%,
9/34; MU: 35%, 12/34; NU: 33% 11/34).
No respondent selected “not sure” when considering
meetings/workshops/conferences and information from testing labs. For the other three
options, “not sure” ranged from 3-9%. “In-house expertise” was an additional source
identified as “other”.

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Figure 35: Usefulness of Information Sources Regarding Lower Vertebrate/
Invertebrate Alternatives
How useful were the following information sources as you explored the use of lower
vertebrates/ invertebrates as an alternative to animal testing?

The respondents were asked about the eventual outcome of their exploration
activities. Forty percent (14/35) implemented these methods but do not use them
routinely, 26% (9/35) use them routinely, 29% (10/35) are still exploring, 3% 1(/35)
abandoned them after initial implementation, and 3% (1/35) decided to not go forward
with these methods (Figure 37). Those who chose “still exploring” were allowed to skip
the remaining questions in this category and were directed to questions described in
section 4.5.4.

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Figure 36: Outcome of Exploration of Lower Vertebrates/Invertebrates-Based
Methods
What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebrates to replace or reduce animal testing?

The single respondent who indicated that the company decided not to go forward
with these alternatives was asked about the factors driving that decision. “Development
would take too long” and “Our team did not have the required know how” were selected
as somewhat important where as “replacement would be too expensive” and “lack of
necessary infrastructure” were considered not important (Table 18).
Table 18: Factors Driving the Decision to not Pursue Using Lower Vertebrates/
Invertebrates Alternatives During Exploration
What factors drove the decision not to go forward with this type of method during the
evaluation/exploration process
Factors Very
Important
Somewhat
Important
Not
Important
Not
Sure
Development would take too long 0 1 0 0
Replacement would be too expensive 0 0 1 0
Lack of necessary infrastructure 0 0 1 0
Our team lacked required know how 0 1 0 0

Although only one respondent indicated that that he/she abandoned the lower
vertebrate/ invertebrate-based alternatives after the initial implementation, two
respondents gave answers when asked to rate the factors contributing to that decision

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(Table 19). “Disappointing results from initial runs” was selected as “very critical” by
both. One noted also that “resistance to change” was critical but the other as not critical.
“Lack of systematic plan” and “insufficient infrastructure” were selected as not critical by
both.
Table 19: Factors Causing Abandonment of Lower Vertebrates/Invertebrates-
Based Methods After Initial Implementation
What factors played a role in your decision to abandon the methods using lower
vertebrates or invertebrates after the initial implementation?
Factors Very
Critical
Critical Not Critical Not Sure
Lack of systematic plan for training 0 0 2
0
Greater comfort with status
quo/resistance to change 0 1 1

0
Disappointing results from initial runs 2 0 0
0
Insufficient infrastructure 0 0 2
0
Other 0 0 0
0
All respondents were presented with potential challenges when developing new
lower vertebrate/ invertebrate-based alternatives that were ranked by WMs, calculated on
a scale of 1 to 3 (1=strongly agree [SA], 2=agree [A], and 3=disagree [DA]) (Figure 37).
Lack of continuous funding (SA: 22%, 5/23; A: 26%, 6/23; DA: 30%, 7/23) and lack of
experts to guide implementation (SA: 22%, 5/23; A: 44%, 10/23; DA: 35%, 8/23) were
the highest ranked with a WM of 2.1. Somewhat lower (WM=2.3) were the other three
options: “lack of leadership” (SA: 17%, 4/23; A: 30%, 7/23; DA: 48%, 11/23;),
“advocates not following through” (SA: 17%, 4/23; A: 26% 6/23; DA: 48%, 11/23), and
“lack of staff to manage changes” (SA: 9%, 2/23; A: 48%, 11/23; DA: 44%, 10/23).

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Between 4-22% of respondents selected “not sure” for 3 challenges, including
lack of continuous funding, lack of leadership support and failure of advocates to follow
through. A few responses, identified as “other” challenges, as described in Table 20.
Figure 37: Challenges to Development of Alternatives Using Lower
Vertebrates/Invertebrates
The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences.

Table 20: Other-Challenges
Need viable regulatory path with the
alternative
Lack of uptake by pharma from the CRO
offering the alternative

Respondents were asked about their interactions with the FDA during
implementation (Figure 38). Thirty percent (7/23) did not interact with the FDA, 26%
(6/23) received encouraging feedback, and 44% (10/23) neither encouraging or
discouraging feedback. No one felt that feedback was discouraging.

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Figure 38: Regulatory Agency Feedback During Implementation of Lower
Vertebrates/Invertebrates-Based Alternatives
During this process of evaluation/implementation of new alternative methods using lower
vertebrates/invertebrates, the feedback from regulatory agencies was:

The group was asked if the implementation of these alternatives resulted in
reduced animal use. Forty-two percent (10/24) reported a reduction, one-third (33%,
8/24) no reduction, and 25% (6/24) did not know (Figure 39).
Figure 39: Impact of Lower Vertebrates/Invertebrates-Based Alternative
Implementation on Animal Use
In retrospect, do you think that these efforts reduced the use of animals?

Respondents implementing lower vertebrate/invertebrate-based alternatives were
asked to describe them. The main themes, in Table 21, are summarized from data in
Appendix E.

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Table 21: Description of Alternatives Using Lower Vertebrates/ Invertebrates
(Main Themes)
If you implemented new methods using lower vertebrates/invertebrates, please
identify/describe the nature of that method:
Zebra fish for teratogenicity, developmental toxicity, early screening, as a disease model for
screening, evaluation of morphology, systemic toxicity evaluation
Fetax (Frog Embryo Teratogenesis Assay Xenopus (FETAX) test
Lower vertebrates, e.g., alligators, chicken 3-4 hours long experiments to measure various
bodily functions, and keeping the animal alive after experimentation.
LLNA Transgenic mice
Zebra fish larvae for screening Toxicity testing using Artemis Salina

4.5.4 Molecular Imaging/Other Novel Methods
When asked about “molecular imaging/other novel models” as alternatives
(Figure 40), one-third (31%, 49/159) stated that they had explored these and were
presented with additional questions related to that exploration. Half (51%, 81/159) had
not explored them and 18% (29/159) did not know; they were directed to the end of the
survey.
Figure 40: Exploration of Molecular Imaging/Other Novel Methods
Has your organization explored alternative "molecular imaging or other novel methods
(e.g., omics technology)" in lieu of or to reduce the size or number of animal studies?

Respondents who had explored molecular imaging/other novel methods were
asked to provide their opinion on the usefulness of various information sources. WMs

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calculated from a scale of 1 to 3 (1=very useful [VU], 2=modestly useful [MU] and
3=not useful [NU]), were used to order the relative usefulness of these sources
(Figure 41).
Scientific publications were ranked highest (WM=1.4). They were viewed as
very useful by more than half of respondents; almost all found them to be at least
modestly useful and no one viewed them as not useful (VU: 57%, 28/49; MU: 33%,
16/49). “Meetings/workshops/conferences” (WM=1.6) were also viewed by all as very
useful or modestly useful and no one viewed them as not useful (VU: 40%, 19/47; MU:
49%, 23/47). “Information from testing labs” had a slightly lower WM of 1.7 (VU: 39%,
19/49; MU: 45%, 22/49; NU: 4%, 2/49). Regulatory agency guidance was ranked lower
(WM=2.0); it was seen as modestly useful by about half of the respondents and the
remaining were mostly split between very useful and not useful (VU: 19%, 9/48; MU:
48%, 23/48; NU: 21% 10/48). Input from consultants was ranked lowest (WM=2.1);
more found them to be not useful compared to those who found them to be very useful
(VU: 16%, 8/49; MU: 39%, 19/49; NU: 25% 12/49).
Between 10-13% of respondents chose “not sure” for all but one option, “input
from consultants”, chosen by 20%. Additional sources were associated with “other” and
are presented in Table 22.

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Figure 41: Usefulness of Information Sources During Exploration of Molecular
Imaging/Other Novel Methods
During your evaluation of molecular imaging or other novel methods as an alternative to
animal testing, how useful were the following information sources?

Table 22: Other-Useful Information Sources
Working with companies developing organs on a chip technology-also university
colleagues
Internal method development and validation
Previous experience from a larger pharma company with incorporation of this technique
early in development.
Internal investigational experiences
Input from sponsors that the testing is useful and accepted by regulatory agencies

The respondents were asked about the eventual outcomes of their exploration
activities. Forty-six percent (23/50) implemented these methods but do not use them
routinely, 28% (14/50) use them routinely, 24% (12/50) are still exploring, and 2% (1/50)
decided to not go forward with these methods. No organization abandoned them after
initial implementation (Figure 24). Those who chose “still exploring” were allowed to
skip the remaining questions in this category and were directed to the end of the survey.

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Figure 42: Outcome of Exploration of Molecular Imaging/Other Novel Methods
What was the outcome of your exploration/evaluation of methods using molecular
imaging/other novel methods to replace or reduce animal testing?

The single respondent who indicated that the company decided not to go forward
with molecular imaging/ other novel methods was asked about the factors driving that
decision. This respondent regarded “lack of necessary infrastructure” as very important
factor and the remaining options as somewhat important (Table 23).
Table 23: Factors that Drove the Decision to not Pursue Molecular Imaging/
Other Novel Methods
What factors drove the decision not to go forward with this type of method during the
evaluation/exploration process?
Factors Very
Important
Somewhat
Important
Not
Important
Not
Sure
Development would take too long 0 1 0 0
Replacement would be too expensive 0 1 0 0
Lack of necessary infrastructure 1 0 0 0
Our team lacked required know how 0 1 0 0

Although no one chose the option, “our company considered them but abandoned
them after initial implementation”, one response was obtained when asked to rate the
factors contributing to that decision (Table 24). “Disappointing results from initial runs”

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was selected as a very critical factor, “resistance to change” as critical, “insufficient
infrastructure” and “lack of systematic plan” as not critical.
Table 24: Factors Causing Abandonment of Molecular Imaging/Other Novel
Methods After Initial Implementation
What factors played a role in your decision to abandon the methods using lower
vertebrates or invertebrates after the initial implementation?
Factors Very
Critical
Critical Not Critical Not Sure
Lack of systematic plan for training 0 0 1
0
Greater comfort with status
quo/resistance to change 0 1 0
0
Disappointing results from initial runs 1 0 0
0
Insufficient infrastructure 0 0 1
0

All respondents were presented with factors that were viewed previously by
others as challenges when developing new molecular imaging/other novel alternatives.
Results were ranked by WMs, from a scale of 1 to 3 (1=strongly agree [SA], 2=agree [A],
and 3=disagree [DA]) (Figure 25). “Lack of experts to guide implementation” (SA: 24%,
9/37; A: 43%, 16/37; DA: 27%, 10/37) and “lack of continuous funding” (SA: 20%, 7/35;
A: 35%, 12/35; DA: 26%, 9/35) were ranked highest with a WM of 2.0 and 2.1,
respectively. For the other three options, the percent of respondents who selected
“strongly agree” was very low (5-17%) compared to those who selected “disagree” (35-
47%). For “lack of leadership support” (WM=2.2), 60% of respondents either chose
“strongly agree” or “agree” whereas more than a third also chose “disagree” (SA: 14%,
5/37; A: 46%, 17/37; DA: 35%, 13/37). Both “advocates not following through” (SA:
17%, 6/36; A: 33% 12/36; DA: 47%, 17/36) and “lack of staff to manage changes” (SA:

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5%, 2/37; A: 54%, 20/37; DA: 38%, 14/37) had the same WM of 2.3. For all options,
respondents selecting “not sure” ranged from 3-20%.
Figure 43: Challenges to Development of Alternatives Based on Molecular
Imaging/Other Novel Methods
The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences.

Respondents were asked about their interactions with FDA during the
implementation of molecular imaging/ other novel methods (Figure 44). Thirty-one
percent (11/35) did not interact, 20% (7/35) received encouraging feedback, 46% (16/35)
neither encouraging or discouraging feedback and 3% (1/35) discouraging feedback.

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Figure 44: Regulatory Agency Feedback During Implementation of Molecular
Imaging/Other Novel Methods
During this process of evaluation/implementation of new Imaging/other novel methods,
the feedback from the regulatory agencies was:

The same group was asked if the implementation of molecular imaging and other
novel methods resulted in the reduced use of animals. Almost half (51%, 19/37) reported
a reduction, one-third (30% 11/37) no reduction, and 19% (7/37) did not know
(Figure 45).
Figure 45: Impact of Implementation of Molecular Imaging/ Other Novel
Methods on Animal Use
In retrospect, do you think that these efforts reduced the use of animals?

Respondents who implemented molecular imaging/ other novel methods were
asked to describe them. The main themes are summarized in Table 25 from data in
Appendix F.

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Table 25: Description of Molecular Imaging/Other Novel Methods Implemented
If you implemented new imaging techniques/other novel methods, please identify/describe
the nature of that method:
LCMS MALDI imaging; MALDI imaging into single dose PK studies in rat
Genomic analyses on primary cells and tissues to understand insertion site distribution for
viral vectors
RNA seq analyses combined with systems biology pathway analysis software
Next-generation sequencing (NGS) of tissue
Cell based omics screening HCS multiparameter screening
PET imaging Single cell imaging techniques
Metabolomics methods Celomic platform
Cell microarray for ligand-receptor
interactions
Multiplex imaging of cell culture toxicity
studies
In vitro MPS with genomics Toxicogenomics in cultured cells

4.6 Potential Policy Recommendations
Respondents were asked to rate initiatives that could help to accelerate the
validation of alternatives (Figure 46). Results were ranked by WMs, from a scale of 1 to
3 (1=very important [VI], 2=important [I], and 3=not important [NI]). “Establishment of
global standards for validation processes” (WM=1.5) was viewed as very important or
important by the large majority of respondents (VI: 58%, 95/165; I: 38%, 63/165; NI:
3%,5/165). Only slightly less important, with WMs of 1.6, were two choices, “increase in
funding for institutions involved in validation, acceptance and implementation of
alternatives” (VI: 48%,79/166; I: 45% 74/166; NI: 5%, 8/166) and “creating stronger
incentives for organizations to validate methods after development” (VI: 44%, 72/166; I:
50%, 83/165; NI: 3%, 5/165). “Incorporation of more active incentives for the use of
alternatives” (WM=1.9) was ranked lowest (VI: 28%, 46/163; I: 46%, 75/163; NI: 15%,

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25/163). For all options, the number of respondents selecting “not sure” ranged from 1-
10%.
Figure 46: Efforts to Accelerate Validation of Alternatives
Do you think that the following efforts may help accelerate the process of validation of
new alternative methods?

Respondents were asked about the recommendations to make implementation of
alternatives more efficient. Results were ranked by WMs, from a scale of 1 to 3 (1=very
important [VI], 2=important [I], and 3=not important [NI]) as shown in Figure 47.
“Establishment of pathway for early involvement of regulators” was viewed by all
respondents (WM=1.3) (VI: 68%, 113/165. I: 32%, 52/165) as very important or
important. “Negotiation of global harmonization of testing requirements” had a similarly
strong WM of 1.4. It was also viewed as very important or important by most (VI: 65%,
108/165; I: 32%, 52/165) with only 2% (3/165) finding it not important. “Creation of
forum for data sharing between stakeholders” (WM=1.5) was viewed by most

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respondents as very important (49%, 80/165) or important (47%, 78/165); only 2%
(4/165) found them not important. “Implementation of legislation in the US similar to
Directive 2010/63/EU” (WM=1.7) was ranked lowest with fewer respondents finding it
very important or important (VI: 34%, 55/164; I: 34%, 56/164) whereas 11% (18/164)
found it to not important and 21% were not sure. A few responses identified “other”
recommendations as shown in Table 26.
Figure 47: Recommendations to Make Implementation More Efficient
Do you agree with the following recommendations to make the adoption and
implementation of alternative methods more efficient?

Table 26: Other-Recommendations
Change can be affected but it takes time and investment
Absolutely need involvement of reg agencies
Significant concordance between animal data and alternative model

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Respondents were presented with arguments used previously to explain failure to
develop new alternatives (Figure 48). Results were ranked by WMs, calculated from a
scale of 1 to 3 (1=Agree [A], 2=Neither agree nor disagree [NAD], and 3=disagree [D]).
Agreement was most commonly for the choices, “animal studies are still viewed as the
essential scientific standard” (A: 91%, 149/164; NAD: 7%, 12/164; D: 2%, 3/164) and
“animal studies will still be required by regulatory agencies over the next decade” (A:
88%;145/165; NAD: 10%, 17/165; D: 2%, 3/165) with WMs of 1.1.
“Replacement of animal studies is more complex than initially thought” (A: 80%,
132/165; NAD: 15%, 24/165; D: 5.45%, 9/165), “database of experience and knowledge
comes from traditional animal methods” (A:79%, 129/164; NAD: 16%, 27/164; D: 5%,
8/164) and “most alternative test methods are designed to monitor a single toxic effect
and cannot test complex systemic effects” (A: 76%, 126/165; NAD: 15%, 25/165; D: 9%,
14/165) were next with very similar WMs of 1.3. “Historical achievements depended on
animal research” (A: 62%, 102/16; NAD: 28%, 46/165; D: 10%, 17/165) and “most in
the scientific community regard the value of animal testing as self-evident” (A: 60%,
99/165; NAD: 29%, 48/165; D: 11%, 18/165) had less common agreement and WMs of
1.5.

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Figure 48: Arguments for Failing to Develop Alternatives
The following views are used as arguments for failing to develop alternative methods. Do
you agree?

Respondents were asked to provide their thoughts about how to address situations
in which an equivalent non-animal option does not exist. The 101 responses to this
question are provided in Appendix G and some representative comments are shown in
Table 27.

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Table 27: Addressing Studies When Non-Animal Alternative Do Not Exist
(Main Themes)
For some type of experiments there may not be an equivalent non-animal option. How
will that be addressed?
1. Animal studies will still be needed for multitude of reasons in many cases
2. There are no real alternatives for many kinds of studies
3. Reduction, reuse and refinement is possible but complete replacement of animal
studies in many cases is not feasible
4. Alternatives are aiding in reduction of animal use primarily but are not ready for
replacement
5. Combination of both approaches (in vivo and in vitro) will be needed to reduce the
number of animals used
6. Regulation will need to change; allow replacement with validated assays;
7. Harmonize globally so to prevent repetition to address small differences replication for
other countries.
8. Multiple approaches needed- Understand mechanism, systems toxicology and
modeling approaches, AOPs, PK/TK and PD/TD, weight of evidence, computer
simulation model, batteries of tests, use of AI once a critical knowledge base threshold
has been reached, use of MOA, development of humanized microtissues/MPS to
enable investigating human biology involving multiple cell types within a tissue
microenvironment, complexing various 'organs on a chip, use of ex vivo assays
9. Eliminate animal studies once lack of relevance to humans is determined
10. Scientific creativity, exchange of ideas, more research with time may lead to possible
alternatives
11. Human risk assessment in humans is risky/unethical, risky clinical trials, poses greater
risk to society
12. Early use of in silico/in vitro studies in discovery to eliminate number of candidates
and advance the most promising candidates and thus limit animal testing
13. Don’t use animals as a default and when its used employ 3Rs, combine endpoints
14. Non-animal options can be risky and
15. The biology is too complex to be replaced by alternatives
16. Uncertain results with alternatives eventually lead back to animal studies resulting in
significant loss of time and resource.

Respondents were asked to provide additional comments on this subject. Their
responses to this question are provided in Appendix H.

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Chapter 5. Discussion
5.1 Summary
The last two decades have seen an enormous investment in the discovery,
development and commercialization of new non-animal technologies and alternative
methods as described in Chapter 2. The scientific community, including industry and the
FDA, are well placed to exploit these opportunities in order to reduce the overall use of
animals for research when developing medical products. The goal of this study was to
explore how these alternatives to animal use are being implemented, by seeking the
experience and views of one principal stakeholder, the pharmaceutical industry. The
responses of their scientists and regulatory professionals give provocative insight into the
roadblocks and opportunities that shape the development and use of alternative methods.
However, the validity of the results presented here must be interpreted carefully by
recognizing certain limitations and delimitations that might affect any conclusions that
we might want to draw.
5.2 Methodological Considerations
5.2.1 Limitations
Several limitations were anticipated in this study. Some were due to the typical
challenges associated with any project/study that utilizes a survey instrument for data
collection.
Sensitivity of Information: The use of animals in research has been a sensitive
subject for many people and many years (Peters, 2012). Thus, I was concerned that some
respondents might be hesitant to share information and candid opinions that they might
consider to be damaging to the company. In the past, many companies and individuals

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engaged in animal research have been targeted by research extremist groups opposed to
animal research (Beversdorf et al., 2015). Concerns about confidentiality might also
affect the willingness of respondents to participate in the survey altogether. To allay such
concerns, special care was taken to (a) assure the participants that responses would be
anonymous and (b) phrase the questions in a way that would not ask for information
about company-specific activities. The large number of respondents who provided frank
and critical responses in open-text comment fields suggests that respondents were
engaged and enthusiastic about the issues considered in this study. In fact, many
respondents expressed interest in knowing the outcome of this study; many emphasized
how important this subject is; and some thanked me for taking on this important project
(personal communications). These unsolicited communications further suggested that the
that the responses were authentic and thoughtful.
Privacy issues: Similarly, many people do not participate in surveys because of
privacy concerns. Technology has enabled researchers to collect personal data and create
detailed profiles of individuals, so potential survey participants are often concerned about
their privacy and the security of the personal information that they share electronically
(Cho and Larose, 1999). Significant effort was made to identify this project as doctoral
research from USC by providing the contact information of my supervisor and me and by
sharing our LinkedIn profiles in the email invitation, to assure the participants that the
study represented legitimate academic research. This approach appeared to assure most
respondents but there is no way of knowing whether the individuals who did not respond
to the survey invitation did not participate due to concerns about losing personal
information.

105
Treatment as “junk” email: Failure to respond to a survey invitation does not
occur only because a respondent is disinterested. It can also occur if the invitation is
filtered as “spam” or “junk” mail (Evans and Mathur, 2018). According to Statista
(Statistica.com, 2021), spam messages accounted for 47.3 percent of e-mail traffic in
September 2020. To counter these intrusions, most companies have developed tools that
automatically filter unsolicited mail. Thus, it is common for survey invitations
disseminated by survey management sites to be intercepted and directed to a spam folder,
invisible to the participant. To overcome this challenge, I reached out to professional
organizations, who helped me by sending duplicate invitations to many of the invitees.
Sampling Issues: Survey validity is driven both by the response rates as well as
the representativeness of its respondents. Representativeness refers to how well the
sample drawn for the study compares with the population of interest (Fincham, 2008). If
the participants are not evenly distributed across companies of different sizes and product
lines, the results may not be fully representative of the larger group that the subset is
purported to represent. A skewed dataset can result more easily if only a small number of
invitees are invited to participate in the survey. To address this concern, I first attempted
to assure that survey invitations were sent to more than 500 potential participants from all
part of North America, as well as to companies of all sizes and to companies that were
engaged in the development of many types of therapeutic products. Additionally, I
personally scrutinized the profiles of most invitees to identify toxicologists engaged in
safety assessment of drugs and biologics. This approach yielded a robust representation
from the target population who worked who worked with companies of different sizes
and locations. The largest subset of respondents was from the east coast followed by the

106
west coast, as might be expected knowing that the two coasts of the continent have the
biggest pharma/biotech hubs (Fochios, 2017).
Declining response rate: For the reasons cited above, most people tend to
mistrust any invitations they receive to participate in online surveys if they do not
recognize their source. Even when a survey is received, a busy professional may be
unwilling to use his or her precious time for a research study. It is well-known from the
literature that many recipients fail to engage in a survey, either because of the perceived
time commitment or more generally because of “survey fatigue” (Ben-Nun, 2008). As a
result, most invitations to participate in surveys are ignored and this reduces the response
rate. For example, one meta-analysis (Manfreda et al., 2008) found that the average
response rate for web surveys was 11 percent. It has been suggested that multiple
contacts, degree of personal interaction and sponsorship can have significant impacts on
survey response rates (Dillman, Smyth and Christian, 2014). Thus, a proactive effort was
made to personally reach out to as many invitees as possible after sending the invitation
and then to send scheduled reminders directly from Qualtrics. I considered that the
completion response rate of 23% for the individually approached participants was
sufficient for the purposes of this exploratory study particularly due to the relative
homogeneity of job functions all related to this type of safety testing. The additional 56
responses received through the anonymous link added to the responses that were
collected more directly, but obviously no response rate could be calculated without
knowing the extent to which anonymous invitations were viewed by potential
respondents.

107
Personal Bias: It is also important to acknowledge my own personal bias because
my professional experience has come from working as a toxicology expert in small
companies. This experience could have unintentionally affected the nature of the survey
questions. Leading questions tend to be aligned with the goals of the researcher and can
unknowingly lead the respondent to support the views of the researcher (Allen, 2017).
Thus, I made every effort to assure that questions were impartial and did not attempt to
lead respondents toward a particular position. The ‘face’ validity of the survey was
further tested by subjecting the survey to critical peer review conducted by a focus group
of experienced industry leaders and faculty members from the Regulatory and Quality
Science department. Such focus groups are seen as a valuable tool to obtain feedback on
areas that are insufficiently explored or are unclear, and to assure that the intent of each
question was interpreted as consistently as possible (mTab, 2021).
5.2.2 Delimitations
This research has also been delimited in ways that could affect its external
validity. First, the primary focus of this dissertation was on alternative models in the
pharmaceutical and biologics industries. The use of animal alternatives is also of concern
to other industries, such as chemical, cosmetic, and food-additive industries. The results
explored here cannot be guaranteed to reflect the views of those other industries.
Nevertheless, it is likely that many of the concerns identified here will be generalizable to
those in other industries because animal testing is also required for safety assessment of
their products. This assumption would have to be tested by examining the responses of
individuals in those other industries in future.

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Further, the survey participants were mostly scientists engaged in safety
assessment of drugs and biologics. Thus, the data gathered does not reflect the
experience of individuals in those same companies but in other areas that also use
animals, such as basic science, translational research or pharmacology. Further they do
not include the experiences and views of highly diverse groups of academic researchers,
whose specialized scientific research may work under different rules within their
respective institutions. Academic laboratories in the US/Canada represent another large
sector for whom animal research has been key to significant discoveries (Carbone, 2021).
For example, the sequencing of the mouse and human genome along with the tremendous
growth in genome-editing technologies has led to a significant growth in the use of
genetically modified mice (Yang, Wang and Jaenisch, 2014). However, academic
laboratories mostly do not conduct regulated safety assessment studies. The federal
government also uses many research animals (National Research Council and Institute of
Medicine, 1988) for their intramural research and testing, but that work is also typically
not directed at the commercial development of drugs. The exclusion of these additional
users will affect the general interpretation of the results but will also reduce the
possibility that confounding information would reduce the clarity of the results.
However, the present study did include participants not only from pharmaceutical
companies but also from CROs. Today, nonclinical drug testing is commonly carried out
in partnerships that involve specialized contracting organizations skilled in the art and
science of safety testing strategies. The growing research and development (R&D)
activities over the years have also led to bigger demand for preclinical CRO services,
particularly for toxicology testing (Grand View Research, 2021). These organizations are

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in a strong position to inform on trends and challenges associated with the adoption of
non-animal alternatives.
The present survey was directed at participants who work for companies in the
United States and Canada. Such a focus seemed justified as a starting point because of
the strong position of the US as an innovative pharmaceutical leader. US companies
dominate the market in drug development; they provide jobs directly to more than
800,000 Americans and support the jobs of 4.7 million Americans indirectly (Select
USA, 2021). According to one study, the FDA approved 170 new therapeutic agents and
a higher percentage of orphan drugs than any other global economy between 2011 and
2015 (Downing, Zhang and Ross, 2017). Whether these US-centric views and
experiences generalize to other countries or regions is still unclear. For example, the EU
and China are also major pharmaceutical suppliers. However, approaches to the use of
animals are more restricted in the EU whereas they are more liberal in China (Deborah,
2018). Thus, it seemed unwise to introduce a potentially confounding factor related to
different rules and practices in other regions of the world at this early stage.
Another delimiting factor that defines the scope of the study has been its use of a
well-validated implementation framework developed by Fixsen and colleagues (2005).
The use of such a framework is viewed as a valuable tool in social-sciences research
because it can systematize and focus the development of survey questions and aid the
interpretation of the results. Implementation frameworks also aid the systematic
exploration of barriers and enablers that have been found to influence implementation
outcomes (Moullin et al., 2019). In this study, the framework was helpful in assuring that

110
the rather complex topic could be systematized in ways that allowed the comparison
between different types of approaches.
5.3 Considerations of the Results
5.3.1 Exploration and Installation
An important goal of a company is to identify an existing or emergent need for
change and then determine what new set of practices are likely to meet that need before
deciding whether and how to move ahead with the implementation process. This
“exploration phase” then transitions to an “installation phase” whose goal is to establish
organizational competencies and make essential changes to support the implementation
of the new practice or approach (Fixsen et al., 2005). In this study, however, the analysis
was complicated because companies have the option to adopt different types of non-
animal alternatives depending on their specific needs and circumstances. Because each
has its own merits and challenges, it seemed prudent to evaluate the approaches to each at
the earliest stages of implementation. Interestingly, a side-by side comparison of certain
key results, shown in Table 28, suggested more similarities than differences in the
exploration and installation of these different options. Certain key takeaways of these
results are discussed below.

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Table 28: Exploration and Installation of Various Categories of Alternatives
In Silico/
Computer-based
Tissue/Cell Culture
Technologies
Imaging/Other
Novel Methods
Lower Vertebrates/
Invertebrates
No. of total responses
166 163 159 162
Percent of respondents that explored new alternatives
58% 49% 31% 22%
Main information sources used during exploration
1.Scientific publication
2. Meetings/conferences
1.Scientific publication
2. Meetings/conferences
1.Scientific publication
2. Meetings/conferences
1.Scientific publication
2. Meetings/conferences
Respondents who decided to not pursue installation of alternatives after exploration
1% 0% 2% 3%
Feedback from FDA during exploration
Encouraging: 30%
Discouraging: 3%
Neither: 27%
Did not interact: 40%
Encouraging: 26%
Discouraging: 8%
Neither: 25%
Did not interact: 42%
Encouraging: 20%
Discouraging: 3%
Neither: 46%
Did not interact: 31%
Encouraging: 26%
Discouraging: 0%
Neither: 44%
Did not interact: 30%
Outcome of exploration leading to installation
Routinely used: 33%
Not used routinely: 36%
Still exploring: 28%
Routinely used: 34%
Not used routinely: 33%
Still exploring: 31%
Routinely used: 28%
Not used routinely: 46%
Still exploring: 24%
Routinely used: 26%
Not used routinely: 40%
Still exploring: 29%
Percent of respondents who abandoned alternatives after initial installation
2% 3% 0% 3%
Main challenges encountered during installation
1.Lack of experts
2.Lack of continuous
funding
1.Lack of continuous
funding
2.Lack of experts
1.Lack of experts
2.Lack of
continuous funding
1.Lack of continuous
funding
2.Lack of experts
Impact of alternatives on animal use
Yes: 48%
No: 29%
Yes: 59%
No: 28%
Yes: 51%
No: 30%
Yes: 42%
No: 33%

5.3.1.1 How commonly are different alternatives explored?
Of the four types of non-animal alternatives investigated in this study, in
silico/computer-based alternatives appeared to be considered most commonly. The rapid
growth in computational technologies has yielded several cost-efficient and high-

112
throughput methods that are now used routinely in drug design and screening (Lin, Li and
Lin, 2020). They include a variety of models encoded with specific software tools to
predict whether a compound is toxic or non-toxic. Programs like the Derek Nexus,
ToxTree, and OECD QSAR toolbox are able of predict several different toxicity
endpoints (Cuffari, 2020). Their use is also driven by initiatives in Europe, North
America and at the OECD to accelerate the use of in silico techniques as alternatives to
animals for regulatory toxicity testing (Taylor and Rego Alvarez, 2020).
Another often-explored set of methods are those involving tissue/cell culture.
These techniques use a broad array of tissue slices, isolated organs, isolated primary cell
cultures, explant cultures, cell lines, and subcellular fractions like mitochondria,
microsomes, and even membranes. The relevance and popularity of such methods have
increased as biotechnological advances yield fast, reproducible, and reliable assays. The
tests are typically easier to set up, faster and cheaper than in vivo studies. Their use in
some specific areas has also been motivated by societal concerns and ethical
considerations (Singh, Khanna and Pant, 2018). An important example is that of ocular
toxicity, the potential of compounds in drugs and cosmetics to induce ocular irritation.
Previously, this assessment was carried out using the Draize test on animals, typically
rabbits. The test was highly criticized in the media and by animal rights proponents
because it can be painful and requires that a new animal be used for each exposure
(Humane Society International, 2013). Today, an alternative approach can use a bovine
corneal organ culture, for example, to evaluate the potential for irritancy in vitro (Xu, Li
and Yu, 2000).

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Less commonly explored by the respondents here are imaging techniques and
alternatives in lower vertebrates or invertebrates. Nonetheless, these promising
approaches may become more popular in future. Lower vertebrates and invertebrates can
be useful because they have more complete physiological systems that bear some genetic
relatedness to higher vertebrates including mammals, but they provoke less ethical
sensitivity. For example, the zebra fish, danio rerio, has been an attractive model because
of its small size and high fecundity. It is used in a variety of applications that can take
advantage of its short life cycle for the toxicological study of chemicals and
pharmaceuticals (Hill et al., 2005). The fruit fly, drosophila melanogaster, is one of the
most widely studied invertebrates in research. It is inexpensive to maintain, propagate
and its short life cycle ensures rapid test results (Gilbert, 2008). However, such model
organisms are typically used in early discovery research and are used less commonly to
test the toxicity of drugs.
Another option to help reduce animal use is the application of non-invasive
imaging techniques such as magnetic resonance imaging (MRI) computed tomography
(Select USA) scanning, positron emission tomography (PET), and optical imaging. These
offer a window into the living body, often in real time, to study, view, diagnose and in
many cases treat disease conditions without causing pain, suffering or termination
(Heindl, Hess and Brune, 2008). Rapid advances in technology have enabled these
methods to become highly sophisticated and have outstanding resolution and precision.
However, the equipment of these modalities is extremely expensive and requires
specialized training to learn and use. They have other limitations as well. For example, a
scan may show a change in the brain, but the drugs under investigation will still need to

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be tested in live animals in order to understand the physiological implications of those
changes. Thus, at least some animals must be tested regardless.
5.3.1.2 What resources were useful during those explorations?
During the exploration stages, respondents were surprisingly consistent in the
sources of information that they used. They appeared to rely most heavily on scientific
publications and meetings or conferences regardless of the type of alternative strategy
that they were considering. This is perhaps not surprising. These sources would provide
the most detailed information on the strengths, weaknesses and especially the logistical
requirements for the alternatives under consideration. It may also reflect the fact that
most funding of alternative animal research has been directed at universities and research
institutions, where academic publications and conference proceedings are considered as
the “gold standard” to communicate scientific matters (Harley et al., 2006). For example,
the Alternatives Research & Development Foundation (ARDF) has been supporting such
research in this area through grants and sponsorships of conferences for academic
scientists (The Alternatives Research & Development Foundation, n.d.). Further, certain
governmental organizations have invested heavily in this type of research. In 2019, for
instance, the EPA awarded $4.25 million to USA universities to develop non-animal
alternatives (The Business Research Company, 2021). This type of support has even
motivated academia to set up specialized programs. For example, the Johns Hopkins
Center for Alternatives to Animal Testing (CAAT) at the Johns Hopkins University was
developed to research alternatives to animals in research, undertake safety testing, and
provide educational opportunities (Johns Hopkins Bloomberg School of Public Health,
n.d.).

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In comparison, regulatory guidances were not considered so useful by the
respondents here. This might be explained, at least in part, by the fact that regulations and
guidances are well known to lag technological advancements. The FDA’s
recommendations regarding innovative ideas are often cautious in order to ensure the
safety of the American public (Ellenberg, 2017). Although both the United States and
European Union have recognized the value of alternative methods to animals, most such
methods still await regulatory acceptance as replacements for animal testing in drug
approval processes. As identified in an ICH guidance,
Although not discussed in this guidance, consideration should be given to
use of new in vitro alternative methods for safety evaluation. These
methods, if validated and accepted by all ICH regulatory authorities, can
be used to replace current standard methods (ICH, 2009, p.2).
The regulatory acceptance of a specific method is a complex process involving
not only scientific considerations, but also policy issues, cost-benefit evaluations,
comparisons to traditional methods and product-specific requirements that may be unique
to each country (Burden, Sewell and Chapman, 2015). For the same reasons as described
above, information from testing laboratories and inputs from consultants were also
considered to be less useful. Because the number of accepted alternatives is still small,
the testing laboratory or consultant may still be inexperienced with alternatives, even if
they are expert in animal testing, so may want to restrict their advice to their area of
expertise.
5.3.1.3 How often did explored alternatives advance to the installation phase?
After exploration, almost all respondents reported that their companies advanced
to installation activities. This rate of adoption is impressive and may suggest that the

116
decision to implement additional non-animal methods was already prioritized even before
the exploration of methods began. That companies are highly motivated to introduce
non-animal alternative methods is also suggested by a recent survey by Goh et al. (2015).
Goh examined the number of compounds that had been screened using in vitro methods
over time and reported a steady increase in the use of in vitro tests by the pharmaceutical
industry. It is apparent that the alternative technologies are gaining popularity not only to
reduce the usage of animals but to also to fast track the drug development process in a
cost-effective manner.
To facilitate the transition to alternative testing, end-use industries, including
cosmetic, pharmaceutical, medical device, chemical, and food companies, are now
collaborating with organizations that are developing alternative testing technologies to
understand the requirements and bottlenecks in the deployment of these alternatives.
Some examples include Unilever’s collaboration with the EPA to develop alternatives
and AstraZeneca’s collaboration with Emulate, Inc. to develop organ-on-chip technology
(The Business Research Company, 2020). This may help to share the costs and risks of
developing alternative technologies and take advantage of the specialized expertise in the
science-based organizations.
5.3.1.4 How often do sponsors seek regulatory guidance from the FDA during the
exploration and installation stages?
In this study, only a small number of respondents sought regulatory
guidance/feedback from the FDA during exploration and installation stages of new
alternative methodologies. This seems surprising because questions must arise as these
novel methods are implemented. The process of validation and regulatory acceptance is

117
still not standardized so companies with new methods are often unsure about the process,
for a variety of reasons. They may not know whether they need to submit their method
for official validation and qualification, and whether it would add value to speak directly
to the regulatory body (Taylor, 2019). However, if they were to talk to regulators, they
may not know who to contact and what information they need to provide. The process of
seeking formal meetings with some FDA offices can be onerous and time-consuming. It
is also possible that the scientists involved with the drug safety testing have outsourced
that specialized testing to a CRO. As a result, they rely on the advice of the CRO,
especially at early stages, and may not see it as their responsibility to talk to the FDA at
that point. Perhaps companies are unsure about the reception that they might get when
proposing a novel method. However, the current results suggest that most of those who
did interact with the regulators rated the feedback as either encouraging or neither
encouraging nor discouraging; only a small number found them to be discouraging.
5.3.1.5 What were the main challenges encountered during installation?
Regardless of the type of non-animal alternative, the two most common
deficiencies during early stages appeared to be the lack of experts to guide the
implementation process and the lack of continuous funding to support the initial
implementations. The challenges of finding experts may in part extend from the fact that
experience and expertise with these new methods are confined to a small group of
individuals and may not be present in all companies. Further, companies that develop
such methods may have no incentive to share the method with the wider scientific
community. They may have invested considerable time and money to create a method
that improves competitiveness and prevent competitors to gain from its use. Once such

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example is the development of the cell-based potency assay by Allergan for botulinum
toxin products to replace the animal based LD50 assay, which must be conducted using
thousands of mice (GenomeWeb, 2011). However, due to the proprietary nature of that
test, other companies must continue to use the animal LD50 assay until they are able to
develop their own in vitro test. For these reasons, access to and dissemination of
information is a topic of discussion and signals a fundamental shift to encourage “open
source” information as suggested, for example, during a meeting of In the Vitro Testing
Industrial Platform (IVTIP) (Ashton et al., 2014).
The other major obstacle is the insufficiency of continuous funding to support the
development of alternative methods. At the outset of such development, it can be unclear
how much the development will cost and whether the new method will be accepted by
the regulators even if that investment is made. Thus, replacing animal tests is likely to be
slow unless there is a significant increase in funding proportionate to the scale of the
problem (Taylor, 2019). One way to approach this problem is through public-private
partnerships where cost- and risk-sharing can be leveraged, as discussed in Chapter 2
with respect to the Critical Path initiative. That initiative, for example, facilitated the
“fit-for-use” regulatory acceptance of a battery of in vitro kidney damage assays that
could replace specific animal testing for kidney damage (Avila et al., 2020).
Interestingly, some of the internal factors, such as lack of leadership, lack of staff
to manage changes or problems when early advocates/champions did not follow through
were not considered as the toughest hurdles, though their contributions as hurdles should
not be discounted in some companies. These results suggest that once decisions have
been made, many companies recognize the need to establish effective and supported

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teams. However, for others, attention needs to be paid to the role of leadership, for
example.
5.3.2 Implementation
The implementation phase has two stages: initial implementation, when a newly
installed innovation is first used by practitioners; and full implementation, when the
practice becomes routine and well-integrated into the repertoire of the practitioners. The
initial phase is important to assess whether the expected advantages of the new method
are achieved and whether improvement strategies can be developed.
Results from this study indicated newly installed methodologies were seldom
abandoned, as noted above. Nevertheless, it was interesting that only about one-third are
being used routinely. Respondents identified many challenges as implementation barriers,
and all deserve further study in future. Amongst the most notable from this study are
three that deserve particular discussion. These include the “lack of validation of existing
alternatives” and “concerns about regulatory acceptance” of newly installed alternatives,
as well as “lack of confidence in scientific adequacy” of these methods.
5.3.2.1 Why is validation such a large concern?
Any new alternative must be validated fully before its widespread uptake and
regulatory acceptance. However, validation is well-recognized to be a complex process
requiring much time and money to reduce various sources of uncertainty. Respondents
here also noted the lengthy and costly nature of validation to prove that the outcomes will
be at least as good as those obtained with current animal-based options. Often companies
are even unclear about what would constitute an adequate level of validation evidence
(Prior et al., 2019). Another related hurdle is the absence of an overarching strategy to

120
assess the predictive capabilities and reproducibility of the test for a specific regulatory
need (Piersma et al., 2018). When respondents were asked directly who should bear the
costs of validating an alternative method, it was interesting that half saw the primary
responsibility to be that of industry. However, most alternative methods for toxicity
testing would be a shared resource for a wide variety of companies and organizations, so
the role of the governments and international standards-setting and guidance-developing
bodies were also seen by many to extend beyond just advocating for their use to actually
assisting with their validation. Developing new methods in partnerships would also
reduce the likelihood that the methods would become proprietary rather than publicly
accessible.
5.3.2.2 How does regulatory acceptance influence alternative model development?
Significant progress has been made in the attitudes of regulatory agencies
regarding the regulatory acceptance of alternative methods over the past 10 years. A
recent report by the “Alternative Methods Working Group” at the FDA described how
FDA scientists are laying the groundwork for integrating alternative approaches into
FDA regulatory programs. This report demonstrates not only FDA’s commitment to
reduced animal testing but also to encouraging partnerships with other stakeholders to
achieve this goal. Results from their research activities add to the body of knowledge
upon which regulators can draw as they set regulatory acceptance criteria for new
alternative methods (FDA, 2021). Nonetheless, the fact that half of the respondents still
appeared to be concerned about regulatory acceptance points to the continued need to
address the gap between foundation-building for qualification and actual acceptance of
methods.

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5.3.2.3 What ways are available to increase acceptance for safety testing?
Data generated from methods that use animal testing alternatives may be
challenging for regulatory bodies to interpret with respect to human risk assessment. The
data in this study suggested a strong support for a multifaceted strategy of
communication to facilitate rapid acceptance of non-animal alternatives that included
weight-of-evidence (WoE) approaches, dissemination of information through
publications and workshops or conferences as well as the use of subject matter experts
(SME)and opinion leaders. The WoE approach, most commonly selected here, relies on a
combination of information from several independent sources, that then must be
evaluated using scientific judgment to assess the quality of the data, consistency of
results, nature and severity of effects, and relevance of the information (ECHA, 2016).
However, it is also important to communicate the information and results of assessments
and new decisions. Respondents recognized that the dissemination of information might
use several channels, including publications and presentations. Adoption was also seen to
depend on champions and experts to spearhead awareness. In the absence of such
information, development teams may continue to fall back on the status quo animal
testing.
5.4 Current Status and Outlook
5.4.1 Will alternative methods reduce animal studies in the near future?
It seems clear from these results that the large-scale replacement of animal testing
by non-animal methods is not likely to happen in the immediate future, at least through
the eyes of at least half of the respondents. It was striking that a majority believed that the
number of currently used animals is reasonable and that drug safety assessment cannot be

122
animal free. This not to say that gains cannot be made. Standards and ICH guidelines are
reviewed and revised periodically to incorporate recent scientific developments and
remove practices that no longer add value providing some opportunity for change. Such
changes have been seen in the past to remove the stand-alone acute toxicity testing and
integrate bone marrow micronucleus endpoints in rodent general toxicity studies (ICH,
2009; Ledwith and DeGeorge, 2011). However, both these changes reduce the number of
animals without relying on alternative methods.
It will be interesting to see whether the promising non-animal toxicity tests now
emerging from various partnerships will result in further modifications to ICH guidelines.
Despite such revisions in those guidelines, more than half of the respondents did not
expect that changes related to non-animal models for testing would occur in ICH
guidances in the next decade. If this is the case it has significant implications. ICH
recommendations are important in driving uptake. Although not legally binding, they do
set the stage for a successful marketing authorization. Even though they address the issue
of animal use directly, they also underline the limitations posed by sufficient validation
and acceptance.
This guidance should facilitate the timely conduct of clinical trials, reduce
the use of animals in accordance with the 3R (reduce/refine/replace)
principles and reduce the use of other drug development resources.
Although not discussed in this guidance, consideration should be given to
use of new in vitro alternative methods for safety evaluation. These
methods, if validated and accepted by all ICH regulatory authorities, can
be used to replace current standard methods (ICH, 2009, p.4).
5.4.2 How much impact have alternative tests really had?
About half of the respondents expressed the view that the alternatives to animal
tests have reduced the use of animals. At the same time, however, one-third had the more

123
pessimistic opinion, that they have had little impact. These later views align with those of
certain other policy analysts who have suggested that replacement of animal testing by
nonanimal alternatives will be difficult to justify for more than the detection of specific
types of organ or tissue damage. As stated by Stokes, “…uncertainties related to
absorption, distribution, metabolism, and excretion must be overcome. Other challenges
include eliminating extrapolation uncertainties for factors such as age, gender, ethnicity,
genetic susceptibilities, and co-morbidities in target populations” (Stokes, 2015, p.1301).
Reduction of animal use in certain areas is more likely than complete replacement
of animals. Additionally, alternatives may help corroborate or refine animal testing
results to strengthen the evidence on which a conclusion can be drawn with confidence.
The implementation of in vitro and in silico models has been growing rapidly, as
suggested by the results here, although many respondents identified that they were often
add-on assays with modest impact on overall animal testing. However, a trend to use
more assays of these types would not be surprising. Only about 1 in 10 compounds that
enter first-in-human studies will eventually be approved by the FDA (Hay et al., 2014).
High-throughput tests to differentiate promising drug candidates earlier in the
development cycle can reduce the number of compounds that progress to regulatory
toxicology studies in animals (Liebsch et al., 2011). Even a small improvement in the
ability to filter compounds with toxic profiles would help to reduce the numbers of
animal-rich toxicology studies that might otherwise be carried out on compounds that are
eventually doomed to fail in the clinic. This would have a significant impact, albeit
indirectly on the overall use of animals in drug development.

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5.5 Areas of Policy Focus
Numerous studies provide evidence that the implementation of effective programs
or practices can lead to desirable outcomes (Fixsen et al., 2001). However, as described
in previous sections, many gaps exist in the widespread implementation of alternative
testing methods, and this makes the views of respondents on how to remediate those gaps
of great value. One area of focus was on the role of the FDA, typically characterized by
the respondents as only modestly successful at best. Although the FDA has expressed
support for alternative methods, to date most drugs still require a stereotypic battery of
animal testing that cannot be replaced by alternative methods. Despite the promise shown
by many different alternatives to facilitate early drug development, most have yet to
make significant inroads as replacements for regulatory animal testing (Van Norman,
2020).
“Validation” appeared to be the biggest hurdle. Respondents pointed to the
potential usefulness of certain programs and policies to accelerate the validation process.
One area in which more activity appeared to be important was that of global standard-
setting to align multiple agencies on common validation principles (Prior et al., 2019).
Not only are global standards for validation approaches important to encourage the
development of testing methods, but also to increase the likelihood that the validated test
will be accepted as fit-for use in multiple jurisdictions. Another area of focus related to
the need for increased funding and incentives to fast-track the validation, qualification
and implementation of alternatives.
The survey responses also recommended other opportunities for improvement.
The respondents suggested that a pathway be put into place to engage with regulators

125
early in development and foster the “creation of forum for data sharing between
stakeholders”. This suggestion aligns with a call by Burden et al. (Burden et al., 2015) for
the industry and regulatory communities to work together so that safety assessment
strategies can be modified and animal usage reduced. Also important were negotiating
the global harmonization of testing requirements and the improved acceptance of a
common data set in different jurisdictions. Most pharmaceutical companies have a global
reach and currently face requirements to conduct redundant animal studies for
registration in different regions. Nevertheless, “implementation of legislation in the US
similar to Directive 2010/63/EU”, an obvious way to begin such alignment, did not seem
very popular with toxicologists in the US/Canada. The reasons for the caution expressed
here would be an excellent subject for further research.
All of these suggestions could usefully be considered as a list of action items that
would greatly enhance the development and implementation of alternatives. What this
study identifies is a field in transition, whose advances depend not only on increased
efforts to validate and accept alternatives but by also collaborative approaches to allow
data sharing. However, the development and implementation of non-animal alternatives
is not an easy task given the need to meet or surpass the outcomes of current animal
methods. The political will to invest sufficiently in needed research and validation of
alternative methods may depend as much on societal pressure and governmental funding
allocations as on the views of toxicologists and regulators. Nevertheless, the suggestions
made here could usefully form a list of action items that could be addressed immediately
by these expert stakeholders, to establish the policies and frameworks that would

126
facilitate the validation principles and harmonized acceptance of publicly available
alternative methods globally.

127
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SURVEY
Implementation of Alternative Methods in Safety Assessment of Drugs
and Biologics
• Note to participants: Q1
• Demography and professional profile of participants (N=6): Q2-Q7
• Current status, trends and outlook (N=6): Q8, Q14-18
• Barriers and drivers (N=5): Q9-Q13
• Exploration and installation of different category of alternatives (N=9/category, 36
total): Q23-Q58
• Potential policy solutions (N=4): Q19-22
• Additional Note: Q59
• Thank you Note: Q60

Start of Block: Babbar Survey
Q1 Implementation of Alternative Methods to Animal Testing for Safety Assessment
of Drugs and Biologics in North America
Thank you for participating in this survey. It is intended for toxicologists and other
individuals involved in evaluating the safety of drugs and biologics. The goal of this
study is to gather your experiences and views related to whether and how the industry is
implementing “alternative methods” to reduce the use of animals when assessing the
safety of drugs and biologics. In the context of this survey, an 'alternative method' is
a new research technique that either (a) replaces the use of animals altogether or (b)
reduces the number of animals. Your responses will be kept anonymous. If you find
that you cannot answer a question, please feel free to skip it or to choose the “not sure/I
don’t know” options.

Demography
Q2 Please describe your organization.
o Contract Research Organization/Testing Facility (1)
o Pharmaceutical/Biotechnology Company (2)
o Consulting Company (4)
o Government (e.g., FDA/Health Canada) (5)
o Other (6) ________________________________________________

140
Q3 What is the size of your company based on the number of employees? If you are a
consultant, please select the main organization for which you provide service and
estimate its size.
o Less than 100 employees (1)
o 100 - 1000 (2)
o 1001 - 10000 (4)
o More than 10000 (5)

Q4 Please describe the approximate age of your organization.
o Less than 10 years old (1)
o Between 10 and 20 years old (2)
o More than 20 years old (3)

Q5 Please indicate the geographical location of your company's North American
office in the US or Canada.
o West Coast (1)
o East Coast (2)
o Mid-West (3)
o Other (4) ________________________________________________

Q6 How many years of personal experience do you have in nonclinical safety testing?
o Less than 5 years (1)
o 5 - 10 years (2)
o 11 - 20 years (3)
o Greater than 20 years (4)

141

Q7 What kind of therapy are you developing for your current organization? If you are
working or consulting for more than one product type, please characterize the area
in which you have had most experience in the last 5 years.
o Small molecules (1)
o Large molecules (therapeutic proteins and monoclonal antibodies etc.) (2)
o Gene therapy, cell therapy, vaccines (6)
o Other (4) ________________________________________________

Current Status, Trends and Outlook

Q8 In your professional opinion, are the number of animals used in nonclinical safety
assessment…
o Excessive? (1)
o Reasonable? (2)
o Insufficient? (3)
o Animals are not used in my work (4)

Q14 Do you think that efforts to implement alternatives to animal testing have helped
to reduce the number of animals used in safety assessment?
o Yes (1)
o Somewhat (2)
o No (3)
o I don't know (5)

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Q15 In the US and the EU, ICH guidance requires extensive animal testing of
drugs/biologics before human exposure. Do you see that changing in the next
decade or so?
o Very likely (1)
o Somwhat likely (2)
o Unlikely (3)
o Not sure (4)

Q16 Research suggests an upward trend in the implementation of in vitro tests for
safety assessment. In your view, are the new alternatives being implemented as
“add-on” methods (to further assist in safety determination) or do they result in
reduction in animal usage?
o Mostly to reduce animal use (1)
o Mostly as add-on without reducing animal use (2)
o A mix of both (4)
o Not sure (5)

Q17 Do you believe that drug safety assessment can be animal-free?
o Yes (1)
o Maybe (2)
o No (3)
o I don't know (4)

143
Q18 Do you believe that the FDA has been successful in implementing alternative
methods to replace/reduce animal testing in the field of safety assessment?
o Very successful (1)
o Modestly successful (2)
o Not successful (3)
o I don't know (4)

Barriers and Drivers

Q9 What do you believe are the barriers that have impeded the implementation of
new alternative methods? Please select all that apply.

Very
Significant (1)
Significant (2)
Not
Significant (3)
Not Sure (4)
Lack of confidence in
scientific adequacy
(1)
o o o o
Concerns about
regulatory
acceptance (2)
o o o o
Risk averse climate
(3)
o o o o
High cost of
development (4)
o o o o
Absence of
leadership/champion
(6)
o o o o
Lack of validation of
existing alternative
(7)
o o o o
Other (8)
o o o o

144
Q10 Please rate the following factors that may have played a role in promoting
development of alternatives to animal use in toxicity testing. Please select all that
apply.

Most
Important (1)
Important (2)
Marginally
Important (3)
Not Important
(4)
Improved
regulatory
agency climate
for use of
alternative
methods (1)
o o o o
Ability to
decrease testing
cost (2)
o o o o
Response to
societal
sensitivities (3)
o o o o
Better scientific
results (4)
o o o o
Ethical
considerations
(5)
o o o o

145
Q11 Lack of validation of a new alternative method is considered as a major hurdle in
adoption of alternative methods. What are the barriers that hold up the validation
of alternatives? Please select all that apply.

Very Important
(1)
Important (2)
Not Important
(3)
Not Sure (4)
Highly time
consuming (1)
o o o o
Large
investment
needed (2)
o o o o
Lack of market
due to limited
regulatory
acceptance (3)
o o o o
Uncertainty in
achieving
successful
outcome (4)
o o o o
Concerns about
opinions of
peer/scientific
community (5)
o o o o
Validation too
complex
technically (6)
o o o o
Alternatives are
not animal-free
(7)
o o o o

146
Q12 The cost associated with the validation process of new alternatives is considered a
barrier to the development of an alternative method. Who should
be responsible for supporting the cost to validate the new methods for regulatory
acceptance? Please select all that apply.

Primarily
Responsible (1)
Somewhat
Responsible (2)
Not
Responsible (3)
Not Sure (5)
Government
Agencies (e.g.
FDA/NIH) (1)
o o o o
Industry
(Pharmaceutical-
biotechnology)
(2)
o o o o
Public-private
consortia {e.g.
John Hopkins
Center for
Alternatives to
Animal Testing,
Critical Path} (3)
o o o o
International
organizations
(e.g. ICH, WHO)
(4)
o o o o
Contract
Research
Organizations (5)
o o o o

147
Q13 Once an alternative method is validated, what strategy should be employed by
stakeholders to work with regulatory agencies to ensure its acceptance? Please
select all that apply.

Most Effective
(1)
Effective (2)
Not Effective
(3)
Not Sure (4)
Dissemination
through
publication/white
paper (1)
o o o o
Weight-of-
evidence
approaches (e.g.
multiple sources
of information)
(2)
o o o o
Use of subject
matter
experts/key
opinion leaders
(3)
o o o o
Dissemination of
information
through specialty
workshops/
conferences (6)
o o o o
Other (7)
o o o o

148
Exploration and Installation Stages

Q23 Has your organization explored alternative methods using “in silico/computer-
based models” (including organ/tissue chips) in lieu of or to reduce the size or
number of animal studies?
o Yes (1)
o No (2)
o I don't know (3)

Skip To: Q24 If Has your organization explored alternative methods using “in silico/computer-based
models” (inclu... = Yes
Skip To: Q32 If Has your organization explored alternative methods using “in silico/computer-based
models” (inclu... = No
Skip To: Q32 If Has your organization explored alternative methods using “in silico/computer-based
models” (inclu... = I don't know

149
Q24 As you tried to learn about the use of in silico/computer-based models as an
alternative to animal testing, how useful were the following information sources?
Please select all that apply.

Very
Useful (1)
Modestly
Useful (2)
Not
Useful (3)
Not Sure
(4)
Input from consultants (1)
o o o o
Meetings/workshops/conferences
(2)
o o o o
Regulatory agency guidance (4)
o o o o
Information from testing labs (5)
o o o o
Scientific publications (7)
o o o o
Other (8)
o o o o

150
Q25 What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to replace or reduce animal testing?
o Our organization decided not to go forward with these types of alternative
methods (1)
o Our company considered them but abandoned them after initial implementation
(2)
o We have implemented such methods but do not use them routinely (3)
o We have implemented such methods for routine use (4)
o Still exploring (5)

Skip To: Q26 If What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to repla... = Our organization decided not to go forward with these types of alternative methods
Skip To: Q27 If What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to repla... = Our company considered them but abandoned them after initial implementation
Skip To: Q28 If What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to repla... = We have implemented such methods but do not use them routinely
Skip To: Q28 If What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to repla... = We have implemented such methods for routine use
Skip To: Q32 If What was the outcome of your evaluation/exploration of in silico/ computer-based
methods to repla... = Still exploring

151
Q26 What factors drove the decision not to go forward with this type of method during
the evaluation/exploration process? Please select all that apply.

Very Important
(1)
Somewhat
Important (2)
Not Important
(3)
Not Sure (4)
Development
would take too
long (1)
o o o o
Replacement
would be too
expensive (2)
o o o o
Lack of
necessary
infrastructure
(3)
o o o o
Our team did
not have the
required know
how (4)
o o o o
Other (5)
o o o o

152
Q27 What factors played a role in your decision to abandon in silico/computer-based
methods after the initial implementation? Please select all that apply.

Very Critical
(1)
Critical (2) Not Critical (3) Not Sure (4)
Lack of
systematic plan
for training (1)
o o o o
Greater comfort
with status
quo/resistance
to change (2)
o o o o
Disappointing
results from
initial runs (3)
o o o o
Insufficient
infrastructure
(4)
o o o o
Other (5)
o o o o

153
Q28 The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences. Please select all that apply.

Strongly Agree
(1)
Agree (2) Disagree (3) Not Sure (4)
Lack of staff to
manage the
changes (1)
o o o o
Lack of experts
capable of
guiding
implementation
(2)
o o o o
Lack of
leadership
support for such
methods (3)
o o o o
Advocates
during
exploration do
not follow
through (4)
o o o o
Lack of
continuous
funding (5)
o o o o
Others (8)
o o o o

154
Q29 During this process of evaluation/implementation of new in silico/computer-based
methods, the feedback from the regulatory agencies was:
o Encouraging (1)
o Neither encouraging nor discouraging (2)
o Discouraging (3)
o Did not interact (4)

Q30 In retrospect, do you think that these efforts reduced the use of animals?
o Yes (1)
o No (2)
o I don't know (3)

Q31 If you implemented new in silico/computer-based method, please
identify/describe the nature of that method:
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

155
Q32 Has your organization explored alternative methods using “tissue/cell culture
technologies” in lieu of or to reduce the size or number of animal studies?
o Yes (1)
o No (2)
o I don't know (3)
Skip To: Q33 If Has your organization explored alternative methods using “tissue/cell culture technologies”
in li... = Yes
Skip To: Q41 If Has your organization explored alternative methods using “tissue/cell culture technologies”
in li... = No
Skip To: Q41 If Has your organization explored alternative methods using “tissue/cell culture technologies”
in li... = I don't know

Q33 As you tried to learn about the use of tissue/cell culture technologies as an
alternative to animal testing, how useful were the following information sources?
Please select all that apply.

Very
Useful (1)
Modestly
Useful (2)
Not
Useful (3)
Not Sure
(4)
Inputs from consultants (1)
o o o o
Meetings/workshops/conferences
(2)
o o o o
Regulatory agency guidance (5)
o o o o
Information from testing labs (6)
o o o o
Scientific publications (7)
o o o o
Other (8)
o o o o

156

Q34 What was the outcome of your evaluation/exploration of tissue/cell culture
technologies to replace or reduce animal testing?
o Our organization decided not to go forward with these types of alternative
methods (1)
o Our company considered them but abandoned them after initial
implementation (2)
o We implemented such methods but do not use them routinely (3)
o We have implemented such methods for routine use (4)
o Still exploring (6)

Skip To: Q35 If What was the outcome of your evaluation/exploration of tissue/cell culture technologies to
replac... = Our organization decided not to go forward with these types of alternative methods
Skip To: Q36 If What was the outcome of your evaluation/exploration of tissue/cell culture technologies to
replac... = Our company considered them but abandoned them after initial implementation
Skip To: Q37 If What was the outcome of your evaluation/exploration of tissue/cell culture technologies to
replac... = We implemented such methods but do not use them routinely
Skip To: Q37 If What was the outcome of your evaluation/exploration of tissue/cell culture technologies to
replac... = We have implemented such methods for routine use
Skip To: Q41 If What was the outcome of your evaluation/exploration of tissue/cell culture technologies to
replac... = Still exploring

157
Q35 What factors drove your decision not to go forward with this type of
method during the evaluation/exploration process? Please select all that apply.

Very Important
(1)
Somewhat
Important (2)
Not Important
(3)
Not Sure (4)
Development
would take too
long (1)
o o o o
Replacement
would be too
expensive (2)
o o o o
Lack of
necessary
infrastructure
(3)
o o o o
Our team did
not have the
required know
how (4)
o o o o
Others (5)
o o o o

158
Q36 What factors played a role in your decision to abandon tissue/cell culture
technologies after the initial implementation? Please select all that apply.

Very Critical
(1)
Critical (2) Not Critical (3) Not Sure (4)
Lack of
systematic plan
for training (1)
o o o o
Greater comfort
with status
quo/resistance
to change (2)
o o o o
Disappointing
results from
initial runs (3)
o o o o
Infrastructure
was insufficient
(4)
o o o o
Others (5)
o o o o

159
Q37 The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences. Please select all that apply.

Strongly Agree
(1)
Agree (6) Disagree (7) Not Sure (8)
Lack of staff to
manage the
changes (1)
o o o o
Lack of experts
capable of
guiding
implementation
(2)
o o o o
Lack of
leadership
support for such
methods (3)
o o o o
Enthusiastic
advocates
during
exploration do
not follow
through (4)
o o o o
Lack of
continuous
funding (5)
o o o o
Others (6)
o o o o

160
Q38 During this process of evaluation/implementation of new tissue/cell culture
technologies, the feedback from the regulatory agencies was:
o Encouraging (1)
o Neither encouraging nor discouraging (4)
o Discouraging (5)
o Did not interact (6)

Q39 In retrospect, do you think that the use of alternative cell culture-based
technologies reduced the use of animals?
o Yes (1)
o No (4)
o I don’t know (5)

Q40 If you implemented a new tissue/cell culture-based method, please
identify/describe the nature of that method:
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

161
Q41 Has your organization explored alternative methods using "lower
vertebrates/invertebrates" in lieu of or to reduce the size or number of animal
studies?
o Yes (1)
o No (4)
o I don't know (5)

Skip To: Q42 If Has your organization explored alternative methods using "lower
vertebrates/invertebrates" in lie... = Yes
Skip To: Q50 If Has your organization explored alternative methods using "lower
vertebrates/invertebrates" in lie... = No
Skip To: Q50 If Has your organization explored alternative methods using "lower
vertebrates/invertebrates" in lie... = I don't know

162
Q42 How useful were the following information sources as you explored the use of
lower vertebrates/ invertebrates as an alternative to animal testing? Please select
all that apply.

Very
Useful (1)
Modestly
Useful (2)
Not
Useful (3)
Not Sure
(4)
Input from consultants (1)
o o o o
Meetings/workshops/conferences
(2)
o o o o
Regulatory agency guidance (5)
o o o o
Information from testing labs (6)
o o o o
Scientific publications (7)
o o o o
Others (8)
o o o o

163
Q43 What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebrates to replace or reduce animal testing?
o Our company/organization decided not to go forward with this type of
alternative method (1)
o Our company considered them but abandoned them after initial
implementation (4)
o We implemented such methods but do not use them routinely (5)
o We have implemented such methods for routine use (6)
o Still exploring (7)

Skip To: Q44 If What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebra... = Our company/organization decided not to go forward with this type of
alternative method
Skip To: Q45 If What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebra... = Our company considered them but abandoned them after initial
implementation
Skip To: Q46 If What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebra... = We implemented such methods but do not use them routinely
Skip To: Q46 If What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebra... = We have implemented such methods for routine use
Skip To: Q50 If What was the outcome of your evaluation/exploration of methods using lower
vertebrates/invertebra... = Still exploring

164
Q44 What factors drove the decision not to go forward with this type of method during
the evaluation/exploration process? Please select all that apply.

Very Important
(1)
Somewhat
Important (4)
Not Important
(5)
Not Sure (6)
Development
would take too
long (1)
o o o o
Replacement
would be too
expensive (4)
o o o o
Lack of
necessary
infrastructure
(5)
o o o o
Our team did
not have the
required know
how (6)
o o o o
Other (7)
o o o o

165
Q45 What factors played a role in your decision to abandon the methods using lower
vertebrates or invertebrates after the initial implementation? Please select all that
apply.

Very Critical
(1)
Critical (2) Not Critical (3) Not Sure (4)
Lack of
systematic plan
for training (1)
o o o o
Greater comfort
with status
quo/resistance
to change (4)
o o o o
Disappointing
results from
initial runs (5)
o o o o
Insufficient
infrastructure
(6)
o o o o
Other (7)
o o o o

166
Q46 The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences. Please select all that apply.

Strongly Agree
(1)
Agree (6) Disagree (7) Not Sure (8)
Lack of staff to
manage the
changes (1)
o o o o
Lack of experts
capable of
guiding
implementation
(2)
o o o o
Lack of
leadership
support for such
methods (3)
o o o o
Enthusiastic
advocates
during
exploration do
not follow
through (4)
o o o o
Lack of
continuous
funding (5)
o o o o
Other (6)
o o o o

167
Q47 During this process of evaluation/implementation of new alternative methods
using lower vertebrates/invertebrates, the feedback from regulatory agencies was:
o Encouraging (1)
o Neither encouraging nor discouraging (4)
o Discouraging (5)
o Did not interact (6)

Q48 In retrospect, do you think that these efforts reduced the use of animals?
o Yes (1)
o No (4)
o I don’t know (5)

Q49 If you implemented new methods using lower vertebrates/invertebrates, can you
identify or describe the nature of that method?
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

168
Q50 Has your organization explored alternative "molecular imaging or other novel
methods (e.g. omics technology)" in lieu of or to reduce the size or number of
animal studies?
o Yes (1)
o No (4)
o I don't know (5)

Skip To: Q51 If Has your organization explored alternative "molecular imaging or other novel methods
(e.g. omics... = Yes
Skip To: Q59 If Has your organization explored alternative "molecular imaging or other novel methods
(e.g. omics... = No
Skip To: Q59 If Has your organization explored alternative "molecular imaging or other novel methods
(e.g. omics... = I don't know

169
Q51 During your evaluation of molecular imaging or other novel methods as an
alternative to animal testing, how useful were the following information sources?
Please select all that apply.

Very
Useful (1)
Modestly
Useful (2)
Not
Useful (4)
Not Sure
(5)
Input from Consultants (1)
o o o o
Meetings/workshops/conferences
(4)
o o o o
Regulatory agency guidance (6)
o o o o
Information from testing labs (7)
o o o o
Scientific publications (8)
o o o o
Other (9)
o o o o

170
Q52 What was the outcome of your exploration/evaluation of methods using molecular
imaging/other novel methods to replace or reduce animal testing?
o Our company/organization decided not to go forward with this type of
alternative method (1)
o Our company considered them but abandoned them after initial
implementation (4)
o We implemented such methods but do not use them routinely (5)
o We have implemented such alternative methods for routine use (6)
o Still exploring (7)
Skip To: Q53 If What was the outcome of your exploration/evaluation of methods using molecular
imaging/other nove... = Our company/organization decided not to go forward with this type of alternative
method
Skip To: Q54 If What was the outcome of your exploration/evaluation of methods using molecular
imaging/other nove... = Our company considered them but abandoned them after initial implementation
Skip To: Q55 If What was the outcome of your exploration/evaluation of methods using molecular
imaging/other nove... = We implemented such methods but do not use them routinely
Skip To: Q55 If What was the outcome of your exploration/evaluation of methods using molecular
imaging/other nove... = We have implemented such alternative methods for routine use
Skip To: Q59 If What was the outcome of your exploration/evaluation of methods using molecular
imaging/other nove... = Still exploring

171
Q53 What factors drove the decision not to go forward with this type of method during
the evaluation/exploration process? Please select all that apply.

Very Important
(1)
Somewhat
Important (5)
Not Important
(6)
Not Sure (7)
Development
would take too
long (1)
o o o o
Replacement
would be too
expensive (4)
o o o o
Lack of
necessary
infrastructure
(5)
o o o o
Our team did
not have the
required know
how (6)
o o o o
Other (7)
o o o o

172
Q54 What factors played a role in your decision to abandon the methods using lower
vertebrates or invertebrates after the initial implementation? Please select all that
apply.

Very Critical
(1)
Critical (2) Not Critical (3) Not Sure (4)
Lack of
systematic plan
for training (1)
o o o o
Greater comfort
with status
quo/resistance
to change (4)
o o o o
Disappointing
results from
initial runs (5)
o o o o
Infrastructure
was insufficient
(6)
o o o o
Other (7)
o o o o

173
Q55 The factors listed below are often identified as challenges when new non-animal
alternatives are being developed. Please identify how strongly these apply to your
experiences. Please select all that apply.

Strongly Agree
(1)
Agree (6) Disagree (3) Not Sure (4)
Lack of staff to
manage the
changes (1)
o o o o
Lack of experts
capable of
guiding
implementation
(4)
o o o o
Lack of
leadership
support for such
methods (5)
o o o o
Enthusiastic
advocates
during
exploration do
not follow
through (6)
o o o o
Lack of
continuous
funding (7)
o o o o
Other (8)
o o o o

174
Q56 During this process of evaluation/implementation of new Imaging or any other
novel methods, the feedback from regulatory agencies was:
o Encouraging (1)
o Neither encouraging nor discouraging (4)
o Discouraging (5)
o Did not interact (6)

Q57 In retrospect, do you think that these efforts reduced the use of animals?
o Yes (1)
o No (4)
o I don’t know (5)

Q58 If you implemented new imaging techniques/other novel methods, please
identify/describe the nature of that method:
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

175
Potential Policy Recommendation

Q19 Do you think that the following efforts may help accelerate the process of
validation of new alternative methods? Please select all that apply.

Very Important
(1)
Important (2)
Not Important
(3)
Not Sure (5)
Incorporate
more ‘active’
incentives for
the use of
alternatives (1)
o o o o
Increase funding
for institutions
involved in
validation,
acceptance and
implementation
of alternatives
(2)
o o o o
Create stronger
incentives for
organizations to
validate
methods after
they have been
developed (3)
o o o o
Establish global
standards for
validation (4)
o o o o

176
Q20 Do you agree with the following recommendations to make the adoption and
implementation of alternative methods more efficient? Please select all that apply.

Very
Important (1)
Important (2)
Not Important
(3)
Not Sure (4)
Create forum for
data sharing
between
stakeholders (1)
o o o o
Establish a
pathway for early
involvement of
regulators to
discuss
acceptance
criteria (2)
o o o o
Negotiate mutual
acceptance/global
harmonization of
testing
requirements (3)
o o o o
Implement
legislation in the
US similar to
directive
2010/63/EU (4)
o o o o
Other (5)
o o o o

177
Q21 The following views are used as arguments for failing to develop alternative
methods. Do you agree? Please select all that apply.

Agree
(1)
Neither Agree
nor Disagree
(2)
Disagree
(3)
Animal studies are still viewed as the essential
scientific standard (1)
o o o
Most in the scientific community regard the
value of animal testing as ‘self-evident’ (2)
o o o
Historical achievements depended on animal
research (7)
o o o
Animal studies will still be required by
regulatory agencies over the next decade (3)
o o o
Database of experience and knowledge comes
from traditional animal methods (4)
o o o
Replacement of animal studies is more
complex than initially thought (5)
o o o
Most alternative test methods are designed to
monitor a single toxic effect and cannot test
complex systemic effects (6)
o o o

Q22 For some type of experiments there may not be an equivalent non-animal option.
How will that be addressed?
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

178
Additional Comment if Any

Q59 Do you have additional comments about this topic ("Implementation of
Alternative Methods for Safety Assessment of Drugs and Biologics") that I may
not have captured?
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________
________________________________________________________________

Thank You Note

Q60 I sincerely thank you for your time in taking this survey. Your responses have
been recorded.

End of Block: Babbar Survey

179

What do you believe are the barriers that have impeded the implementation of new
alternative methods?

1. There have been translatability disconnects that have caused skepticism and doubt into
current approaches. There are alternative solutions but they need a champion and
leadership to partner with regulatory agencies.
2. Specialized equipment that may be needed for the assay
3. most alternative animal methodologies have lacked concordance to data from in vivo
animal models
4. willingness to adopt a new technology
5. An intact immune system cannot (yet) be modeled in vitro/ex vivo.
6. Concerns about translatability of the in vitro findings to humans.
7. Alternative methods not time or cost effective
8. Resistance to change
9. lack of unbiased evaluation of application of alternative models in safety assessment
10. Relevance to human testing
11. Feds are in love with the rodent model and they will not give it up
12. Concerns about ability to replicate effects in a whole animal with multiple organ
systems and complex interactions
13. right experiments not done to prove the model is equal or better
14. #1 is fear. Risk aversion/Reg acceptance are a big part of that although there's more in
terms of anticipated impact of "something going wrong." Usually that means time,
which translates to cost and potentially losing first to market, and most importantly
delaying therapies to patients in need. Fear drives us too much. Lawyers and litigation
is also an ancillary part of this.
15. Big pharma commitment to explore new methodologies
16. Habit and established testing infrastructure
17. More applicable to cell and gene therapy for earlystage investigations
18. Translation issues
19. lack of support from FDA reviewers
20. Historical failures in replacing standard methods
21. Lack of interest of Grant Institutions/Organizations coupled with low volume of grant
applications towards developing new & improving existing alternative methods

180

If you implemented new in silico/computer-based method, please identify/describe the
nature of that method:
1. we've used in silico mutagenicity screening, iPSC models and cell cultures for early
screening, or determining if toxicity is on or off target.
2. early screening to pick better molecules
3. Derek for dermal irritation and sensitization
4. genetic toxicity; off-target receptor binding; computational toxicology methods to
assess different toxicities
5. Used machine learning method to identify new prodrugs that would be stable in
subcutaneous space and be metabolized in the liver by microsomes and hepatocytes.
6. (Q)SAR evaluation for impurity assessment.
7. Use of DEREK and other in silico approaches for compound prioritization, routine
evaluation of potential toxicity.
8. I have not personally implemented computer-based methods, but they are routinely
used throughout discovery & drug development phases in my company.
9. For indemnification of gene tox liabilities
10. Lung on a chip etc
11. Sarah Derek
12. These types of methods are typically used for small molecule analog screening to
remove any compounds with a high liability for a toxicity. We do not currently use an
in silico method to replace a regulatory mandated animal tox study. However, by
removing molecules with potential liabilities through computer based methods, it is
ensured that these molecules are not taken forward and will not fail due to in vivo
toxicity. In this way, these types of analyses reduce the number of animals used in the
discovery phase.
13. In silico models for cardiac arrythmia risk are widely adopted, predicting cardiac
electrophysiology responses based on input of in vitro ion channel activities or
compounds. CIPA initiative has been helpful in driving adoption.
Other more complex systems pharmacology/toxicology models (e.g. DILIsym) have
been used on a limited, case by case way.
14. multiple: off-target interactions with precandidiate drugs, virtual screening of lead
binding to secondary pharmacology targets (cardiac ion channels)
15. Used Multicase and DEREK as early screens for new drug candidates
16. Biologically based dose response modeling (aka quantitative system toxicology
modeling) and other "off-the-shelf" applications.
17. cardiac muscle on chip. iPSC derived.

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18. We are leaders in the use of in silico methods to predict toxicity, which has led to a
reduction of animal studies needed to explain some results. We are still exploring the
use of microphysiological systems to replace animal studies, with a small amount of
implementation so far.
19. organ on a chip
20. mutagenicity prediction
21. Wide use of QSAR
22. Use Derek and LeadScope.
23. In silico assessment of general toxicity and organ-specific toxicity (e.g., kidney,
cardiovascular).
24. I use Derek Nexus and Leadscope, OECD QSAR toolbox, EPA TEST, Vega and
ToxTree
25. Predictive models of various nature.
26. In support of genetic tox specifically.
27. Not for use on developmental, reproductive, or juvenile toxicology studies
28. Currently, Lhasa has an excellent suite of software. Leadscope/Instem isn't far behind.
In the past, I've used toxicogenomics and adverse outcome pathway software that
hasn't truly been impactful either for data interpretation or reduction of animal studies.
29. genotoxicity alerts; tox tree; sara derek
30. Skin sensitization, and acute oral toxicity testing
31. Read across methodology
32. use of in silico methods to predict genotoxicity
33. SAR modeling and Population PKPD
34. Use SAR methods, more frequently than in the past to identify potential toxicities of
impurities/leachables as opposed to MTD studies.
35. Computational toxicology methods to rank order molecules in terms of toxicologic
potential
36. various methods to predict key organ system functions, physical chemical properties,
genotoxicity, ADME, etc
37. proprietary
38. Leadscope software
39. Integrated systems using organ imaging. Chip to serially aggregate data For use with
coupled systems.

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If you implemented new tissue/cell culture-based methods, please identify/describe the
nature of that method
1. early studies to identify better molecules
2. various models (single cell and microphysiologic models) for various investigative
toxicities or as lead optimization screening for various known toxicities, eg hepatotoxicity,
cardiotoxicity, neurotoxicity, genetic toxicity, secondary pharmacology, cytokine release
syndrome.
3. THLE in vitro toxicity assay utilized to rank order compounds in order to select
compounds to go forward into animal studies to increase chances of success.
4. My company is developing a number tissue/cell-based CIVM independently and in
collaboration with vendors.
5. Organ on a chip
6. Cell-based toxicity assays are used to screen early stage molecules to remove any analogs
that have an apparent liability for toxicity.
7. hepatocyte model- raises flag if positive, but doesn't cause early termination of leads
8. Various fit-for-purpose cell cultures using human tissues including tissue organoids.
9. CNS in vitro assays were developed and are routinely used. We developed cardiac assays
which showed reasonable value to predict human response.
10. Organoids
11. We have developed kidneys on a chip to explore renal toxicity. Have also developed a
liver on a chip connected to a kidney on a chip to assess hepatic metabolism on test
compound and its "downstream" effect on kidney. Most work has been done with human
primary cells but currently working on developing canine and rat kidneys on a chip to test
compounds that have failed in development due to renal tox in one species but not the
other and to compare results with human cells. Hopefully these canine and rat kidney chips
can be used for screening drugs for renal tox. This low to mid- thoughput technology but
hopefully will catch on. Very labor intensive. Could possible set up canine/rat liver:kidney
coupled chips to test species specific hepatic metabolisms effects on renal tox but this
would require a lot of resources not usually found in a university research lab.
12. non-regulated receptor binding pharmacology assays.
13. 3-dimensional organoids as well as fluid-based systems
14. tissue in a chip, mostly used for mechanistic studies and not for routine screening
15. Not used for developmental, reproductive or juvenile toxicology studies; used primarily in
the discovery space
16. reconstructed skin, MPS
17. We did not implement a new method, rather we used more extensive screening of tumor
cells and tumor organoids before proceeding to in vivo animal models. in other words, we
used in vitro screening to limit the number of in vivo studies we conducted.
18. we have heavily relied on such systems for engineered T cell therapies. We used a
combination of approaches.
19. so far all we have been using is some degree of skin / eye irritation
mostly non glp-studies not for regulatory submission
20. evaluation of differentiation of embryonic stem cells

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21. Multi parameter high content screening. See O’Brien PJ et al., 2006 Arch Toxic 80, 580-
604 for a nice validation paper on HCS vs animal models.
22. extensive tissue reactivity studies and assessment of translation across human and animals
that could be used for in vivo safety studies.
23. Toxicity Testing using R blood cells
24. I'n unclear on the details. Organ on a chip technology was evaluated.
25. Use of Bovine eyes to replace eye testing in rabbits. Use of skin membranes to replace
dermal testing in some cases
26. 3D liver microspheres from Insphero

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If you implemented new methods using lower vertebrates/invertebrates, please
identify/describe the nature of that method
1. Inducing hypnosis in anesthesia-sensitive lower vertebrates, e.g., alligators, chicken, to
perform 3-4 hours long experiments to measure various bodily functions, and keeping the
animal alive after experimentation.
2. Zebrafish (formerly also used FETAX but no longer in use)
3. The efforts were mainly to use rodents to better understand pharmacology in the human
context. This approach is not used for safety assessment.
4. Zebra fish are used for teratogenicity screening, but I am unsure how routinely this system
is used within our organization
5. Zebra fish larvae used to screen for developmental toxicity of early discovery compounds
6. Zebrafish assay for developmental toxicity.
7. no; proprietary
8. Zebra fish model for teratogenicity.
9. We used zebrafish as a disease model to test mechanisms of our drug. The FDA did not
accept our mechanism data for the mechanism statement in the label, although the EMA
did. We were told by consultants that the FDA did not accept zebrafish data as readily as
rodent data so we are conducting some studies now in rodents.
10. This was specifically in support of EcoTox / Environmental fate studies.
11. 4 or 5 day screens using zebrafish (evaluation of morphology following exposure)
12. LLNA. Transgenic mouse carcinogenicity study.
13. Validation of zebrafish for developmental toxicity
14. Toxicity testing using Artemis Salina
15. Zebrafish model of systemic toxicity
16. Zebrafish studies to utilize in early screening.
17. Zebra fish

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If you implemented new imaging techniques/other novel methods, please identify/describe
the nature of that method:
1. early screening to pick better compounds
2. NGS on tissues from non-clinical species used as tox species to compare target expression
to humans; in vitro kinetic viability assays; genomic analyses on primary cells and tissues
to understand insertion site distribution for viral vectors
3. Incorporated MALDI imaging into single dose PK studies in rat following 24 hour
continuous subcutaneous infusion to determine if the prodrug being administered was
being metabolized at the site of infusion. If it was then that molecule would not be
progressed further into development.
4. Metabolomics (may be some others that I am not aware of)
5. I have n little direct involvement in the use of imaging methodologies.
6. cell based omics screening has been used in our organization to evaluate changes in gene
expression that may signal toxicity or activation of pathways that may be seen as a safety
liability for future development.
7. Combination of in vitro MPS with genomics. Smaller scale in vivo experiments combined
with genomic analysis
8. Multiplex imagaing of cell culture toxicity studies.
9. Toxicogenomics in cultured cells. Was not found to be very useful compared to
toxicogenomics in animals.
10. We are able to extract RNA transcripts from our kidney-on a chip and can perform
RNAseq analyses combined with systems biology pathway analysis software and have
found this to be very useful and reproducible. We can fix and stain chips and perform
immunocytochemistry techniques to identify and quantify many cellular protein targets
(drug transporters, tox signals etc.). The chips can also be evaluated with imaged by
confocal microscopy to produce stunning 3-D images of our kidney tubules within the
chip. Genomic analysis of our kidney cell donors allows us to explore genetic variants of
many tox related genes in our chip system.
11. Drug targeting, distribution, ADME, etc.
12. Used primarily in the discovery space using single cell imaging techniques
13. HCS multiparameter screening
14. Use cell microarray for ligand-receptor interactions. Method is being increasingly used and
FDA does accept results in lieu of in vitro binding assays and tissue cross-reactivity
studies, particularly for biologics. However, cost can be greater than existing methods and
in most recent attempt the contract lab was unable to run the assays in a timely manner.
15. Cellomic platform
16. PET imaging; metabolomics methods
17. LCMS MALDI imaging to make the most of tissues when options are not available.
Integrated systems are complex and can be de convoluted but not to he complete exclusion
of intact invivo systems just yet.

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For some type of experiments there may not be an equivalent non-animal option. How
will that be addressed?
1. Animal testing will be needed.
2. "This has to be done in animals. Where no alternative exists - human risk assessment
in humans will not be considered ethical.
3. Can we tolerate or accept another T-Genero"
4. It cannot be appropriately addressed. Hence easier to run animal studies than to pursue
an alternative path that is uncertain and could ultimately cost the Sponsor time/
resources and you are back to animal studies as your viable option.
5. Decreased number of animals required and refinement of testing, but still use animals
6. Increase spending on PK/TK and PD/TD models so that information obtained can be
synthesized.
7. The animal model will still be used
8. Currently this is best addressed via reduction and reuse of animals until technology
catches up. Microsampling, microdosing and novel analytical techniques will continue
to drive our ability to get more out of a limited number of animals. If we cannot get
away from use, we owe it to our world to greatly reduce what we use while increasing
inferences made from each study.
9. Don't know
10. Will be a great opportunity to show how alternative methods can help out
11. I believe that there will be a need to conduct animal studies for many endpoints.
However, use of available technology and innovative study designs can be
implemented to minimize the number of animals needed in the study.
12. some work can be done my complexing various 'organs on a chip'
13. In circumstances where there are no options for non-animal safety studies, the study
designs should be challenged to find ways to reduce the numbers of animals used.
14. Keep existing animal models
15. Until the FDA allows 'validated' non-animal the need for animal tests will persist,
whether or not there is a non-animal option
16. "Focus more on ex vivo assays (including human where ethically possible). You still
need animals but they can often be reused or used for multiple purposes and the
suffering is less.
17. Harmonize globally so no animal studies need to be repeated to test a slight difference
for another country.
18. Let sponsors use more WOE when the outcome of an animal experiment can
reasonable be predicted based on prior studies or literature."
19. computer simulation model
20. Accept higher risk to society
21. I believe the goal currently is to reduce the number of animals needed. Inevitably,
there will be experiments that will require the use of animals, but this doesn't take
away from the overall goal to reduce animal use.
22. Alternatives will not work in toxicology. You need the interplay between all systems
to truly evaluate toxicity, a most critical component that is lacking from non-animal
options.

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23. Apply as many early in silico/in vitro tests to advance only the most promising
candidates to limited animal testing.
24. Continue with animal studies
25. There will always be a need for animal experiments to account for the situation you
reference above.
26. I don't believe whole animal experimentation will ever be completely replaced without
major advancements in AI to allow for computational modeling of complex, inter-
related simultaneous physiologic processes.
27. Computational toxicology research programs
28. unknown
29. Mutual recognition that alternative testing cannot replace all animal testing and that
some alternative testing can replace animal testing.
30. Animal models in this case will continue to be needed to address safety in humans
31. If there's no non-animal option, then animal tests should be used. While the idea of
promoting a non-animal alternative is noble, if no option exists then the animal test
should not be abandoned.
32. Eventually in silico, but that is even further in the future
33. Requires lot of research and interest of scientific community to bring new alternatives
for non-animal testing methods. In case of no option for alternative testing, then
animals are better option, but it is important reduce the number of animals used for
study than usual
34. Most alternatives are based on mechanistic understanding of a toxicological effect and
assays for some part of that mechanism. Toxic effects of novel drugs, which have yet
unknown toxicological mechanisms, have safety risk for toxicology without benefit of
knowledge to target alternatives that makes sense instead of using a systemwide (i.e.
animal) general safety assessment.
35. The only way is to reduce the number of animals used in the study
36. The feds do not want to adopt alternative models. They are in love with the rodent
model and have been since the 60’s. High content screening can return multi parameter
results quickly and can at least be used to screen compounds. The people in charge of
scientific thought in the US are closed minded.
37. Models that include information human exposure and complete as possible adverse
outcome pathways to allow for a prediction of hazard and a risk assessment for
reliance to humans
38. we can potentially use weigh of evidence, in some cases, animal studies still will need
to be done
39. If there is no alternative option then animals will be used. Alternative options can
however be a weight-of-evidence approach based on multiples complementary
assessments. Note that we already file animal-free INDs for some cellular therapies
where in silico + in vitro data inform risks
40. In my opinion, non-animal methods are to reduce animal testing to the extent possible
rather than replacing it all together
41. animals will likely always be needed in some capacity. It is likely not possible to
address with invitro only.
42. No need to address if there is only 1 option
43. This will be true for lots of models. We will continue to use the studies that use
animals.

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44. Great question. For that scenario, a paper risk assessment could be conducted if
enough relevant historical information was available (read across from related
compounds, known pharmacology/monitorability, in silico testing, etc). And even with
that, it is a clinical experiment where monitoring/reversibility based on
pathophysiology would guide a slow development plan.
45. Surrogate molecules may be an option and some safety testing will still be required
46. In those instances, animal testing should be continued.
47. Can not be addressed as in some cases you need in vivo environment
48. I don't think it will be possible to completely eliminate animals from research. The
biology is too complex to be replaced by in silico or in vitro systems. Animal use can
be improved by increased animal care, and improvements in housing, social needs,
reduction of stress and pain.
49. It isn’t
50. I believe that complex in vitro systems are a possible future tool to consider for these
options. Although we are seeing more and more cases where no relevant animal model
exists and alternative approaches need to be the basis for the risk assessment.
51. Not sure.
52. Traditional use of one (or two) species of animal selected with justification which are
(combined) most appropriate for risk identification prior to clinical trials assessing
human safety.
53. Combine assessments in a few in vivo studies, e.g. in a 3-month tox, include CV, CNS,
RESP, ERG, sperm analysis etc. = clever design
54. Run the animal study, while looking to employ the 3Rs to the best of our ability.
55. It is unlikely in the next decade that animal use will be completely eliminated. Human
data will be the next option for experiments that can't be done in animals or addressed
in vitro.
56. Animals will still need to be used until a non-animal option is available.
57. Tough question- support in vivo studies with in silico/vitro studies in parallel to
possibly reduce animal numbers overall. Hard to do PK studies without animals-
modeling may help but hard to work around this.
58. Much more research with clear early regulatory prodding
59. Depends on the importance and if it absolutely needs to be done to assess human or
animal (vet products) safety
60. Minimize animal use, understand the mechanism of toxicity
61. Animal testing for such experiments may still be needed. Using fewer animals if
possible, can be an option. Carcinogenicity study even for rat can be avoided in cases
where a positive outcome is expected anyway based on genotoxicity, pharmacology,
hormonal/immune profile and tissue hyperplasia data from the 3-month and 6-month
GLP tox studies. This can reduce the overall number of animals used for test
compound.
62. Dont know
63. It is probable that completely eliminating animal research will not be possible. The
translational value of in vitro tests to predict clinical responses in humans remains
imperfect. In vitro is increasingly efficient at testing for selected safety liabilities but
identifying the liabilities that are relevant for a given molecule still requires the use of
animal models to include the complex multi-organ interactions.
64. support and publication of validation as with alternative methods recommended in ICH
S5(R3) for DART EFD

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65. Perhaps we can use historical control data coupled with data gathered from alternative
testing methods.
66. Animals will continue to be used.
67. As time moves on, new methods will be developed for a variety of toxicity endpoints.
Creativity will eventually solve all problems.
68. Use the least amount of animals possible in order to achieve study objectives.
69. I don't agree with this statement if one has a braod, ambitious view of the potential for
alternative methods. It should be noted that already (although exceptionally rare), so
experimental therapeutics are entering clinical trials without in vivo toxicology data.
Eg.g Harper et al PlosOne https://doi.org/10.1371/journal.pone.0205491
70. Use of a combination of alternate methods and minimal testing in animals could reduce
the animal usage
71. Use animals as needed but not as a default option.
72. will still require animal testing, but possibly an abbreviated animal requirement
73. Multiple approaches needed. Complex MPS, systems toxicology and modeling
approaches, AOPs, read across
74. Non-animal based studies will be part of the overall toxicology program but there will
likely still be a need for some animal studies to be conducted. Hopefully in the future,
there will be a standard battery of in vitro based methods (such as in vitro genetox) or
in silico assessments that can be used to reduce the number of traditional repeat-dose
or specialty (DART, carc) toxicology studies.
75. Animal work
76. Refinement is a more plausible option than replacement.
77. trying to reduce the total amount of animals used
78. A combination approach
79. Carcinogenicity studies
80. Development of humanized microtissues/MPS to enable investigating human biology
involving multiple cell types within a tissue microenvironment.
81. Any use of models in evaluating potential risk to human safety from drugs in
development involves a weight of evidence approach. That has been true with in vivo
animal testing and will continue to be true as alternatives become more accepted and
available. Complex In Vitro Models using human tissues are increasingly being used
to bridge gaps in translatability between in vivo animal efficacy and safety models and
human clinical trials. These CIVM will continue to grow in utility and value in
developing weight of evidence positions.
82. Batteries of tests, use of AI once a critical knowledge base threshold has been reached,
use of MOA and Key Events modeling to examine MIEs (molecular initiating events).
EPAs TOX21 and TOXCAST are good examples. Caution must be exercised to avoid
over-simplification of complex biological processes and to not overlook redundancies
and/or thresholds for toxicity (or to place too much emphasis on them).
83. Validation against gold standard to show predictive power.
84. By using an animal model
85. I believe that some routine animal testing will continue to be required for drug safety
assessment, however if alternative models can reduce this to a limited number of key
studies, that will still be a big improvement
86. Development of in vitro methods to help reduce but not replace animal usage
87. By continuing to do the appropriate experiments in vivo -- your own point above about
the fact that in vitro models cannot fully replicate complex organisms supports this.

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88. Will likely continue to address via animal studies unless it is decided that the
information gleaned from those experiments is no longer necessary.
89. Time will tell and hopefully other equivalents will be possible with more research.
90. In this circumstance an animal model will need to be employed.
91. I think the biggest advance in the field of toxicology has emerged in the development
of novel modality therapeutics which have been the first space in which regulators
have not applied check-the-box toxicology programs. This type of approach has
greatly reduced overall animal use, for example, by eliminating the need for
carcinogenicity studies for most biologics due to lack of human relevance.
92. Unsure
93. Explore the unknown & the impossible through brilliant insights, careful thinking,
exchange of ideas & even through currently existing animal experimentation with a
view to discover equivalent non-animal options
94. Will depend on the gravity of the toxicity being evaluated - if it can be monitored and
mitigated in the clinic, then an alternative non-animal model may be acceptable.
95. This is the crux of the issue for me personally. I work in abuse liability assessments
and there is no way in which one can model or predict animal or human behavior
without a live animal. Receptor binding and structure-activity relationship modeling
cannot predict abuse potential. There is no alternative to using live animals.
96. clinical trials become more risky
97. I don't believe that animal experimentation can be removed entirely any time in the
foreseeable future. If we can make progress to reduce the number of animals needed
particularly for early-stage discovery work where it may be easier to develop
alternatives and reduce numbers, that is where we should focus.
98. In those cases animal testing can not be replaced.
99. By continuing to use animal models in those experiments
100. If it is a regulatory requirement, I find it highly unlikely regulatory agencies
would deem the animal study unnecessary
101. At this point the alternative methods primarily serve as early stop signals and
reduce animal use on projects that are doomed to failure. Except for some validated
methods like dermal testing, photo tox alternatives and in silico mutagenicity
screening, the follow up will still be an animal study

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Do you have additional comments about this topic ("Implementation of Alternative
Methods for Safety Assessment of Drugs and Biologics") that I may not have captured?
1. Implementing this early in the process, before formally selecting a compound for clinical
development, is free from regulatory oversight. We are convinced it results in us selecting
better compounds that subsequently enter the formal regulatory safety testing in animals.
So it does not always reduce animal use, but it allows us to invest in compounds less likely
to attrite due to a subsequent animal toxicity
2. There have been improvement in animal reduction for ex: 2-year CARC studies are no
longer required for life-threatening drugs, embryofetal studies not mandatory in monkeys
etc.
There must be a legislative change to remove many not so useful animal testing just to
satisfy drug labelling; gather the required safety from human subjects in trials, when some
info is missing state so in the drug label instead of trying to prove a negative outcome in
animals.
3. The refinement is more realistic goal than "alternatives to animal methods". The survey
could have put more emphasis on that angel.
4. It will be a slow process. Animal use may possibly be decreased for specific cases and
situation but it seems bulk of the testing would still continue using animals.
5. While replacement of animals may be less successful, the reduction of animal numbers
used seems more successful.
6. The outcome of a safety assessment study depends on an interplay between all live and
active organs/systems/tissues in the body. The selection of the response of one cell or
tissue type or even modeling to assess the safety of drugs is true a fallacy promoted by
individuals who do not approve of the use of live animals in research.
7. (1) I think biology is much more complex than generally recognized. In silico is based on
what we know, so gaps in our knowledge result in failure of in silico to be completely
accurate. (2) Biology is an interaction of systems, not individual receptors or cells or
tissues. Assays based on cells or tissues may not be good predictors of in vivo
response(Krewski et al., 2010). In vitro assays - cells come from animals or humans, so
don't necessarily eliminate animal use. I think many people don't think about that.
8. I think there's a lot of effort put forward in this space, but I think it will be a while before
animals are replaced. Some useful models have been generated and, when used in the
proper context, can address some mechanisms of toxicity, especially the most frequent
attrition systems (CV and liver). An alternate advantage of in vitro models is addressing
gaps in nonclinical-clinical safety translation e.g. CNS effects.
9. I think we should consider alternatives to the standard requirement for a rodent and non-
rodent in toxicology studies with the goal to reduce the use of non-rodents on a case by
case basis
10. As said earlier, non-animal methods are to reduce animal testing to the extent possible
rather than replacing it. So, these methods should be used to determine all MoA of toxicity
and then conduct only a few animal studies to prove in a complete biological system.
Having said this, I think long-term animal studies do not provide any useful information
that cannot be obtained from short-term and/or in vitro studies, e.g., conducting
carcinogenicity study is not needed as indications are always in short-term animal studies
and/or in vitro mutation studies.

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11. The major hurdle to implementation in the US pharmaceutical industry is the barriers to
validation and acceptance by the US FDA. Greater chance of success exists in the EU due
to the higher level of societal pressure on in vivo animal testing.
12. My company has used in silico, in vitro, and imaging methods in the past for safety
assessments but not in lieu of animal studies. Instead, they were used as part of a
screening cascade/decision tree.
13. Regulatory acceptance is critical to success. Resources required to develop these methods
are significant and shared processes are more likely to be successful. Global data sharing is
important to accelerate the implementation of alternatives.
14. in my view, the main barrier in implementing alternative to animal models is regulatory
acceptance and questioning the lack of animal data
15. Regulatory agencies could provide incentives to biopharmaceutical companies, which
would greatly expand usage of animal-free safety assessments. These incentives could be
in the form of accelerated reviews.
16. Animal usage in safety is purely regulatory driven. If you succeed in convincing
regulators, the industry will follow.
17. Alternatives such as studies with liver cells are good for screening of molecules early on -
so I've seen alternatives used early in development but once development begins, then ICH
M3(R2) is followed and there is no mention of alternatives to speak of in that guidance. A
fairly recent success though is the invitro phototoxicity assay - that is one that is routinely
used as an alternative to in vivo.
18. To predict potential toxicity and first in human dosing, I'm not sure the industry will ever
move completely away from animal testing. Other endpoints, i.e., pharmacology, PD/PK,
have had better success at in vitro modeling.
19. A key problem among toxicologists is fear. We are afraid that the models will not fully
capture the harm that a substance does, and this will come back to bite us. If you find ways
to make us more confident that we haven't missed anything, the rest will follow suit.
20. One of the goals of animal research in filing an IND is to choose a safe starting dose for
human clinical trials. At this time, it is difficult to understand how an alternative test
method can accomplish that goal.
21. Public concern for animal suffering has become a major issue and the concern will
continue to increase. Industry, National & International Regulatory Agencies should be
proactive and plan ahead of public pressure to work towards implementation of Alternative
Methods in future decenniums.
22. In order for this to be successful one would need to consider exposure compared to the
clinic and complete adverse outcome pathways (AOP). There are many initiatives in
progress and lots of good science available related to these topics, but the perspective of
implement alternatives in safety assessment has lagged behind
23. In terms of 'omics, I recorded "no" based on the current activities. Plenty of that in the past,
it has just seen it's time for broad utility and is only situationally useful.
24. The issue that I typically encounter when I am evaluating a potential in vitro animal
alternative is that most for profit 2-D and 3-D technology companies that are developing
these alternatives do not want to do the crucial experiments to show if there model is as
good or better than the current animal models. My favorite example of this is models some
for profit organizations are trying to develop for liver tox. All us toxicologists know the
handful of compounds that made it to market and were shown to be hepatotoxic and
withdrawn. There were no nonclinical signals of liver toxicity in the development
programs. We want a model that would have predicted these withdrawn molecules as
toxic AND also will pick up more classical hepatotoxicants. Essentially, no one wants to

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do the experiment with their model and the withdrawn materials because if there is low
predictability, the model is dead as a replacement for animal work.
25. There are issues with alternative methods that are developed by for-profit laboratories, as
the techniques are often proprietary which results in little cost savings and the CRO
controls timing. Not all alternative methods, even those with regulatory acceptance, are
used because a positive result in an in vitro method may trigger an in vivo study anyway
(e.g. if photosensitivity in 3T3 assay is positive this is managed by one or more in vivo
studies; screening genotox studies that are positive generally requires in vitro, ex vivo and
often in vivo assays; hERG assay is now pretty useless and cardiovascular studies will be
needed in vivo. The biggest impact on the 3R's in pharma research would be in reducing
the number of carcinogenicity assays, and not focusing on early compound development.
26. Global acceptance of ICH is still lacking in Asia
27. I believe that the current unprecedented acceleration of development of COVID-related
biotherapeutics (including vaccines) will ultimately help to re-shape and hopefully
streamline animal safety testing requirements for some (but certainly not all)
biotherapeutics. At the very least, the current situation with the global supply of nonhuman
primates for research purposes will hopefully influence seeking alternatives to these
models for some (but not all) small and large molecule nonclinical safety programs.
28. No- You did a good job covering the area.
29. Thank you for doing this. It is an important issue that needs to be addressed
30. This survey has captured essential questions.
Best information can be obtained from Contract research organization
31. No, the survey was very well thought out
32. This is a complex topic and you did a good job in covering the major factors influencing
the development of animal alternatives. As a consultant I see many opportunities for the
toxicology community to suggest ways to reduce animal usage, especially nonrodents, and
reach out to drug regulatory agencies for discussion and hopefully some day, concurrence
and implementation.
33. No specific comments.
34. Fish are also great models...and I have used them before. They fit on a slide and are a
whole animal model. Another overlooked model with robust results. Guppy and zebrafish
good choices here.
Good luck!
35. we are constantly exploring alternatives to animal testing.
36. This is a bit tangential to your scope, but I believe many of the animal reduction practices
are currently being seen in the discovery, hit to lead and candidate selection space right
now. The speed of new technology adoption and the lack of regulatory approval in this
space has led to a reduction of animals in the early stages of drug discovery and
development, it has also lead to better candidates being nominated with a better chance of
success. Though it does not reduce current requirements to reach safety/tox approvals, it
does reduce overall numbers used and hopefully leads to better results regarding the ones
that are used.
37. You appear to have confused in silico and in vitro/MPS approaches earlier on. I assume
you meant purely in silico for the first section.

Abstract (if available)

Abstract Animal studies have been an essential part of drug development to evaluate the efficacy and safety of biopharmaceutical products. Notably, they are a prerequisite for the conduct of human clinical trials and marketing authorization for pharmaceuticals, but their use adds significant cost to the drug development process, cannot always predict effects in humans and can raise significant ethical concerns. Thus, efforts to replace animal studies with “alternative methods” have been ongoing for several decades. The objective of this study was to explore the current state of alternative methods and identify barriers to their adoption and acceptance for the safety assessment of pharmaceuticals. The research was conducted using a survey tool and explored the status of the “3Rs” (Replacement, Reduction, and Refinement) with a focus on the implementation of alternative methods by two key stakeholdersㅡpharmaceutical industry and contract research organizations in North America. A total of 170 respondents with a background in toxicology and animal studies completed the survey. The results suggest a field in transition. The implementation of alternative methods has grown steadily in the early screening stage of drug development but the field of safety assessment still mostly depends on animal studies. However, significant regulatory barriers exist, including those related to the validation of the alternative tests and to requirements that the tests are as good or better than comparable animal experience. Further, uncertain costs and outcomes make companies wary of pursuing alternative methods. Thus, most respondents do not expect that the implementation of alternative methods will greatly change the nature of drug safety testing in the next decade.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Babbar, Sunita Singh (author)

Core Title Challenges to implementation of alternative methods to animal testing for drug safety assessment in North America

School School of Pharmacy

Degree Doctor of Regulatory Science

Degree Program Regulatory Science

Degree Conferral Date 2021-12

Publication Date 09/30/2021

Defense Date 07/14/2021

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alternative methods,animal testing,challenges,drug safety assessment,non-animal alternatives,North America,OAI-PMH Harvest,regulatory toxicology,survey,Toxicology,validation of alternative methods

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Richmond, Francis J. (committee chair), Church, Terry D. (committee member), Cosenza, Mary Ellen (committee member), Davies, Daryl L. (committee member)

Creator Email sbabbar@usc.edu,sbabbar716@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC16021815

Unique identifier UC16021815

Legacy Identifier etd-BabbarSuni-10124

Document Type Dissertation

Format application/pdf (imt)

Rights Babbar, Sunita Singh

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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