S5(R3) Detection of 
Reproductive and 
Developmental Toxicity for 
Human Pharmaceuticals 
 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
May 2021 
ICH 
 
Revision 3 
    
S5(R3) Detection of 
Reproductive and 
Developmental Toxicity for 
Human Pharmaceuticals 
 
Guidance for Industry 
 
 
 
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https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances     
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
May 2021 
ICH 
 
Revision 3
  Contains Nonbinding Recommendations 
 
FOREWORD 
 
The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for 
Human Use (ICH) has the mission of achieving greater regulatory harmonization worldwide to 
ensure that safe, effective, and high-quality medicines are developed, registered, and maintained 
in the most resource-efficient manner.  By harmonizing the regulatory expectations in regions 
around the world, ICH guidelines have substantially reduced duplicative clinical studies, 
prevented unnecessary animal studies, standardized safety reporting and marketing application 
submissions, and contributed to many other improvements in the quality of global drug 
development and manufacturing and the products available to patients.  
 
ICH is a consensus-driven process that involves technical experts from regulatory authorities and 
industry parties in detailed technical and science-based harmonization work that results in the 
development of ICH guidelines.  The commitment to consistent adoption of these consensus-
based guidelines by regulators around the globe is critical to realizing the benefits of safe, 
effective, and high-quality medicines for patients as well as for industry.  As a Founding 
Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a major role in the 
development of each of the ICH guidelines, which FDA then adopts and issues as guidance to 
industry.  
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION AND GENERAL PRINCIPLES (1) ............................................... 1 
A.  Aim of Studies (1.1) ........................................................................................................................ 2 
II.  SCOPE OF THE GUIDANCE (2) ................................................................................... 3 
III.  GENERAL CONSIDERATIONS ON REPRODUCTIVE TOXICITY 
ASSESSMENT .............................................................................................................................. 3 
A.  Target Patient Population/Therapeutic Indication Considerations (3.1) ................................. 4 
B.  Pharmacology Considerations (3.2) ............................................................................................. 4 
C.  Toxicity Considerations (3.3) ........................................................................................................ 4 
D.  Timing Considerations (3.4) .......................................................................................................... 5 
E.  Toxicokinetics (3.5) ........................................................................................................................ 5 
IV.  DESIGN AND EVALUATION OF IN VIVO MAMMALIAN STUDIES (4) ............ 5 
A.  Strategy To Address Fertility and Early Embryonic Development (FEED) (4.1) ................... 6 
1. Considerations for Biopharmaceuticals (4.1.1) ............................................................................... 6 
B.  Strategies To Address Embryo-Fetal Development (EFD) (4.2) ............................................... 7 
1. Considerations for Biopharmaceuticals (4.2.1) ............................................................................... 7 
2. Alternative Approaches for Addressing EFD Risk (4.2.2) ............................................................... 8 
a.  Use of alternative assays (4.2.2.1)............................................................................................. 8 
3. Potential Approaches To Defer Definitive In Vivo Testing as Part of an Integrated Testing 
Strategy (4.2.3) ..................................................................................................................................... 8 
C.  Strategy To Address Effects on Pre- and Postnatal Development (PPND) (4.3)...................... 9 
1. Considerations for Biopharmaceuticals (4.3.1) ............................................................................... 9 
V.  TEST SYSTEM SELECTION (5) ................................................................................... 9 
A.  Routine Test Species (5.1) .............................................................................................................. 9 
1. Selection of Species for DART Testing (5.1.1) ............................................................................... 10 
2. Species Selection for Preventative and Therapeutic Vaccines (5.1.2) ........................................... 10 
B.  Nonroutine Test Species (5.2) ...................................................................................................... 10 
C.  Use of Disease Models, Genetically Modified Models, and Surrogate Molecules (5.3) .......... 11 
VI.  DOSE-LEVEL SELECTION, ROUTE OF ADMINISTRATION, AND 
SCHEDULE (6) ........................................................................................................................... 11 
A.  Dose Selection (6.1) ...................................................................................................................... 11 
1. Toxicity-Based Endpoint (6.1.1) .................................................................................................... 12 
2. Saturation of Systemic Exposure Endpoint (6.1.2) ........................................................................ 12 
3. Exposure Margin-Based Endpoint (6.1.3) ..................................................................................... 12 
a.  Exposure-based approach for biopharmaceuticals (6.1.3.1) ................................................... 13 
4. Maximum Feasible Dose Endpoint (6.1.4) .................................................................................... 13 
5. Limit Dose Endpoint (6.1.5)........................................................................................................... 13 
i  Contains Nonbinding Recommendations 
 
6. Selection of Lower Dose Levels (6.1.6).......................................................................................... 13 
B.  Route (6.2) ..................................................................................................................................... 13 
C.  Schedule (6.3) ............................................................................................................................... 14 
D.  Dose Selection and Study Designs for Vaccines (6.4) ................................................................ 14 
VII.   POSSIBLE COMBINATION STUDY DESIGNS IN RODENTS (7) ....................... 15 
VIII.  DATA REPORTING AND STATISTICS (8) .............................................................. 16 
A.  Data Reporting (8.1) .................................................................................................................... 16 
B.  Statistics (8.2) ............................................................................................................................... 16 
IX.  PRINCIPLES OF RISK ASSESSMENT (9) ................................................................ 16 
X.  ENDNOTES (10) ............................................................................................................. 18 
XI.  GLOSSARY/ABBREVIATIONS .................................................................................. 19 
XII.  REFERENCES (12) ........................................................................................................ 22 
ANNEX 1   IN VIVO STUDY DESIGNS ................................................................................ 23 
1.1  In Vivo Study Design Considerations ........................................................................................ 26 
1.1.1  Fertility and Early Embryonic Development (FEED) Study .................................................. 26 
1.1.2  Embryo-Fetal Development (EFD) Toxicity Study ................................................................ 28 
1.1.2.1  Study dose-range-finding embryo-fetal developmental (EFD) toxicity study ................ 28 
1.1.2.2  Preliminary embryo-fetal development (pEFD) toxicity study ...................................... 28 
1.1.2.3  Definitive embryo-fetal developmental (EFD) toxicity study ........................................ 28 
1.1.3  Pre- and Postnatal Developmental (PPND) Toxicity Study ................................................... 31 
1.1.3.1  Enhanced pre- and postnatal developmental (ePPND) toxicity study in the nonhuman 
primate  32 
1.1.4  Combination Studies............................................................................................................... 34 
1.1.4.1  FEED and EFD ............................................................................................................... 34 
1.1.4.2 Male fertility and repeated-dose toxicology study ............................................................. 34 
ANNEX 2   ALTERNATIVE ASSAYS ................................................................................... 35 
1.1  Qualification of Alternative Assays for Prediction of MEFL .................................................. 35 
1.2  Examples of EFD Testing Strategies Utilizing Alternative Assays .......................................... 37 
1.2.1  Potential Approach To Defer In Vivo Testing as Part of an Integrated Testing Strategy ...... 37 
1.2.2  Pharmaceuticals Expected To Be Embryo-Fetal Toxicants ................................................... 37 
1.2.3  Pharmaceuticals Intended To Treat Severely Debilitating or Life-Threatening Diseases ..... 38 
1.2.4  Pharmaceuticals Intended to Treat Late-Life-Onset Diseases ............................................... 39 
1.3  Reference Compound List ........................................................................................................... 40 
1.3.1  Positive Control Reference Compounds ................................................................................. 43 
1.3.2  Negative Control Reference Compounds ............................................................................. 110 
ii  Contains Nonbinding Recommendations 
 
S5(R3) Detection of Reproductive and Developmental Toxicity  
for Human Pharmaceuticals 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
 
I.  INTRODUCTION AND GENERAL PRINCIPLES (1)2 
 
The purpose of this guidance is to recommend international standards for, and promote 
harmonization of, the assessment of nonclinical developmental and reproductive toxicity 
(DART) testing necessary to support human clinical trials and marketing authorization for 
pharmaceuticals. The guidance describes potential strategies and study designs to supplement 
available data to identify, assess, and convey risk. General concepts and recommendations are 
also provided that should be considered when interpreting study data. 
  
This guidance revises the ICH guidance S5 Detection of Toxicity to Reproduction for Medicinal 
Products (September 1994).3 This revision brings the guidance into alignment with other ICH 
guidances, elaborates on the use of exposure margins in dose-level selection, incorporates a 
section on risk assessment, and expands the scope to include vaccines and biopharmaceuticals. It 
also describes qualification of alternative assays, describes potential scenarios of use, and 
provides options for deferral of developmental toxicity studies. 
 
To assess a human pharmaceuticals effect on reproduction and development, there should 
generally be information available that addresses the potential impact of exposure to a 
 
1 This guidance was developed within the Expert Working Group (Safety) of the International Council for 
Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been 
subject to consultation by the regulatory parties, in accordance with the ICH process.  This document has been 
endorsed by the ICH Assembly at Step 4 of the ICH process, February 2020.  At Step 4 of the process, the final draft 
is recommended for adoption to the regulatory bodies of the ICH regions. 
 
2 Arabic numbers reflect the organizational breakdown of the document endorsed by the ICH Assembly at Step 4 of 
the ICH process, February 2020. 
 
3 This final guidance replaces earlier versions, including the draft guidance S5(R3) Detection of Toxicity to 
Reproduction (November 2017); the final guidance S5A Detection of Toxicity to Reproduction for Medicinal 
Products (September 1994); and the final guidance S5B Detection of Toxicity to Reproduction for Medicinal 
Products: Addendum on Toxicity to Male Fertility (April 1996).  For the most recent version of a guidance, check 
the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents.   
  1  Contains Nonbinding Recommendations 
 
pharmaceutical and, when appropriate, its metabolites (ICH M3 (ref. 1), ICH S6 (ref. 2)) on all 
stages of reproduction and development. No guidance can provide sufficient information to 
cover all possible cases, and flexibility in testing strategy is warranted. 
 
The contents of this document do not have the force and effect of law and are not meant to bind 
the public in any way, unless specifically incorporated into a contract. This document is intended 
only to provide clarity to the public regarding existing requirements under the law. FDA 
guidance documents, including this guidance, should be viewed only as recommendations, unless 
specific regulatory or statutory requirements are cited.  The use of the word should in Agency 
guidances means that something is suggested or recommended, but not required.  
 
A.  Aim of Studies (1.1) 
 
The aim of DART studies is to reveal any effect of the pharmaceutical on mammalian 
reproduction relevant for human risk assessment. As appropriate, the set of studies conducted 
should encompass observations through one complete life cycle (i.e., from conception in one 
generation through conception in the following generation) and permit detection of immediate 
and latent adverse effects. The following stages of reproduction are generally assessed: 
 
A)  Premating to conception (adult male and female reproductive functions, development 
and maturation of gametes, mating behavior, fertilization). 
 
B)  Conception to implantation (adult female reproductive functions, preimplantation 
development, implantation). 
 
C)  Implantation to closure of the hard palate (adult female reproductive functions, 
embryonic development, major organ formation). 
 
D)  Closure of the hard palate to the end of pregnancy (adult female reproductive 
functions, fetal development and growth, organ development and growth). 
 
E)  Birth to weaning (parturition and lactation, neonate adaptation to extrauterine life, 
pre-weaning development and growth). 
 
F)  Weaning to sexual maturity (post-weaning development and growth, adaptation to 
independent life, onset of puberty and attainment of full sexual function, and effects 
on second generation). 
 
The risks to all stages should be assessed, unless the stage is not relevant to the intended 
population. The stages covered in individual studies are left to the discretion of the sponsor, 
although the timing of studies within the pharmaceutical development process is dependent on 
study populations and phase of pharmaceutical development (see ICH M3, ICH S6 and ICH S9 
(ref. 3)).  
 
 
 
  2  Contains Nonbinding Recommendations 
 
II.  SCOPE OF THE GUIDANCE (2) 
 
This guidance applies to all pharmaceuticals, including biopharmaceuticals, vaccines (and their 
novel constitutive ingredients) for infectious diseases, and novel excipients that are part of the 
final pharmaceutical product. For the purposes of this guidance, the term pharmaceutical is 
used to encompass all of these treatment modalities. This guidance does not apply to cellular 
therapies, gene therapies and tissue-engineered products. The methodological principles (e.g., 
study design, dose selection and species selection, etc.) outlined in this guidance apply to all 
compounds for which the conduct of reproductive and/or developmental toxicity studies is 
appropriate. This guidance should be read in conjunction with ICH M3, ICH S6, and ICH S9 
regarding whether and when nonclinical DART studies are warranted.  
 
III.  GENERAL CONSIDERATIONS ON REPRODUCTIVE TOXICITY 
ASSESSMENT  
 
The majority of pharmaceuticals being developed should be assessed for all stages of the 
reproductive cycle identified above, although there can be some exceptions, which should be 
justified, as indicated below. To support clinical development, these stages have typically been 
evaluated using three in vivo study types: (1) a fertility and early embryonic development study 
(FEED - stages A and B), (2) embryo-fetal development studies in two species (EFD - stages C 
and D), and (3) a pre- and a postnatal development study (PPND  stages C through F). For each 
compound, the stages that are to be evaluated should be determined and the most appropriate 
studies to conduct should be identified. Key factors to consider when developing an overall 
integrated testing strategy to evaluate effects on reproduction and development include: 
 
  The targeted patient population and conditions of use (especially in relation to 
reproductive potential and severity of disease) 
  
  The formulation of the pharmaceutical and route(s) of administration intended for 
humans 
 
  Relevant data on toxicity (which can also include data from in vitro, ex vivo and 
nonmammalian studies, and structure-activity relationships), pharmacodynamics, 
pharmacokinetics, and pharmacological similarity to other pharmaceuticals 
  
  Aspects of the general biology of the pharmaceutical target, or known roles of the target 
in reproduction or development 
 
These concepts are discussed in more detail throughout the guidance. 
 
To the extent that it does not diminish the overall risk assessment, the experimental strategy 
should minimize the use of animals.4 Approaches towards this goal can include the conduct of 
 
4 We support the principles of the 3Rs, to reduce, refine, and replace animal use in testing when feasible.  We 
encourage sponsors to consult with us if they wish to use a non-animal testing method they believe is suitable, 
  3  Contains Nonbinding Recommendations 
 
studies that combine typical study types (see section VII (7)), as well as appropriately qualified 
alternative assays for risk assessment (see Annex 2). Since many clinical development programs 
are terminated prior to Phase 3, animal use can also be reduced by appropriately timing studies to 
support ongoing clinical development (e.g., embryo-fetal developmental toxicity data to support 
enrollment of women of childbearing potential) as per ICH M3. 
 
DART studies should, in general, be conducted according to good laboratory practice (GLP) 
regulations, as they will contribute to the risk assessment. However, if a relevant DART risk is 
identified in a non-GLP study, repetition of the study to confirm the finding(s) under GLP 
conditions is not necessarily warranted. A relevant risk is one that occurs at or near intended 
clinical exposures and is of a nature that is reasonably likely to translate to humans (see section 
IX (9)). It is recognized that GLP compliance is not expected for some study types, or aspects of 
some studies, employing specialized test systems or methods. However, high quality scientific 
standards should be applied with data collection records readily available. Areas of 
noncompliance should be identified within the study report, and their impact on study 
results/data interpretation should be considered relative to the overall safety assessment. 
 
A.  Target Patient Population/Therapeutic Indication Considerations (3.1) 
 
The intended patient population or therapeutic indication can influence the extent of DART 
testing. Studies evaluating all stages of reproduction and development are not warranted if the 
disease indicates that DART will have minimal impact on the risk of the pharmaceutical in the 
target population. For example, studies covering all stages are not necessarily appropriate for an 
exclusively postmenopausal female patient population, for use in the pediatric or juvenile 
prepubescent population, or for patient populations in hospitalized settings where pregnancy can 
be excluded.  
 
B.  Pharmacology Considerations (3.2) 
 
Before designing a testing strategy, it should be determined if the intended pharmacologic effects 
of a pharmaceutical are known to be incompatible with fertility, normal EFD, or assessment of 
particular endpoints (e.g., a general anesthetic and assessment of mating behavior). This 
assessment can be based on data with other pharmaceuticals with similar pharmacology, known 
effects of target engagement, or on knowledge of effects in humans with related genetic diseases. 
For example, it would be appropriate to modify the design of a PPND study for a pharmaceutical 
developed to prevent preterm labor. If the intended pharmacologic effects are incompatible with 
the study endpoints, testing for a particular reproductive endpoint is not warranted, with 
justification.  
C.  Toxicity Considerations (3.3) 
 
Repeated-dose toxicity studies with sexually mature animals can provide important information 
on toxicity to reproductive organs that can affect the design of a DART study. The existing 
toxicology data for the compound should always be considered, taking into account the dose 
 
adequate, validated, and feasible.   We will consider if such an alternative method is adequate to meet the regulatory 
need. 
 
  4  Contains Nonbinding Recommendations 
 
levels, toxicokinetic profile, and dosing duration. For example, the standard fertility study design 
can be modified to alter the duration of dosing, or the start of cohabitation, for a compound that 
affects testicular tissue. 
 
D.  Timing Considerations (3.4) 
 
General guidance on the timing for conduct of studies assessing reproductive and developmental 
endpoints is described in ICH M3, ICH S6, and ICH S9. The timing for when to conduct specific 
DART assessments should take into consideration the need for these data to support the safe use 
of the pharmaceutical in clinical trials or the intended patient population. Consequently, it can be 
appropriate to consider altering the timing of the assessment of specific reproductive stages. 
Additional options are discussed in sections IV.B.2 (4.2.2) and IV.B.3 (4.2.3). 
 
E.  Toxicokinetics (3.5) 
 
Exposure data can be generated in either reproductive (dose-range finding (DRF) or pivotal) or 
repeated-dose toxicity studies. However, given the potential for meaningful changes in 
toxicokinetic (TK) parameters induced by pregnancy, it is recommended to determine if 
pregnancy alters exposure. If dose selection is based on exposure ratio (see section VI.A.3 
(6.1.3)), GLP-compliant TK data in pregnant animals is expected. Sampling day(s) should be 
justified. 
  
When warranted, determination of the pharmaceuticals concentration in the embryo or fetus can 
facilitate interpretation of discordant or equivocal evidence of developmental hazard. This 
information can be collected in a separate study to determine the actual exposure. However, a 
direct comparison to the potential levels in the human conceptus is not appropriate. 
  
Evidence of lactational excretion can be obtained, when warranted, by sampling milk or by 
demonstrating exposure in offspring during the preweaning period. 
  
General concepts regarding TK data collection are discussed in ICH S3A (ref. 4).  
 
 
IV.  DESIGN AND EVALUATION OF IN VIVO MAMMALIAN STUDIES (4) 
 
The strategy to evaluate the potential reproductive and developmental risk of a pharmaceutical 
generally includes one or more in vivo studies. The key factor is that, in total, they leave no gaps 
between stages and allow for evaluation of all stages of the reproductive process, although in 
some species (e.g., the nonhuman primate (NHP)) it is not possible to evaluate all stages. For 
most pharmaceuticals, the three-study design will usually be appropriate, although various 
combinations of these study designs can be conducted to address specific product needs and to 
reduce animal use. Study details for the FEED, EFD, and PPND studies, and combinations 
thereof, can be found in Annex 1. The stages covered in individual studies are left to the 
discretion of the sponsor. All available pharmacological, toxicokinetic, and toxicological data for 
the pharmaceutical should be considered in determining which study design(s) should be used. 
 
  5  Contains Nonbinding Recommendations 
 
 
A.  Strategy To Address Fertility and Early Embryonic Development (FEED) 
(4.1) 
 
The aim of the FEED study is to test for adverse effects resulting from treatment initiated prior to 
mating of males and/or females and continued through mating and implantation; this comprises 
evaluation of stages A and B of the reproductive process. Results from repeated-dose toxicity 
studies of at least two weeks duration can often be used to design the fertility study without 
conducting further dose-ranging studies, although studies of such short duration can be 
insufficient to reveal all adverse effects. 
  
A mating phase is expected in most cases when a FEED study is warranted to support exposure 
of the target population. Such studies are typically performed in rodents. If no adverse effects on 
fertility are anticipated, both sexes can be treated and cohabited together in the same study. If 
effects on fertility are identified in the study, the affected sex should then be determined. In 
contrast, if adverse effects are anticipated based on mode of action or on the results of repeated-
dose studies, each treated sex can be cohabited with untreated animals of the opposite sex. This 
can be achieved using separate treatment arms within a single study or by the conduct of two 
separate FEED studies. Reversibility of adverse effects on fertility and early embryonic 
development can have an important impact on risk assessment. 
  
The FEED study design in female rodents (see Annex 1) allows for the detection of effects on 
the estrous cycle, tubal transport, implantation, and development of preimplantation stages of the 
embryo. When estrous/menstrual cycles are evaluated, it is important to obtain baseline cycle 
data (over 2 or 3 cycles minimum) to distinguish between treatment-related effects and inter/intra 
animal variability. The monitoring of estrous cyclicity should continue through the time of 
confirmation of mating. 
 
The FEED study design for male rodents that includes 2 to 4 weeks of treatment prior to 
cohabitation allows for the detection of effects on spermatogenesis and epididymal transport. 
When data from repeated-dose studies suggest toxicity to the testis, it can be appropriate to 
extend the duration of pre-cohabitation treatment to 10 weeks; this extension permits assessment 
of effects on the full spermatogenic cycle as well as epididymal transport. The FEED study 
additionally permits detection of functional effects (e.g., on libido, epididymal sperm maturation, 
ejaculation) that cannot be detected by histological examinations of the male reproductive 
organs. 
   
When there is cause for concern based on mode of action or data from previous studies, 
additional examinations can be included in repeated-dose toxicity and/or fertility studies (e.g., 
collecting sperm for counts and morphology/motility assessments, measuring hormone levels, or 
monitoring of the estrous/menstrual cycle) to further characterize potential effects on fertility.  
 
1.  Considerations for Biopharmaceuticals (4.1.1) 
 
If the biopharmaceutical is pharmacologically active in rodents or rabbits, a FEED study in one 
of these species is recommended. Mating evaluations are not generally feasible in nonrodents 
  6  Contains Nonbinding Recommendations 
 
such as dogs and NHPs. For example, if NHPs are the only pharmacologically relevant species 
(as for many monoclonal antibodies, see ICH S6), histopathological examinations of the 
reproductive tissues from the repeated-dose toxicity studies of at least three months duration can 
serve as a substitute for the fertility assessments. Such an approach should include a 
comprehensive histopathological examination of the reproductive organs from both male and 
female animals (Note 1). Unless the biopharmaceutical is intended to treat advanced cancer, in 
which case FEED studies are not warranted, animals should be sexually mature at study 
initiation in order for an adequate evaluation of the reproductive tissues to be made. These data 
would only provide information on the structure of the reproductive tissues, as no functional 
assessment of fertility can be made and predicting effects on fertility and early embryonic 
development is not always possible based solely on the results of histopathology assessments.  
 
B.  Strategies To Address Embryo-Fetal Development (EFD) (4.2) 
 
The aim of the EFD studies is to detect adverse effects on the pregnant female and development 
of the embryo and fetus following treatment (Stage C) of the pregnant female during 
organogenesis. EFD studies include evaluation of fetal development and survival (stages C 
through D). 
 
For most small molecules, effects on EFD are typically evaluated in two species (i.e., rodent and 
nonrodent (typically rabbit)). At least one of the test species should exhibit the desired 
pharmacodynamic response. If the pharmaceutical is not pharmacodynamically active in any 
routinely used species (see section V.A (5.1)) then use of nonroutine species (see section V.B 
(5.2)), genetically modified animals, or a species-specific surrogate molecule (section V.C (5.3)) 
(e.g., in the case of oligonucleotides) can be considered, provided there is sufficient 
characterization of the model to ensure pharmacologic relevance. Genetically modified animals 
and surrogate molecules are generally most useful for hazard identification but have limitations 
when used for risk assessment. Even when there are no relevant models (e.g., the 
pharmacological target only exists in humans, either normally or in the diseased state), EFD 
studies should be conducted in two species to detect the adversity of off-target effects or 
secondary pharmacology. 
  
Clearly positive results for the induction of malformations or embryo-fetal lethality (MEFL), in a 
single species, at exposures similar to that at the projected clinical exposure at the maximum 
recommended human dose (MRHD) can be sufficient for risk assessment. 
  
Under limited circumstances, other approaches can be used in place of definitive EFD studies 
(see Annex 2). Alternatively, there can be adequate information to communicate risk without 
conducting EFD studies. Evidence suggesting an adverse effect of the intended pharmacological 
mechanism on EFD (e.g., mechanism of action, phenotypic data from genetically modified 
animals) can be sufficient to communicate risk. 
 
1.  Considerations for Biopharmaceuticals (4.2.1) 
 
The effect of biopharmaceuticals on EFD should typically be assessed in two species (one rodent 
and one nonrodent) if both are pharmacologically relevant. However, the rodent is often not 
  7  Contains Nonbinding Recommendations 
 
pharmacologically relevant, in which case EFD assessment in a single pharmacologically 
relevant nonrodent species can be conducted. In cases where the NHP is the only relevant 
species, an enhanced pre- and post-natal development (ePPND) study can be conducted instead 
of an EFD study.  Biopharmaceuticals intended for the treatment of advanced cancer typically 
can be assessed in a single pharmacologically relevant species (ICH S9). 
  
When no relevant species can be identified because the biopharmaceutical does not interact with 
the orthologous target in any species relevant to reproductive toxicity testing, use of surrogate 
molecules or transgenic models can be considered, as described in ICH S6. Calculating safety 
margins relative to human exposures with surrogate molecules is not appropriate. If there are no 
relevant species, genetically modified animals or surrogates available, in vivo reproductive 
toxicity testing is not meaningful. In this case, the approach used for risk assessment, or rationale 
for not conducting studies, should be justified. 
 
2.  Alternative Approaches for Addressing EFD Risk (4.2.2) 
 
a.  Use of alternative assays (4.2.2.1) 
 
A number of alternative in vitro, ex vivo, and nonmammalian in vivo assays (alternative assays) 
have been developed to detect potential hazards to embryo-fetal development. They have been 
used as drug discovery screens for adverse effects on EFD and have assisted in the understanding 
of the mechanism of toxicity, which can be useful for translating nonclinical data to human risk 
(especially for human-specific targets). The continued use of alternative assays for these 
purposes is encouraged.  
 
If properly qualified, alternative assays have the potential to defer or replace (in certain 
circumstances) conventional in vivo studies. This has the added benefit of potentially reducing 
animal use. Concepts to consider when qualifying these assays, and examples when the use of 
such assays could be appropriate, appear in Annex 2. Approaches that incorporate alternative 
assays should provide a level of confidence for human safety assurance at least equivalent to that 
provided by the current testing paradigms described above. Based on the direction of scientific 
development as of the writing of this document, it is expected that for regulatory purposes, 
multiple alternative assays will be used within a tiered or battery approach. These testing 
strategies will be qualified within a certain context of use, which is defined by the chemical 
applicability domain of the assay, and by characterization of the biological mechanisms covered 
by the assay.  
 
3.  Potential Approaches To Defer Definitive In Vivo Testing as Part of an Integrated 
Testing Strategy (4.2.3) 
 
The design of an appropriate testing strategy relies on a cumulative weight-of-evidence 
approach. ICH M3 allows preliminary embryo-fetal developmental (pEFD) toxicity data from 
two species to support the limited inclusion of women of childbearing potential (WOCBP) (up to 
150 WOCBP for up to 3 months) before conducting definitive EFD studies. Based on these 
considerations, this guidance expands on ICH M3 by allowing two additional options to support 
inclusion of WOCBP prior to Phase 3 clinical trials:  
  8  Contains Nonbinding Recommendations 
 
 
(1) Qualified alternative assays that predict the outcome in one species (see Annex 2), 
can be combined with a pEFD study from a second species to enable the limited 
inclusion of WOCBP (up to 150 WOCBP for up to 3 months). The alternative assay 
and the second species should generally cover both a rodent and a nonrodent species. 
 
(2) Additional endpoints incorporated into at least one GLP pEFD study (specifically 
increasing the group size of evaluable litters with inclusion of skeletal examinations) 
performed in a pharmacologically relevant species, if available, combined with a 
pEFD study in a 2nd species allows all regions to include an unlimited number of 
WOCBP in clinical trials through Phase 2. 
 
C.  Strategy To Address Effects on Pre- and Postnatal Development (PPND) 
(4.3) 
 
The aim of the PPND study is to detect adverse effects following exposure of the maternal 
animal from implantation through weaning to evaluate effects on the pregnant or lactating female 
and development of the offspring. Since manifestations of effects induced during this period can 
be delayed, development of the offspring is monitored through sexual maturity (i.e., stages C to 
F). The rodent is usually used to assess PPND; however, other species can be used as appropriate 
(see Annex 1). 
 
In most cases, a preliminary (dose-range finding) PPND study is not warranted, because the 
appropriate information is generally available from prior studies. However, a preliminary PPND 
study with termination of the pups before or at weaning can be used to select dose levels or 
inform study design and/or to provide pup exposure data. 
 
If a modified PPND/ePPND study design is being considered to support pediatric development, 
see ICH S11 (ref. 5). 5 
 
1.  Considerations for Biopharmaceuticals (4.3.1)  
 
For pharmaceuticals that can only be tested in the NHP, the ePPND study can provide a limited 
assessment of postnatal effects, but it is not generally feasible to follow the offspring through 
maturity (see Annex 1 and ICH S6).  
 
 
V.  TEST SYSTEM SELECTION (5) 
 
A.  Routine Test Species (5.1) 
 
Mammalian species should be used to detect DART. The use of the same species and strain as 
that in already completed toxicity studies can eliminate the need to use additional animals or 
conduct additional studies to characterize pharmacokinetics and metabolism, and/or for dose-
 
5 When final, this guidance will represent FDAs current thinking on this topic. 
  9  Contains Nonbinding Recommendations 
 
range finding. The species used should be well characterized and relevant for detecting effects on 
the endpoints in a particular study (e.g., with respect to health, fertility, fecundity, background 
rates of malformation and embryo-fetal death).   
 
1.  Selection of Species for DART Testing (5.1.1) 
 
The rat is generally appropriate for DART testing and is the most-often used rodent species for 
reasons of practicality, general knowledge of pharmacology in this species, the extensive 
toxicology data usually available for interpretation of nonclinical observations, and the large 
amount of historical background data. The mouse is also often used as the rodent species for 
many of the same reasons. 
 
For assessment of EFD only, a second mammalian nonrodent species is typically evaluated, 
although there are exceptions (e.g., vaccines and biopharmaceuticals (see sections V.A.2 (5.1.2) 
and V.B (5.2), respectively)). The rabbit has proven to be useful in identifying human teratogens 
that have not been detected in rodents and is routinely used as the nonrodent species based on the 
extensive historical background data, availability of animals, and practicality. 
 
2.  Species Selection for Preventative and Therapeutic Vaccines (5.1.2) 
 
The animal species selected for testing of vaccines (with or without adjuvants) should 
demonstrate an immune response to the vaccine. The type of developmental toxicity study 
conducted, and the choice of the animal model, should be justified based on the immune 
response observed and the ability to administer an appropriate dose. Typically, rabbits, rats, or 
mice are used in developmental toxicity studies for vaccines. Even though quantitative and 
qualitative differences can exist in the responses (e.g., in humoral and cellular endpoints) 
between species, it is usually sufficient to conduct developmental toxicity studies in a single 
species. Although the degree and time course of transfer of maternal antibodies across the 
placenta varies between species, a developmental toxicity study in rabbits, rats, or mice can still 
provide important information regarding potential embryo-fetal toxicity of the vaccine 
components/formulation and safety of the product during pregnancy. NHP should be used only if 
no other relevant animal species demonstrates an immune response. 
 
When there is a lack of an appropriate animal model (including NHP), an EFD toxicity study in 
rabbits, rats, or mice can still provide important information regarding potential embryo-fetal 
toxicity of the vaccine components/formulation and safety of the product during pregnancy. 
 
B.  Nonroutine Test Species (5.2) 
 
Species other than the rat, mouse or rabbit can be used to evaluate the effects of pharmaceuticals 
on various reproductive stages. When considering the use of other species, their advantages and 
disadvantages (summarized in Table 1 of Annex 1) should be considered in relation to the 
pharmaceutical being tested, the study design and selected endpoints, and the ability to 
extrapolate results to the human situation. 
 
  10  Contains Nonbinding Recommendations 
 
NHPs should be considered a nonroutine test species. They are most typically used for 
evaluating effects on embryo-fetal development and early postnatal development for 
biopharmaceuticals that are only pharmacologically active in primates, as described in ICH S6. 
However, there are additional considerations that limit the utility of studies in NHPs for 
assessing some endpoints for DART risk assessment (see Annex 1 and ICH S6). 
 
C.  Use of Disease Models, Genetically Modified Models, and Surrogate 
Molecules (5.3) 
 
Animal models of disease, genetically modified models, and surrogate molecules can be valuable 
for investigating the effect of the intended pharmacology on development and reproduction. 
Studies in disease models can be of value in cases where the data obtained from healthy animals 
could be misleading or otherwise not apply to the disease conditions in the clinical setting. The 
model should be pharmacologically relevant and appropriate for the development and 
reproductive endpoints being assessed. The pathophysiology of the disease course in the model 
should be characterized. Some differences from the human pathophysiology would not preclude 
its use if these are unlikely to confound data interpretation. Animal-to-animal variability should 
be characterized and appropriate within the context of the study. If historical control information 
is limited, reference data for the study endpoints should be available or should be generated 
during the study to aid data interpretation. 
  
Genetically modified models can be used to provide information about on-target effects of a 
pharmaceutical on DART parameters through permanent or conditional alterations in target 
activity. Such models can inform on whether the biology of the target is closely linked to adverse 
effects on reproduction and development in routine test species. 
  
When the pharmaceutical does not have adequate activity against the target in the routine test 
species, surrogate molecules can be used to assess potential adverse effects on reproduction and 
development.  
 
 
VI.  DOSE-LEVEL SELECTION, ROUTE OF ADMINISTRATION, AND 
SCHEDULE (6) 
 
The choice of dose levels, schedule and route of administration are important study design 
considerations and should be based on all available information (e.g., pharmacology, repeated-
dose toxicity, pharmacokinetics, and dose-range-finding studies). Guidance on the principles of 
dose selection for small molecules and biopharmaceuticals is given in ICH M3 and ICH S6, 
respectively. When sufficient information on tolerability in the test system is not available, dose-
range-finding studies are advisable. 
 
A.  Dose Selection (6.1) 
 
There are a number of dose-selection endpoints that can be used for DART studies. All endpoints 
discussed in this section are considered equally appropriate in terms of study design. The high 
dose in the definitive studies should be one that is predicted to comply with one or more of the 
  11  Contains Nonbinding Recommendations 
 
concepts set forth in sections VI.A.1 (6.1.1) to VI.A.5 (6.1.5) below. The selected doses should 
take into account observations made in previous studies (e.g., repeated-dose, TK, DRF). There 
can be instances where fewer than three dose levels are sufficient to provide the information for 
risk assessment. 
 
Justification for high-dose selection using endpoints other than those discussed below can be 
made on a case-by-case basis. 
 
1.  Toxicity-Based Endpoint (6.1.1) 
 
This endpoint is based on inducing a minimal level of toxicity in the parental animals at the high 
dose. Factors limiting the high dose determined from previously conducted studies could include, 
but are not limited to: 
 
  Alterations in body weight (gain or absolute; either reductions or increases). Minor, 
transient changes in body weight gain or body weight are not appropriate for dose 
selection. When assessing weight change effects, the entire dosing duration of the study 
should be considered. 
  
  Exaggerated pharmacological responses (e.g., excessive sedation or hypoglycemia). 
 
  Toxicological responses (e.g., convulsions, excessive embryo-fetal lethality, clinical 
pathology perturbations). Specific target-organ toxicity that would interfere with the 
study endpoints within the duration of the planned DART study. 
 
2.  Saturation of Systemic Exposure Endpoint (6.1.2) 
 
High dose selection based on saturation of systemic exposure measured by systemic availability 
of pharmaceutical-related substances can be appropriate. There is little value in increasing the 
administered dose if it does not result in increased plasma concentration of parent or metabolites.  
 
3.  Exposure Margin-Based Endpoint (6.1.3) 
 
It can be appropriate to select doses based on predicted exposure margins relative to the exposure 
at the MRHD. For small molecules, a systemic exposure representing a large multiple of the 
human area under the curve (AUC) or maximum plasma concentration (C ) at the MRHD can 
max
be an appropriate endpoint for high dose selection. Doses providing an exposure in pregnant 
animals > 25fold the exposure at the MRHD are generally considered appropriate as the 
maximum dose for DART studies (Note 2). The 25-fold exposure margin should be established 
in a GLP-compliant dose-range-finding/pEFD or definitive study. Usually, this multiple should 
be determined based on parent drug levels; however, consideration should also be given to 
ensuring an adequate exposure margin to major human metabolites (see ICH M3 and ICH M3 
Q&A (ref. 1)). In the case of prodrugs, it can be more appropriate to establish the exposure 
multiple on the basis of the active metabolite, particularly if the test species has a lower ratio of 
active metabolite to prodrug, compared to humans. The basis for the moiety used for comparison 
(parent drug or metabolite) should be justified. 
  12  Contains Nonbinding Recommendations 
 
  
For pharmaceuticals that have demonstrated pharmacodynamic activity in the test species only at 
exposures > 25-fold that projected at the MRHD, higher doses can be warranted to assess 
adverse effects of exaggerated pharmacology. However, irrelevant off-target effects are more 
likely to be observed. 
 
When exposure-based endpoints are used as the basis for selection of the dose levels for EFD 
studies, TK data from pregnant animals in a GLP-compliant study is expected. The choice for 
using total versus fraction unbound pharmaceutical exposures should be justified and consistent 
with the entire nonclinical development program as outlined in ICH S3A (ref. 4). 
 
a.  Exposure-based approach for biopharmaceuticals (6.1.3.1) 
 
Exposure-based margins can be appropriate to select doses for biopharmaceuticals as per ICH 
S6.  Generally, the dose should provide the maximum intended pharmacological effect in the 
preclinical species or provide an approximately 10-fold exposure multiple over the maximum 
exposure to be achieved in the clinic, whichever is higher. ICH S6 should be consulted with 
regard to dose adjustment for differences in target-binding affinity and other relevant factors. 
 
4.  Maximum Feasible Dose Endpoint (6.1.4) 
 
The maximum feasible dose (MFD) can be used for high dose selection when the 
physicochemical properties of the pharmaceutical (or formulation) associated with the 
route/frequency of administration and the anatomical/physiological attributes of the test species 
limit the amount of the pharmaceutical that can be administered. Use of the MFD should 
maximize exposure in the test species, rather than maximize the administered dose, as per ICH 
M3 Q&A (ref. 1). Note that changes to the frequency of dose administration can be considered to 
increase the total feasible daily exposure (see section VI.C (6.3)). 
 
5.  Limit Dose Endpoint (6.1.5) 
 
A limit dose of 1 gram (g)/kilogram (kg)/day can generally be applied when other dose-selection 
factors have not been attained with lower dose levels (see also ICH M3 for other considerations).   
 
6.  Selection of Lower Dose Levels (6.1.6) 
 
It is generally desirable to establish a no observed adverse effect level (NOAEL) for DART. The 
selection of lower dose levels should take into account exposure, pharmacology, and toxicity, 
such that the dose-response of findings can be established when appropriate. The low dose 
should generally provide a low multiple (e.g., 1- to 5-fold) of the human exposure at the MRHD. 
Dose levels that yield exposures that are subtherapeutic in humans should be justified. 
 
B.  Route (6.2) 
 
In general, the route of administration should be the clinical route. If, however, sufficient 
exposure cannot be achieved using the clinical route or the clinical route is not feasible, a 
  13  Contains Nonbinding Recommendations 
 
different route should be considered. When multiple routes of administration are being evaluated 
in humans, a single route in the test species can be adequate provided that sufficient systemic 
exposure is achieved compared to that of all clinical routes and that there is adequate coverage 
for the metabolites. 
 
C.  Schedule (6.3) 
 
Dosing schedules used in the toxicity studies determine the exposure profile, which can be 
important in the risk assessment. Although mimicking the clinical schedule is often sufficient, a 
more or a less frequent schedule can be appropriate. For example, twice daily dosing can be 
warranted with compounds that are quickly metabolized in the test species, although pragmatic 
factors (e.g., study logistics, stress on animals) should be considered when a more frequent 
schedule is contemplated. It can also be important to alter the dosing schedule to ensure that 
adequate exposure is obtained at all critical stages of reproduction and/or development being 
evaluated in a given study. 
 
D.  Dose Selection and Study Designs for Vaccines (6.4) 
 
This guidance covers vaccines (adjuvanted or not) used in both preventative and therapeutic 
indications against infectious diseases. While not within the scope of this guidance, the 
principles outlined can be applicable to the nonclinical testing of vaccines for other indications as 
well (e.g., cancer). 
  
The types of reproductive and/or developmental toxicity studies used for preventative and 
therapeutic vaccines depend on the target population for the vaccine and the relevant 
reproductive risk. Generally, DART studies are not warranted for vaccines being developed for 
neonates, prepubertal children, or geriatric populations. 
  
For reproductive toxicity studies of vaccines, it is typically sufficient to assess a single dose level 
capable of eliciting an immune response in the animal model (section V.A.1 (5.1.2)), using the 
clinical route of administration. This single dose level should be the maximum human dose 
without correcting for body weight (i.e., 1 human dose = 1 animal dose). If it is not feasible to 
administer the maximum human dose to the animal because of a limitation in total volume that 
can be administered, or because of dose-limiting toxicity, whether local or systemic, a dose that 
exceeds the human dose on a milligram (mg)/kg basis can be used. To use a reduced dose, 
justification as to why a full human dose cannot be used in an animal model should be provided. 
  
The vaccination regimen should maximize maternal antibody titers and/or immune response 
throughout the embryonic, fetal, and early postnatal periods. Timing and number of doses will 
depend on the onset and duration of the immune response of the particular vaccine. When 
developing vaccines to be given during pregnancy, a justification should be provided for the 
specific study design, based upon its intended use (e.g., protecting the mother during pregnancy 
or protecting the child early postnatally). 
  
Daily dosing regimens can lead to overexposure to the vaccine constituents. Episodic dosing of 
pregnant animals rather than daily dosing is recommended. Also, episodic dosing better 
  14  Contains Nonbinding Recommendations 
 
approximates the proposed clinical immunization schedule for most preventive and therapeutic 
vaccines. Considering the short gestational period of routine animal species, it is generally 
recommended to administer a priming dose(s) to the animals several days or weeks prior to 
mating in order to elicit peak immune response during the critical phases of pregnancy (i.e., the 
period of organogenesis). The dosing regimen can be modified according to the intended 
vaccination schedule in humans. 
 
At least one dose should be administered during early organogenesis to evaluate potential direct 
embryotoxic effects of the components of the vaccine formulation and to maintain a high 
antibody response throughout the remainder of gestation. If embryo-fetal toxicity is observed, 
this can be further assessed using subgroups of animals that are dosed at certain time points. 
  
In cases where a vaccine includes a novel active constitutive ingredient (including novel 
adjuvants), consideration of additional testing strategies similar to those for non-vaccine products 
can be appropriate.  
 
 
VII.   POSSIBLE COMBINATION STUDY DESIGNS IN RODENTS (7) 
 
Although three separate study designs, i.e., FEED (stages A and B), EFD (stages C through D) 
and PPND (stages C through F) have been employed to develop the majority of pharmaceuticals, 
various combinations of these study designs can be conducted to reduce animal use. The main 
advantage of combination designs is that all relevant stages of the reproductive process can be 
assessed using fewer animals. Combination studies can also better mimic the exposure duration 
in the clinic, especially for drugs with long half-lives. A common combination study design is a 
combined Fertility and EFD study (stages A through D) with a separate PPND study (stages C 
through F). 
 
Designs and study details for FEED, EFD, and PPND studies, and combinations thereof, can be 
found in Annex 1. 
 
In cases where no effects on male or female fertility are anticipated, or where extending the 
dosing period is appropriate due to observation of reproductive organ toxicity in a repeated-dose 
toxicity study, a combination design of repeated-dose and fertility studies can be considered. 
After a defined dosing period within the repeated-dose toxicity study, males can be paired with 
sexually mature females (whether untreated or dosed for at least two weeks prior to mating). This 
combination study can reduce the number of animals used, but the number of mating pairs per 
group should be at least 16. Furthermore, if treated, dosing of females can be extended until the 
end of organogenesis, thereby allowing evaluation of EFD endpoints (Annex 1).  
 
 
 
 
 
 
 
  15  Contains Nonbinding Recommendations 
 
VIII.  DATA REPORTING AND STATISTICS (8) 
 
A.  Data Reporting (8.1) 
 
Individual values should be tabulated in a clear, concise manner to account for all animals in the 
study. The data tables should allow ready tracking of individual animals and their conceptuses, 
from study initiation through study conclusion. 
  
Fetal morphologic abnormalities should be described using industry-harmonized terminology. 
All findings for each litter should be clearly listed by conceptus. Summary listings should be 
prepared by type of abnormality. The inclusion or exclusion of data from nonpregnant animals in 
summary tables should be clearly indicated. 
  
Interpretation of study data relies primarily on comparison with the concurrent control group. 
Historical control/reference data can be used to assist data interpretation. Recent historical 
control data from the performing laboratory is preferable. Contemporary data typically from a 
five-year period is desirable and permits identification of genetic drift.  
 
B.  Statistics (8.2) 
 
Statistical testing to assess the significance of differences between the treated and control groups 
is expected in definitive studies. Many of the datasets from DART studies do not follow a 
normal distribution, necessitating the use of non-parametric statistical methods. Cesarean, fetal, 
and postnatal data summary statistics should be calculated using the litter as the unit of analysis. 
Statistical significance need not convey a positive signal, nor lack of statistical significance 
impute absence of effect. Determination of biological plausibility, based on all available 
pharmacologic and toxicologic data, is often useful.  
 
 
IX.  PRINCIPLES OF RISK ASSESSMENT (9) 
 
As described in the preceding sections of this guidance, all available data garnered from the 
pharmaceutical, related compounds, human genetics, and knowledge of the role of target biology 
in human reproduction should be used to address potential reproductive risks in humans under 
the conditions of use, both during clinical trials and after marketing authorization. Any 
limitations (e.g., test system relevance, achieved exposure), uncertainties and data gaps in the 
available nonclinical DART data package should be addressed and their impact assessed. 
Generally, the results from definitive in vivo studies in an appropriate species with adequate 
exposures carry more weight than those from alternative assays or preliminary studies. Risk 
assessment is a continuous process through product development as more information becomes 
available. 
  
Not all findings reported in DART studies are adverse. When a finding is deemed adverse, 
several factors should be considered in a weight-of-evidence evaluation for risk assessment. 
These factors can include exposure margins, biological plausibility, evidence of a dose-response 
relationship, potential for reversibility, the potential for confounding parental toxicity, and 
  16  Contains Nonbinding Recommendations 
 
evidence for cross-species concordance.  For rare malformations, the absence of increased 
frequency with dose does not always alleviate concern. 
  
Comparison of pharmaceutical exposure at the NOAEL in the test species to the exposure at the 
MRHD is an important component of the risk assessment. This comparison should be based on 
the most relevant metric (e.g., AUC, C , minimum plasma concentration (C ),body surface 
max min  
area-adjusted dose). In general, there is increased concern when the NOAEL occurs at exposures 
less than 10-fold the human exposure at the MRHD; above this threshold, concern is reduced. 
Effects that are limited to occurrence at more than 25-fold the human exposure at the MRHD are 
usually of minor concern for the clinical use of the pharmaceutical. The most relevant margin is 
generally the exposure metric in the most sensitive species, unless appropriately justified 
otherwise. Biological plausibility is assessed by comparison of pharmacologic mechanism of 
action with the known role of the target in reproduction or development. A finding that can be 
interpreted as a consequence of pharmacology suggests that it will be of concern for humans. 
This relationship is further strengthened by evidence that the finding is dose related, whether 
characterized as increasing incidence or severity. Absence of biological plausibility does not 
preclude off-target toxicity, particularly if the finding is characterized by a dose-response 
relationship. 
 
Understanding the potential for reversibility will alter the risk assessment. Effects on male and 
female fertility that are reversible after cessation of treatment are of less concern. Conversely, 
critical irreversible developmental endpoints, such as death or malformation, are of increased 
concern. Other forms of developmental toxicity (e.g., growth retardation, functional deficits), 
may or may not be reversible. Generally, transient findings (e.g., skeletal variations, such as 
wavy ribs in rodents) are of less concern when they occur in isolation. Similarly, variations that 
are indicative of growth retardation in the presence of reduced fetal weight are of less concern. 
However, an overall increase in the incidence of variations (qualitatively similar or not) can 
suggest increased concern for dysmorphogenesis in the presence of an equivocal increase in 
malformations. 
  
The role of parental toxicity should be considered in determination of the relevance of findings. 
Embryo-fetal toxicity observed in the presence of maternal toxicity should be considered 
carefully to determine the likelihood that the finding is relevant for humans. Specifically, 
evaluation of the concordance between individual litter findings and the severity of maternal 
toxicity in the dam could be helpful in this assessment. It should not be assumed that 
developmental toxicity is secondary to maternal toxicity, unless such a relationship is 
demonstrated de novo, or relevant published literature can be cited. 
  
Consistency of findings reported among studies or between species can strengthen the concern 
for an adverse effect. Increased fetal lethality seen in a rodent EFD study that is consistent with 
decreased live litter sizes in the PPND study is an example of cross-study concordance. 
Observations of increased post implantation loss in rats and rabbits is an example of cross-
species concordance. Further knowledge of the mechanism of reproductive or developmental 
effects identified in animal studies can help to explain differences in responses between species 
and provide information on the human relevance of the effect (e.g., corticosteroid-induced cleft 
palate in mice). 
  17  Contains Nonbinding Recommendations 
 
  
A specific risk assessment conducted for breastfeeding would be predicated on hazards identified 
by the in vivo littering study (PPND or ePPND). These hazards can include adverse effects on 
offspring growth and development that are attributed to excretion of the pharmaceutical in the 
milk. Systemic exposure data in the pups from the littering study, if available, can also be 
compared with projected lactational exposures in the human infant. While interspecies 
differences in milk composition preclude a direct quantitative correlation of animal milk levels to 
human milk levels of a pharmaceutical, the presence of pharmaceutical in animal milk generally 
indicates the presence of pharmaceutical in human milk. 
  
Lastly, available human data can influence the overall assessment of human reproductive risk. 
 
 
  18  Contains Nonbinding Recommendations 
 
X.  ENDNOTES (10) 
   
Note 1: In particular, the testes and epididymides should be sampled and processed using 
methods that preserve the tissue architecture of the seminiferous epithelium. A detailed 
qualitative microscopic evaluation with awareness of the spermatogenic cycle is a sensitive 
means to detect effects on spermatogenesis. While generally not warranted, additional 
experimental endpoints (e.g., immunohistochemistry, homogenization resistant spermatid counts, 
flow cytometry, quantitative analysis of staging) can be incorporated into the study design to 
further characterize any identified effects. In females, a detailed qualitative microscopic 
examination of the ovary (including follicles, corpora lutea, stroma, interstitium, and 
vasculature); uterus; and vagina should be conducted with awareness of the reproductive cycle 
and the presence of primordial and primary follicles. 
   
Note 2: An analysis of 22 known human or presumed human teratogens showed that if MEFL 
was observed, exposure at the lowest observed adverse effect level (LOAEL) in at least one 
species was < 6-fold the exposure at the MRHD (Andrews et al. (ref. 6)). This indicates that 
using a > 25-fold exposure ratio for high-dose selection in the EFD toxicity studies would have 
been sufficient to detect the teratogenic hazard for all these pharmaceuticals. The analysis also 
showed that for human teratogens that were detected in animal species, the exposure at the 
NOAEL in at least one species was < 4-fold the exposure at the MRHD. 
 
In addition, a survey was conducted on EFD toxicity studies by the IQ DruSafe Leadership 
Group (Andrews et al. (ref. 7)). This survey identified 153 and 128 definitive rat and rabbit EFD 
studies, respectively, that achieved  15-fold animal to human parent drug exposure ratios (using 
human exposure at the intended therapeutic dose) in the absence of confounding (i.e., dose-
limiting) maternal toxicity. These data show that dosing animals to achieve exposures  25-fold 
human exposures when there is no maternal toxicity (that would otherwise limit the high dose) 
only infrequently detects MEFL. In all these cases, MEFL findings were not observed until 
exposures exceeded 50-fold and findings at such high exposures are not believed to be relevant 
to human risk assessment. In the absence of confounding maternal toxicity, the selection of a 
high dose for EFD and PPND studies that represents a > 25-fold exposure ratio to human plasma 
exposure of total parent compound at the intended maximal therapeutic dose is therefore 
considered pragmatic and reasonably sufficient for detecting outcomes relevant for human risk 
assessment. 
 
 
  18  Contains Nonbinding Recommendations 
 
XI.  GLOSSARY/ABBREVIATIONS 
 
Disclaimer:  The definitions in this glossary are specific for their use within this guidance. 
 
Alternative assay(s):  In vitro, ex vivo or nonmammalian in vivo assay(s) intended to predict 
malformations or embryo-fetal lethality; see MEFL. 
 
Applicability domain:  refers to the definition of the physicochemical properties of the 
substances that can be reliably tested in the assay and the biological mechanisms of action 
covered by the assay. 
 
Assay qualification (for regulatory use):  Confirmation of the predictivity of an alternative 
assay(s) to identify MEFL, as observed in vivo. 
 
AUC:  Area under the curve. 
 
C :  Maximum plasma concentration. 
max
 
C :  Minimum plasma concentration. 
min
 
Constitutive ingredients:  Chemicals or biologic substances used as excipients, diluents, or 
adjuvants in a vaccine, including any diluent provided as an aid in the administration of the 
product and supplied separately. 
 
DART:  Developmental and reproductive toxicity.  
 
DRF:  Dose-range finding. 
 
Developmental toxicity:  Any adverse effect induced prior to attainment of adult life. It includes 
effects induced or manifested from conception to postnatal life. 
 
EFD:  Embryo-fetal development. 
 
ePPND:  Enhanced pre- and postnatal development. 
 
FEED:  Fertility and early embryonic development. 
 
GD:  Gestation day. 
 
GD 0:  The day on which positive evidence of mating is detected (e.g., sperm is found in the 
vaginal smear/vaginal plug in rodents or observed mating in rabbits). 
 
GI:  Gastrointestinal.  
 
GLP:  Good laboratory practice. 
 
  19  Contains Nonbinding Recommendations 
 
ICH:  International Council for Harmonization of Technical Requirements for Pharmaceuticals 
for Human Use. 
 
IV:  Intravenous. 
  
LOAEL:  Lowest observed adverse effect level. 
 
LLO:  Late-life onset. 
 
Malformation:  Permanent structural deviation that generally is incompatible with or severely 
detrimental to normal development or survival. 
 
MOA:  Mechanism of action. 
 
MEFL:  Malformation or Embryo-fetal lethality. 
 
MFD:   Maximum feasible dose. 
 
MRHD:  Maximum recommended human dose. 
 
NHP:  Nonhuman primate. 
 
NOAEL:  No observed adverse effect level. 
 
PD:  Pharmacodynamic. 
pEFD:  Preliminary embryo-fetal development. 
PK: Pharmacokinetic. 
 
PND:  Postnatal day 
 
PPND: Pre- and postnatal development.  
 
Preliminary EFD (pEFD) toxicity study: An embryo-fetal developmental toxicity study that 
includes exposure over the period of organogenesis, has adequate dose levels, uses a minimum of 
6 pregnant animals per group, and includes assessments of fetal survival, fetal weight, and 
external and soft tissue alterations (see ICH M3). 
 
SDLT: Severely debilitating or life-threatening.  
 
Surrogate molecule: A molecule showing similar pharmacologic activity in the test species as 
that shown by the human pharmaceutical in the human. 
 
TK: Toxicokinetic. 
 
  20  Contains Nonbinding Recommendations 
 
Vaccine: For the purpose of this guidance, this term refers to preventative or therapeutic 
vaccines for infectious diseases. Vaccine (inclusive of the term vaccine product) is defined as the 
complete formulation and includes antigen(s) (or immunogen(s)) and any additives such as 
adjuvants, excipients or preservatives. The vaccine is intended to stimulate the immune system 
and result in an immune response to the vaccine antigen(s). The primary pharmacological effect 
of the vaccine is the prevention and/or treatment of an infection or infectious disease. 
 
Variation: Structural change that does not impact viability, development, or function (e.g., 
delays in ossification) which can be reversible, and are found in the normal population under 
investigation. 
 
WOCBP: Women of child bearing potential. 
 
 
  21  Contains Nonbinding Recommendations 
 
XII.  REFERENCES (12) 
 
1.  ICH guidance for industry, M3(R2) Nonclinical Safety Studies for the Conduct of 
Human Clinical Trials and Marketing Authorization for Pharmaceuticals (January 
2010); ICH guidance for industry, M3(R2) Nonclinical Safety Studies for the Conduct 
of Human Clinical Trials and Marketing Authorization for Pharmaceuticals  
Questions & Answers (R2) (February 2013). 
 
2.  ICH guidance for industry, S6(R1) Preclinical Safety Evaluation of Biotechnology-
Derived Pharmaceuticals (May 2012). 
 
3.  ICH guidance for industry, S9 Nonclinical Evaluation for Anticancer 
Pharmaceuticals (March 2010). 
 
4.  ICH guidance for industry, S3A Toxicokinetics: The Assessment of Systemic Exposure 
in Toxicity Studies (March 1995); ICH guidance for industry, S3A Guidance: Note for 
Guidance on Toxicokinetics: The Assessment of Systemic Exposure in Toxicity 
Studies: Focus on Microsampling  Questions and Answers (May 2018). 
 
5.  ICH draft guidance for industry, S11 Nonclinical Safety Testing in Support of 
Development of Pediatric Medicines (February 2019). 
 
6.  Andrews PA, Blanset D, Lemos Costa P, Green M, Green ML, Jacobs A, et al. 
Analysis of exposure margins in developmental toxicity studies for detection of 
human teratogens.  Regul Toxicol Pharmacol. 2019a;105:62-68. 
 
7.  Andrews PA, McNerney ME, DeGeorge JJ. Reproductive and developmental toxicity 
testing: An IQ-DruSafe industry survey on current practices.  Regul Toxicol 
Pharmacol. 2019b;107:104413. 
 
 
  22  Contains Nonbinding Recommendations 
 
 
ANNEX 1   IN VIVO STUDY DESIGNS 
 
Outlined below are advantages and disadvantages to the use of various species utilized in 
DART studies. 
  
Table 1: Principle Advantages and Disadvantages of Various Species for 
Developmental and Reproductive Toxicity Testing  
 
Routine Species 
Species  Advantages  Disadvantages 
Rat    Well-understood biology    Different placentation to 
  Widely used for  human (e.g., timing, 
pharmacodynamics and drug  inverted yolk sac) 
discovery     Dependence on prolactin as 
  Robust reproductive capacity  the primary hormone for 
with short gestation  establishment and 
  Large group sizes and litter  maintenance of early 
size  pregnancy, which makes 
  Data available from repeated- them sensitive to some 
dose toxicity study  pharmaceuticals (e.g., 
  Suitable for all stages of  dopamine agonists) 
testing    Highly sensitive to 
  Widespread laboratory  pharmaceuticals that disrupt 
parturition (e.g., 
experience and availability 
nonsteroidal anti-
  Extensive historical data 
inflammatory drugs in late 
pregnancy) 
  Less sensitive than humans 
to fertility perturbations 
  Limited application for 
foreign proteins 
  Limited or no 
o
pharmacologic activity 
  Potential impact of 
o
immunogenicity 
 
 
23  Contains Nonbinding Recommendations 
 
 
Rabbit    Similar advantages to rats    Limitations similar to rat for 
  Nonrodent model  foreign proteins 
  Suitable for serial semen    Limited historical data for 
sampling and mating studies   fertility and pre-/postnatal 
  Placental transfer of antibodies  studies 
more closely approximates    Sensitive to gastrointestinal 
primates than rodents, an  disturbances; (e.g., some 
advantage for DART testing of  antibiotics)  
vaccines     Prone to spontaneous abortion 
  General physical condition 
difficult to monitor using 
clinical signs 
  Should generate 
pharmacodynamic (PD), 
toxicity, and toxicokinetic 
(TK) data as not generally 
used for toxicology programs 
(except for vaccines) 
Mouse    Similar advantages to rats    Similar limitations to rats  
  Genetically modified models    Small fetus size and tissue 
available or can be generated  volumes 
  Surrogate molecules are often    Stress sensitivity 
available    Malformation clusters are 
  Uses small amounts of test  known to occur 
material   
 
Nonroutine Species 
Species  Advantages  Disadvantages 
Cynomolgus    Generally, more    Small group size, hence low 
Monkey  phylogenetically and  statistical power and wide 
physiologically similar to  variability across groups 
(NHP) 
humans than other species    Low fecundity 
  More likely than rodents to    Single offspring 
o
show similar pharmacology to    High background pregnancy 
humans  loss 
  Placentation similar to human     Limited availability of 
  Data available from repeated- breeding animals  
dose toxicity study    Long menstrual cycle (30 
  Transfer of antibodies across  days) and gestation (165 
the placenta similar to humans   days) 
  Impractical for fertility 
(mating) studies 
  F1 reproduction function not 
practical to evaluate due to 
24  Contains Nonbinding Recommendations 
 
 
late sexual maturity (around 3 
to 6 years of age) 
  Sexual maturity cannot be 
determined by age and body 
weight 
  Ethical considerations 
  Less historical control data 
and laboratory 
experience/capability 
  Highly variable age, weight 
and pregnancy history at the 
start   
Mini-pig    Alternate nonrodent for general    Limited number of 
toxicity testing  experienced laboratories 
  Short period of organogenesis    Long gestation (114 days) 
(gestation day (GD) 11-35)    Uses a large amount of test 
  Defined genetic background  material  
and specific-pathogen-free    Minimal to no prenatal 
animals   transfer of antibodies 
  Sexual maturity by 7 months 
  Larger litter size compared to 
NHP 
  Suitable for serial semen 
sampling and mating studies   
  Sufficient historical 
background data on 
reproductive endpoints  
 
 
Limited Use Species (primarily used for investigative purposes) 
Species  Advantages  Disadvantages 
Hamster    Alternate rodent model that can    High postnatal loss due to 
be pharmacologically relevant   cannibalization 
  Limited historical control data 
and laboratory experience 
  Limited availability of 
postnatal behavioral and 
functional tests 
  Intravenous (IV) route 
difficult 
  Aggressive 
  Sensitive to gastrointestinal 
(GI) disturbances 
  Should generate PD, toxicity, 
and TK data as not generally 
used for toxicology programs  
  Blood sampling is difficult 
25  Contains Nonbinding Recommendations 
 
 
Dog    Usually have repeated-dose    Long gestation (63 days) 
toxicity data    Limited historical control data 
  Readily amenable to semen  and laboratory experience 
collection    Limited availability of 
postnatal behavioral and 
functional tests 
  Uses a large amount of test 
material  
Other mammalian species not listed here can also be used to evaluate the effects of 
pharmaceuticals on DART endpoints.  
 
1.1  In Vivo Study Design Considerations  
 
Generally, within and between reproductive studies, animals should be of comparable 
age, weight and parity at the start. The easiest way to fulfill these factors is to use animals 
that are young, sexually mature adults at the time of the start of dosing.  The number of 
animals per group specified in individual studies is a balance based on scientific judgment 
from many years of experience with these study designs, and ethical considerations on the 
appropriate use of animals. Smaller group sizes can be sufficient to demonstrate 
anticipated adverse effects on reproduction or development at clinically relevant 
exposures of the pharmaceutical. 
  
Evaluation of 16 to 20 litters for rodents and rabbits provides a degree of consistency 
among studies. If fewer than 16 litters per group are evaluated, inter-study results become 
inconsistent. If more than 20 to 24 litters per group are evaluated, consistency and 
precision are not greatly enhanced. These numbers refer to litters available for evaluation.  
If groups are subdivided for different evaluations, the number of animals starting the 
study should be adjusted accordingly.  
 
The suggested study designs below can be modified, particularly with respect to 
parameters, timings, and assessments, and still meet the study objectives. Expert 
judgment should be used for adapting these framework designs for individual laboratories 
and purposes. 
 
1.1.1  Fertility and Early Embryonic Development (FEED) Study  
 
The FEED study is designed to assess the maturation of gametes, mating behavior, 
fertility, preimplantation development of the embryo, and implantation. For females, this 
includes effects on the estrous cycle and tubal transport. For males, it includes detection 
of functional effects (e.g., epididymal sperm maturation) that cannot be detected by 
histological examinations of the male reproductive organs. 
  
A combined male/female FEED study, in which both sexes are administered test article, 
is commonly used (see Table 2). However, separate male only or female only studies can 
be conducted by substituting the appropriate number of untreated females or males in the 
study designs.     
26  Contains Nonbinding Recommendations 
 
 
Table 2: FEED Study Design: Rodents, Combined Male and Female Study 
     
Parameter     
Group size   At least 16 of each sex.   
Number of dose groups  4 (including 1 control).   
Administration perioda  M:  2 weeks prior to cohabitation through at   
least confirmation of mating.  
F:  2 weeks prior to cohabitation through 
implantation (GD6). 
Mating ratio  1 male:1 female.   
Mating periodb   2 weeks.   
Estrous cycle evaluation  Daily, commencing 2 weeks before   
cohabitation and until confirmation of 
mating.  
Clinical  At least once daily.   
observations/mortality 
Body weight  At least twice weekly.   
Food consumption  At least once weekly (except during mating).   
Male necropsyc  Preserve testes and epididymides for possible   
histological examination; and evaluate on a 
case by case basis. 
Perform macroscopic examination and preserve 
organs with findings for possible histological 
evaluation; keep corresponding organs of 
sufficient controls for comparison.  
 
 
Sperm analysisd  Optional   
Female necropsye  On a case-by-case basis, preserve ovaries and   
uteri for possible histological examination 
and evaluation. 
Perform macroscopic examination and preserve 
organs with findings for possible histological 
evaluation; keep corresponding organs of 
sufficient controls for comparison.  
 
 
Scheduled cesarean section:   Cesarean sections typically performed mid-  
Uterine implantation data  gestation; corpora lutea counts, number of 
implantation sites, live and dead embryos. 
__________________ 
a 
Available data from repeated-dose toxicity studies and genotoxicity studies should be used to justify 
dosing duration, especially for detecting effects on spermatogenesis. A premating treatment interval of 2 
weeks for females and 2 weeks for males can be used provided no effects have been found in repeated-dose 
toxicity studies of at least 2 weeks duration that preclude this. Treatment of males should continue 
throughout confirmation of mating, although termination following confirmation of female fertility can be 
valuable.  Treatment of females should continue through at least implantation. This will permit evaluation 
of functional effects on fertility that cannot be detected by histopathological examination in repeated-dose 
toxicity studies and effects on mating behavior.  
27  Contains Nonbinding Recommendations 
 
 
b Most rats or mice will mate within the first 5 days of cohabitation (i.e., at the first available estrus), but in 
some cases, females can become pseudopregnant. Leaving the female with the male for longer than 2 weeks 
can allow these females to restart estrous cycles and become pregnant. 
c It can be of value to delay euthanasia of the males until the outcome of mating is known. In the event of 
an effect on fertility, males could be mated with untreated females to ascertain any potential male-mediation 
of the effect. A more complete evaluation of toxicity to the male reproductive system can be achieved if 
dosing is continued beyond mating and euthanasia delayed so that the males are exposed for the total 
duration of a spermatogenic cycle (e.g., 10 weeks).  
d Sperm analysis (e.g., sperm counts, motility, and/or morphology) sometimes can be useful if issues arise 
to support risk assessment. 
e Termination of females around days 13 to 15 of pregnancy in general is adequate to assess effects on 
fertility and reproductive function (e.g., to differentiate between live implantations and resorption sites). 
There is an option to terminate females near the end of gestation. 
 
1.1.2  Embryo-Fetal Development (EFD) Toxicity Study  
 
The EFD toxicity study is designed to assess maternal toxicity relative to that in 
nonpregnant females, and to evaluate potential effects on embryo-fetal survival, 
intrauterine growth, and morphological development. 
 
Suggested study designs for rodents, rabbits, and cynomolgus monkeys are described 
below.  
 
1.1.2.1 Study dose-range-finding embryo-fetal developmental (EFD) 
toxicity study  
 
Dose-range-finding studies in mated females are most often used to select appropriate 
dose levels, or dose schedules, for the definitive rodent and rabbit EFD studies. 
Tolerability and TK data from existing repeated-dose toxicity studies can, however, be 
sufficient for this purpose. 
 
1.1.2.2 Preliminary embryo-fetal development (pEFD) toxicity study  
 
The pEFD toxicity study (Table 3) is similar in design to the definitive EFD toxicity 
study. A typical pEFD toxicity study design includes dosing over the period of 
organogenesis, has adequate dose levels, evaluates a minimum of 6 pregnant females per 
group, and includes assessments of fetal survival, fetal weight, external fetal 
abnormalities and soft tissue abnormalities (see ICH M3 (ref. 1)). 
 
1.1.2.3 Definitive embryo-fetal developmental (EFD) toxicity study  
 
The females are submitted to cesarean section near term. Assessments of fetal survival, 
fetal weight, external fetal abnormalities, soft tissue abnormalities and skeletal 
examinations are performed (Table 3). The timing given in Table 3 is for rodent, rabbit, 
and cynomolgus monkeys; for other species, appropriate timing should be used.
28  Contains Nonbinding Recommendations 
 
 
Table 3: EFD Toxicity Study Designs for Rodent, Rabbit and Nonhuman Primate 
 
  pEFD                                   EFD   
Parameter  Rodent/Rabbit  Rat (Mouse)  Rabbit  Nonhuman Primate 
(NHP)a 
GLP Status  Optionalc  Yes  Yes  Yes 
Minimum number of 
6   16  16  16b 
pregnant females 
4 (including 1  At least 2 (including 1 
Number of dose groups  4 (including 1 control)  4 (including 1 control) 
control)  control) 
Approximately GD20 
Administration periodd  Species appropriate  GD6/7-17 (6/7-15)  GD6/7-19 
to at least GD50 
Antemortem endpoints         
Clinical 
At least once daily  At least once daily  At least once daily  At least once daily 
observations/mortality 
At least twice 
Body weight  At least twice weekly  At least twice weeklye  At least once weekly 
weeklye 
Food consumption  At least once weekly  At least once weekly  At least once weekly  Optional 
Toxicokinetics   Optionalc  Yes  Yes  Yes 
 
Postmortem endpoints       
Cesarean section f  Species appropriate  GD20/21 (17/18)  GD28/29  GD100 
Macroscopic examination   Yes   Yes   Yes   Optional 
Gravid uterine weight  Optional   Optional   Optional   NA 
Corpora lutea  Yes   Yes   Yes   NA 
Implant sites  Yes  Yes  Yes  NA 
Live and dead conceptuses  Yes  Yes  Yes  Yes 
Early and late resorptions  Yes  Yes  Yes  NA 
Gross evaluation of  Yes  Yes  Yes  Yes 
placenta 
Weight of placenta  Optional  Optional  Optional  Optional 
Fetal body weight  Yes  Yes  Yes  Yes 
Fetal sex  Yes  Yes  Yes  Yes 
29  Contains Nonbinding Recommendations 
 
 
Fetal external evaluationsg  Yes  Yes  Yes  Yes 
Fetal soft tissue 
Yes  Yesg  Yes  Yes 
evaluationsg 
Fetal skeletal evaluationsh  Optionalc  Yesg  Yes  Yes 
__________________ 
a If a NHP other than the Cynomolgus monkey is used, the study design should be adapted. 
b Group sizes in EFD studies should yield a sufficient number of fetuses in order to assess potential adverse effects on morphological development. 
c If the pEFD is used to defer a definitive EFD study, then the pEFD should be done in accordance with GLP regulations, TK data in pregnant animals should be 
collected, and skeletal evaluations should be performed. 
d For rodents and rabbits, females are dosed with the test substance from implantation to closure of the hard palate (i.e., stage C of the reproductive process, see 
section 1.1 of this annex).  For NHP, females are dosed from confirmation of pregnancy (approximately GD20) to at least Day 50 (end of major organogenesis). 
e Daily weighing of pregnant females during treatment can provide useful information.  
f For rodents and rabbits, cesarean sections should be conducted approximately one day prior to expected parturition.  Preserve organs with macroscopic findings 
for possible histological evaluation; keep corresponding organs of sufficient controls for comparison. For NHP, cesarean sections should be conducted on 
approximately GD100. 
g All fetuses should be examined for viability and abnormalities. To permit subsequent assessment of the relationship between observations made by different 
techniques, fetuses should be individually identified. 
h Although it is preferable to examine all rodent fetuses for both soft tissue and skeletal alterations (if methods allow), it is acceptable to submit 50% of fetuses in 
each litter to separate examinations. 
30  Contains Nonbinding Recommendations 
 
 
1.1.3  Pre- and Postnatal Developmental (PPND) Toxicity Study  
 
The PPND toxicity study is designed to assess enhanced toxicity relative to that in nonpregnant 
females, pre- and postnatal viability of offspring, altered growth and development, and 
functional deficits in offspring, including sexual maturation, reproductive capacity at maturity, 
sensory functions, motor activity, and learning and memory. 
 
The females are permitted to deliver and rear their offspring to weaning at which time at least 
one male and one female offspring per litter are selected for rearing to adulthood and mating to 
assess reproductive competence (see Table 4). 
 
Table 4: PPND Toxicity Study Design: Rats 
     
Parameter     
Group size  At least 16 litters    
Number of dose groups  4 (including 1 control)   
Administration period  From implantation (GD6/7) through weaning   
(postnatal day (PND) 20) 
     
F0 Females     
Clinical observations/  At least once daily   
mortality 
Body weight  At least twice weekly    
Food consumption  At least once weekly until mid-lactation   
Parturition observations  GD21 until complete   
Necropsy  PND 21   
At necropsy, preserve and retain tissues with   
macroscopic findings and corresponding control 
tissues for possible histological evaluation, 
count uterine implantation sites 
F1 Preweaning     
Clinical observations/  Daily from PND 0   
mortality 
Preweaning and postweaning  Daily from PND 0   
survival 
Body weight and sex   PND 0/1 and then at least twice per week   
Optional Standardization of    PND 4, to 4 or 5 pups per sex    
   litter size 
Physical developmenta  Preweaning landmarks of development and reflex   
ontogeny (e.g. eye opening, pinna unfolding, 
surface righting, auditory startle, air righting, 
and response to light) 
     
 
 
F1 Postweaning   
31  Contains Nonbinding Recommendations 
 
 
Selection for postweaning      PND 21, at least 1 male and 1 female/litter where 
  evaluation and group sizeb  possible to achieve 16 animals per group/sex 
Clinical observations/  Daily 
mortality 
Body weight  Weekly 
Optional Food consumption  Weekly 
Sexual maturationc  Females: vaginal opening 
Males: preputial separation 
Other functional testsd  Assess sensory functions, motor activity, and learning 
and memory 
Reproductive performance  At least 10 weeks old, paired for mating (1M:1F) 
within the same group (not siblings) 
__________________ 
a The best indicator of physical development is body weight; however, measurement of body weight alone is not an 
acceptable substitute for the evaluation of other developmental parameters.    
b At least one animal per sex per litter should be retained to conduct behavioral and other functional tests, and to 
assess reproductive function.  There can be circumstances where more animals per litter can be retained for 
independent functional assessments.   
c Body weight should be recorded at the time of attainment to determine whether any differences from control are 
specific or related to general growth.   
d Learning and memory should be evaluated in a complex learning task. Assessments of locomotor activity and 
startle reflex with prepulse inhibition (if conducted) should be evaluated over a sufficient period of time to 
demonstrate habituation.   
 
 
1.1.3.1 Enhanced pre- and postnatal developmental (ePPND) toxicity study in the 
nonhuman primate  
 
The ePPND toxicity study (Table 5) is a study in NHP that combines the endpoints from both the 
EFD and PPND studies. In this study, dosing is extended throughout the gestation period to 
parturition (e.g., GD20 to parturition). See ICH S6 (ref. 2) for information on timing and 
additional parameters to be evaluated.  
32  Contains Nonbinding Recommendations 
 
 
Table 5: ePPND Toxicity Study Design: for Cynomolgus Monkeya 
     
Parameter 
Group sizeb  Approximately 16 pregnant females    
Number of dose groups  At least 2 (including 1 control)    
Administration period  From confirmation of pregnancy (approximately   
GD20) to parturition 
     
F0 Females     
Clinical observations/  At least once daily   
mortality 
Body weight  At least weekly   
Parturition observations  Document day of completion   
Placenta  Collect and preserve if possible   
Necropsy and tissue  Only as warranted   
evaluation   
Exposure Assessment  TK profiles and/or systemic drug levels should be 
measured, as appropriate 
     
F1  
Clinical observations/  Daily from PND 0   
mortality 
Body weights  Weekly   
Morphometry/physical and/or  At regular intervals, as appropriate    
functional assessment 
Neurobehavioral test battery At least 1 interval during the first 2 weeks   
    postpartum 
Grip strength    PND 28 
Mother-infant interaction  Minimally in early postnatal period to confirm 
  nursing; as appropriate thereafter  
Exposure assessment  Systemic drug levels should be measured, as 
appropriate 
External evaluation  At regular intervals   
Skeletal evaluation  Approximately PND 28 or later   
Visceral evaluation  At necropsy   
Necropsy  At minimum 1 month, depends on aim of the   
evaluations 
Preserve and retain tissues for possible 
histological evaluation   
__________________ 
a If an NHP other than the Cynomolgus monkey is used, the study design should be adapted. 
b Group sizes in ePPND studies should yield a sufficient number of infants in order to assess potential adverse 
effects on pregnancy outcome, as well as dysmorphology and postnatal development, providing the opportunity for 
specialist evaluation if warranted (e.g., immune system). Most ePPND studies accrue pregnant animals over several 
months.  
 
33  Contains Nonbinding Recommendations 
 
 
1.1.4  Combination Studies  
 
The possibility also exists to combine study types to meet the goals of the development program.  
This is accomplished by incorporating appropriate endpoints measured in the separate studies 
summarized above into a single study.  Concepts for various combination studies are provided 
below. 
 
1.1.4.1 FEED and EFD  
 
The aim of the combined FEED/EFD study is to test for toxic effects resulting from treatment 
from before mating (males/females) through mating, implantation and until the end of 
organogenesis. This comprises evaluation of stages A through D of the reproductive process (see 
section 1.1 of this annex). This study design is most often used with rodents, although it could be 
used with nonrodents. 
 
A combined male/female FEED/EFD can be used, but a separate female-only option is possible 
where male fertility is assessed in a separate study such as a repeated dose study of suitable 
duration. The study would then use untreated males for mating purposes only. For specific study 
design and observational parameters, see sections 1.1.1 and 1.1.2 of this annex. 
 
 1.1.4.2 Male fertility and repeated-dose toxicology study  
 
It is also possible to evaluate male fertility during a rodent repeated-dose toxicity study.  In this 
combination study, males that have been dosed for a defined number of weeks are paired with 
untreated females.  Following cohabitation, the males continue to be dosed until the scheduled 
termination of the repeated-dose toxicity study. The untreated females are subjected to cesarean 
section approximately two weeks after evidence of mating.  The study endpoints collected are 
identical to those outlined in section 1.1.1 of this annex. To adequately assess effects, at least 16 
males per group should be included in the study. Female fertility and other FEED endpoints 
would need to be evaluated in a separate study. 
 
34  Contains Nonbinding Recommendations 
 
 
ANNEX 2   ALTERNATIVE ASSAYS 
 
Data generated from qualified alternative assays (see glossary) conducted alone or in conjunction 
with one or more in vivo studies can be utilized to support hazard identification and risk 
assessment under limited circumstances.   
 
Potential uses can include: 
  
  Circumstances where there is evidence suggesting an adverse effect on EFD (e.g., a 
mechanism of action affecting fundamental pathways in developmental biology, 
phenotypic data from genetically modified animals, class effects) (see section 1.2.2 and 
Figure 1 of this annex) 
 
  Toxicity in animal species precludes attaining systemic exposures relevant to the human 
exposures under conditions of use 
 
  As support for a weight of evidence assessment when there are equivocal findings in 
animal studies 
 
  As partial support for clinical trials including up to 150 WOCBP for up to 3 months 
duration (see section IV.B.3 (4.2.3) of guidance) 
 
  Pharmaceuticals being developed for certain severely debilitating or life-threatening 
diseases or late-life-onset diseases (see section 1.2.3, section 1.2.4, and Figure 2 of this 
annex). 
 
When alternative assays are used to support risk assessment, incorporation of these assays into 
an integrated testing strategy should be justified. Assay(s) used for risk assessment should be 
conducted in accordance with GLP and qualified for context of use (i.e., applicability domain 
and regulatory conditions under which assay results are reliable). Strategies incorporating 
alternative assays should also assess the effects of drug metabolites when warranted (ICH M3). 
This annex does not recommend specific assays; instead, basic scientific principles are included 
to assist in assay qualification for regulatory use. Alternative assays used to explore mechanism 
of action, or otherwise not intended to substitute for in vivo-derived EFD endpoints, are not 
expected to be qualified in this rigorous manner.  
 
1.1  Qualification of Alternative Assays for Prediction of MEFL  
 
Test methods must be appropriate in order for test results to be of value.  Accordingly, the 
endpoints measured should be scientifically justified with respect to assay objectives and 
predictions.  The relationships among the assays predictions, endpoint(s) assessed, and the 
applicability domain, should be supported empirically.  To qualify6 an alternative assay or a 
 
6 Qualified alternative assays within the context of this guidance have not been subject to formal validation as those 
can only be applied under certain specific circumstances.  
35  Contains Nonbinding Recommendations 
 
 
combination of assays for use in risk assessment for regulatory purposes, a comprehensive 
description of the methodology and findings should be provided, including the following: 
 
  A thorough description and justification of the predictive model, including which species 
(e.g., rat, rabbit, and/or human) and endpoint(s) it is predicting. The currently available in 
vitro alternative assays used for evaluating potential hazards to development are designed 
to detect MEFL.  
 
  An evaluation of the biological plausibility of the model, including a description of the 
mechanisms of embryo-fetal development (e.g., cell migration, differentiation, 
vasculogenesis, neurulation, gastrulation) and subsequent developmental adverse effects 
studied with the model. In addition, any limitations of each of the individual assays 
should be discussed. The description should include a discussion and supporting data to 
show that the duration and timing of exposure supports the prediction of MEFL in vivo. 
 
  An assessment of the accuracy and ability for the alternative assay to detect MEFL. The 
performance of the assay is compared to the data generated from in vivo studies with 
compounds that induce MEFL in the absence of confounding maternal toxicity. If the 
compound is not a marketed pharmaceutical, then in vivo data should be provided. 
 
  A discussion determining whether an effect is negative or positive in the assay. 
  
  Definition and justification of the threshold for molecular and metabolic markers 
predicting MEFL. 
  
  The details of the algorithm employed for determining positive and negative outcomes in 
vivo. The predictive model should correlate concentrations tested in the alternative 
assay(s) to the in vivo exposure, preferably in pregnant animals, that results in an adverse 
outcome in the species being predicted. 
  
  The list of compounds in each of the training sets (data used to discover potentially 
predictive relationships) and test sets (data used to assess the strength and utility of a 
predictive relationship) for qualification of the assay and the basis for selection of these 
compounds. 
  
  Data sources (e.g., literature, study reports, regulatory reviews) for all in vivo exposure 
and MEFL data used for compounds in the qualification data set, if not obtained from the 
Reference Compound List (section 1.3 of this annex). 
   
  Data demonstrating the test methods performance covering an appropriate range of 
biological and chemical domains that are justified for the intended use of the alternative 
assay (context of use). 
   
  Data demonstrating the sensitivity, specificity, positive and negative predictive values, 
and reproducibility of an assay or battery of assays to predict in vivo developmental 
36  Contains Nonbinding Recommendations 
 
 
outcomes. The performance of the training and test sets can be evaluated separately 
and/or together, provided the selected approach is justified. 
 
  In cases when more than one assay is conducted, a separate description of the 
performance of each assay, in addition to the integrated assessment used for the 
predictive model. A clear description of how the results of individual assays are 
integrated into the final prediction. 
 
  Historical data for assay development and use (e.g., viability, numbers and types of 
malformations), including positive controls. 
 
The sponsor should state to which health authorities (if any) the assay qualification has been 
previously been submitted. Note that acceptance of an assay by one regulatory authority does not 
bind other health authorities to accept the assay. Evaluation of human teratogens not detected in 
vivo by rat and/or rabbit is encouraged since some alternative assay(s) might predict MEFL that 
are not detectable by in vivo studies. 
  
1.2  Examples of EFD Testing Strategies Utilizing Alternative Assays  
 
This section provides illustrative examples of integrated testing strategies into which alternative 
assays are incorporated to test for adverse effects on EFD. 
 
1.2.1  Potential Approach To Defer In Vivo Testing as Part of an Integrated Testing 
Strategy  
 
See section IV.B.3 (4.2.3) of the guidance. 
 
1.2.2  Pharmaceuticals Expected To Be Embryo-Fetal Toxicants  
 
For pharmaceuticals that are expected to adversely affect embryo-fetal development based on 
mechanism of action, pharmacologic class or target biology, it can be appropriate to confirm this 
activity in a qualified alternative assay(s) (see Figure 1 of this annex).  
 
When a qualified alternative assay clearly predicts MEFL at clinically relevant extrapolated 
exposures, this can be sufficient to identify the compound as an EFD risk, and further testing 
would generally not be warranted. If the alternative assay does not predict MEFL, this should be 
confirmed in definitive in vivo EFD studies in two species. Conducting the studies in series, as 
shown in Figure 1 of this annex, can allow for reduction in animal use, as the second in vivo 
assay would not be warranted if the first one is positive. Under this scenario, since the 
pharmaceutical is expected to adversely affect embryo-fetal development, there is no merit in 
using in vivo EFD studies to attempt to negate a positive alternative assay response. 
 
37  Contains Nonbinding Recommendations 
 
 
Figure 1: Use of Alternative Assays for Pharmaceuticals Expected To Be EFD Toxicants 
 
 
 
__________________ 
1  No additional assessment is warranted if unequivocal MEFL signal is observed at clinically relevant 
extrapolated exposures. 
2  Alternatively, pEFD studies can be used; however, negative results should be confirmed by a definitive study 
in the relevant species. 
3 Conducting in vivo EFD studies in series, as shown, can permit reduction in animal use, as 2nd in vivo assay 
is not warranted if the first study is positive. 
 
 
1.2.3  Pharmaceuticals Intended To Treat Severely Debilitating or Life-Threatening 
Diseases  
 
Considering the risk/benefit for pharmaceuticals intended to treat severely debilitating or life-
threatening conditions (compared to less severe chronic diseases) where the likelihood of 
pregnancy is low, the use of qualified alternative assay(s) can be considered an appropriate 
component of the EFD risk assessment (see Figure 2 in this annex). 
 
38  Contains Nonbinding Recommendations 
 
 
When a qualified alternative assay clearly predicts MEFL in the first species (e.g., rat) at 
clinically relevant extrapolated exposures, this finding can be considered, on a case-by-case 
basis, to sufficiently characterize the EFD risk. However, if the results are equivocal or thought 
to represent a false positive, definitive in vivo studies in one or two species should be conducted 
to assist human risk assessment. If no EFD signal is observed in the two definitive in vivo studies 
at appropriate exposure margins, the results of the alternative assay could be considered of 
minimal concern for human risk.  However, for alternative assays that have been qualified to 
predict human MEFL (i.e., not predicting only animal MEFL), additional data (e.g., mechanistic 
or genetic) should be provided to support a conclusion that the alternative assay results represent 
a false positive finding.  If one or both of the in vivo studies are positive for EFD toxicity, the 
compound is considered to be positive for EFD risk. Conducting the studies in series, as shown 
in Figure 2 of this annex, can allow for reduction in animal use, as the second in vivo assay 
would not be warranted if the first one is positive. 
 
If the alternative assay for the first species predicts a negative outcome (i.e., no MEFL), a 
definitive in vivo EFD study in the second species should be conducted to confirm the 
assessment.  If positive, the compound is considered positive for EFD risk.  If negative, the 
compound is considered negative for EFD risk, and no further testing is generally warranted, 
unless it is judged that additional studies would significantly alter the risk assessment.   
 
1.2.4  Pharmaceuticals Intended to Treat Late-Life-Onset Diseases  
 
Some diseases are typically only diagnosed at a later age but may nonetheless be diagnosed in 
reproductively capable women at a low incidence (e.g., bullous pemphigoid, which is typically 
diagnosed after age 60).  Given the generally low rate of fertility in the female population with 
such late-life-onset diseases, there is a diminished likelihood that a pharmaceutical used 
exclusively in this population will lead to an increase in the incidence of birth defects.  Whether 
an EFD assessment is warranted under this scenario should be determined on a case-by-case 
basis.  This scenario is not intended for situations where the treatment population is 
presumptively infertile (e.g., postmenopausal osteoporosis), for which no EFD assessment would 
typically be warranted.  
 
The testing strategy under this scenario is similar to that depicted for severely debilitating or life-
threatening diseases, with the exception that the first in vivo assessment in the second species 
can be conducted as a pEFD study. 
 
39  Contains Nonbinding Recommendations 
 
 
Figure 2: Use of Alternative Assays for Severely Debilitating or Life-threatening or Late-
Life-Onset Diseases 
 
 
__________________ 
1 A clearly positive MEFL signal at clinically relevant extrapolated exposures can be sufficient to consider a 
pharmaceutical positive for EFD toxicity, without further assessment, on a case-by-case basis.  
2 While pEFD studies can be used, negative results from definitive in vivo EFD studies in two species are warranted 
to establish that alternative assay results represent a false positive. 
3 For late-life-onset diseases, given low likelihood of pregnancy in this patient population, a pEFD study in the 2nd 
species can generally be sufficient. 
4 Conducting in vivo EFD studies in series, as shown, can permit reduction in animal use, as 2nd in vivo assay is not 
to be conducted if the first is positive. 
5 Same species as the alternative assay is intended to predict. 
 
 
1.3  Reference Compound List  
 
The Reference Compound List contains 29 compounds that have been shown to induce MEFL in 
nonclinical studies (in the absence of overt maternal toxicity) and/or humans (Table 1 of this 
annex).  
 
Only findings of MEFL were recognized for NOAEL and LOAEL determinations. Doses 
associated with the induction of reversible or minor manifestations of developmental toxicity 
40  Contains Nonbinding Recommendations 
 
 
(e.g., changes in fetal weight, growth suppression, and skeletal variations) were not used for this 
assessment. (see section IX (9) of the guidance). 
  
The general robustness of the studies (e.g., compliance with GLP regulations, the number of 
animals in the study, number of dose levels) was considered when determining which NOAEL 
and LOAEL values to use. When multiple sources were available, the data from a study designed 
in a manner consistent with the design recommended in the ICH S5(R2) guidance (November 
2005) was accepted as the definitive data. When there were multiple robust sources of data that 
did not closely align, the highest NOAEL (to avoid bias towards claiming a low margin) and 
lowest LOAEL (as is routinely done in regulatory assessments) were generally used, even if the 
data were from different studies. 
  
The compounds in this list, as well as others, can be used to support qualification of an 
alternative assay or battery of assays. 
 
Compounds not causing MEFL (negative compounds) should also be used to assess assay 
specificity. Such compounds would lack MEFL regardless of additional effects on embryo/fetus 
such as fetal body weight changes, structural variations, or delayed/reduced ossification. These 
compounds can be negative at all in vivo doses tested, or can be positive (MEFL observed) at 
higher doses/exposures provided the alternative assay within its context of use predicts the 
transition from negative to positive.  That is, the alternative assay should predict a negative result 
at some extrapolated level under the conditions for which the in vivo study yielded a negative 
result (no MEFL). In the Reference Compound List, three compounds are provided as an 
example for negative controls (Cetirizine, Saxagliptin, Vildagliptin). These compounds did not 
induce MEFL in rat and rabbit at an exposure multiple (AUC and Cmax) of >25 fold at the 
MRHD. 
 
Table 1: Reference Compound Positive Control Examples for Qualifying Alternative 
Assays 
Human  Rat MEFL  Rabbit MEFL 
Positive Controls 
Teratogen 
Acitretin  X  X  X 
Aspirin  X  X   
Bosentan    X   
Busulfan  X  X  X 
Carbamazepine  X  X  X 
Cisplatin    X   
Cyclophosphamide  X  X  X 
Cytarabine  X  X   
Dabrafenib    X   
Dasatinib    X   
Fluconazole  X  X  X 
5-Fluorouracil  X  X  X 
Hydroxyurea  X  X  X 
Ibrutinib    X  X 
41  Contains Nonbinding Recommendations 
 
 
Human  Rat MEFL  Rabbit MEFL 
Positive Controls 
Teratogen 
Ibuprofen  X  X   
Imatinib    X   
Isotretinoin (13-cis-retinoic acid)  X  X  X 
Methotrexate  X  X  X 
Pazopanib    X  X 
Phenytoin (Diphenylhydantoin)  X  X  X 
Pomalidomide  presumed  X  X 
Ribavirin    X  X 
Tacrolimus    X  X 
Thalidomide  X  X  X 
Topiramate  X  X  X 
Tretinoin (all-trans-retinoic acid)  X  X  X 
Trimethadione  X  X   
Valproic acid  X  X  X 
Vismodegib  presumed  X   
 
42  Contains Nonbinding Recommendations 
 
 
 
  1.3.1  Positive Control Reference Compounds  
 
 
ACITRETIN (ETRETIN) 
CAS No.:  55079-83-9 
 
Rat  Rabbit  Rabbit  Human  Margins 
Rat LOAEL  
NOAEL   NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Rat 
Dose  Dose  Dose  Rabbit Findings  C   LOAEL/Human Notes 
max
C   Findings 
max
C   C   C   AUC 
max max max
AUC 
AUC  AUC  AUC 
7.5 mg/kg  15 mg/kg  15 mg/kg:  0.2 mg/kg  0.6 mg/kg  0.6 mg/kg: cleft  50 mg  NOAEL:  Acitretin is the 
oral   oral   malformed oral GD7- oral GD7- palate, open eyelid,  (0.83 mg/kg,   rat  major 
GD7-16   GD7-16   humeri,  19  19  skeletal  29.4 mg/m2)  C  = 1.9  metabolite 
max
(Kistler)  (Kistler)  dilated  (Kistler)  (Kistler)      (1.5/0.79)  (free acid) of 
    renal      2 mg/kg: cleft  Exposure  AUC = 1.8  etretinate 
C  = 1.5  C  = 3.0  pelvis  no PK  no PK  palate, skull and tail  values at  (6.6/3.6)  (ethyl ester) 
max max
g/mLa  g/mLa    data  data  malformations,  steady state:  rabbitc   
AUC = 6.6  AUC = 13.2  30 mg/kg:  available   available  ectrodactyly of the  C  = 0.79  C  = 0.2 
max max
gh/mLa  gh/mLa  cleft  fore- and hindfeet  g/mLb  (0.2/0.83) 
palate;  and malformations  AUC : 3.6 AUC = 0.08 
(0-24h)
malformed  of the long bones  gh/mLb  (2.4/29.4) 
humeri,   
radii and  LOAEL: 
ulnae  rat 
C  = 3.8 
max
(3.0/0.79) 
AUC = 3.7 
(13.2/3.6) 
rabbitc 
C  = 0.7 
max
(0.6/0.83) 
43  Contains Nonbinding Recommendations 
 
 
Rat  Rabbit  Rabbit  Human  Margins 
Rat LOAEL  
NOAEL   NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Rat 
Dose  Dose  Dose  Rabbit Findings  C   LOAEL/Human Notes 
max
C   Findings 
max
C   C   C   AUC 
max max max
AUC 
AUC  AUC  AUC 
AUC = 0.2 
(7.2/29.4) 
a Extrapolated from reported values at 5 mg/kg (Brouwer):  C  = ~1.0 g/mL from visual inspection of graph, AUC = 4.4 gh/mL. 
max
b Steady state values after 21 daily doses administered with food (FDA, United States):  C  = 0.786 g/mL, AUC  = 3.569 gh/mL. 
max (0-24h)
c In the absence of rabbit PK data, C  ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 
max
 
References 
Brouwer KR, McNamara PJ. Influence of pregnancy on the pharmacokinetic disposition of two aromatic retinoids (etretinate and 
acitretin) in the rat. II. Single and multiple oral dosing studies. Drug Metab Dispos. 1989;17:652-655. 
FDA, United States. Approval package review of NDA 019821, part 01 (28 Oct 1996), page 86. 
Kistler A, Hummler H. Teratogenesis and reproductive safety evaluation of the retinoid etretin (Ro 10-1670). Arch Toxicol. 
1985;58:50-56. 
Additional References Evaluated 
FDA, United States. Pharm/tox review of NDA 019821 (08 Jun 1988), page 13. [There were no details provided for study findings, 
study appears to be the same as reported by Kistler and Hummer.] 
 
44  Contains Nonbinding Recommendations 
 
 
ACETYLSALICYLIC ACID (ASPIRIN) 
CAS No.:  50-78-2 
 
Rat  Rat  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
NOAEL   LOAEL  NOAEL   LOAEL  Findings Dose  NOAEL/Human 
Dose  Dose  Dose  Dose  C   LOAEL/Human 
max
C C   C C   AUC 
max  max max  max
AUC  AUC  AUC  AUC 
125 mg/kg  200 mg/kg  Nakatsuka (200  350  Not  None  650 mg (10.8  Aspirin  The aspirin 
oral GD6- oral GD7-17 mg/kg):  mg/kg  Applicable:  mg/kg) q4h  NOAEL:  metabolite, 
17 (n=20  (n=20  malformations  oral  no MEFL  3900 mg daily  rat  salicylate 
Sprague  Sprague  including  GD7-19  findings in  oral (2294  C  =  3.5  (salicylic acid) 
max
Dawley)  Dawley)   craniorachischisis, (n=20  rabbits up  mg/m2 daily)  (25/7.08)  has much higher 
[Gupta]a  [Nakatsuka]e abdominal hernia,  NZW)  to a    AUC = 0.1  0.5  concentrations in 
    exencephaly, club  [Cappon]f maternally  aspirin  (6.6/48.3 to  comparison to the 
aspirin  aspirin   foot, open eyelid,    toxic dose  C = 7.08  25.3/48.3)  parent and is 
max
C  = ~25  C  = ~40  severe defects of  aspirin:  g/mLh  rabbitj  pharmacologically 
max max
g/mLb  g/mLb  vertebral and  aspirin  AUC  =  C  = 32.4  active. Since 
(0-24h) max
AUC = 6.6  AUC = 10.5  costal bones;  PK data  48.3 gh/mLh  (350/10.8)  aspirin 
 25.3   40.5  increased  in rabbits    AUC = 1.8  concentrations 
gh/mLb  gh/mLb  resorptions   is not  salicylic acid   (4200/2294)  were often BLQ, 
      available  C  = 45.2    salicylate 
max
salicylate  salicylate  Gupta (250    g/mLi  LOAEL:  exposure data are 
C  = 132  C  = 211  mg/kg):  salicylate   AUC = 1448  rat  also reported. 
max max
g/mLc  g/mLc  ablepharia,  C  =  gh/mLi  C  = 5.6   
max max
AUC =  AUC =  cranio- 490  (40/7.08)  salicylic acid 
8333  13,333  rachischisis,  g/mLg  AUC = 0.2  0.8  MW = 138.12 
gh/mLd   gh/mLd  exencephaly,  AUC =  (10.5/48.3 to  g/mol 
various low  4865  40.5/48.3)   
occurrence head  gh/mLg  rabbit    aspirin  
malformations,  LOAEL not   MW = 180.16 
bent fore and hind  identified  g/mol 
paw, kinked tail,   
45  Contains Nonbinding Recommendations 
 
 
protruding  Salicylate  
tongue,  NOAEL: 
gastroschisis,  rat 
ectopic adrenal,  C :  2.9 
max
various low  (132/45.2) 
occurrence  AUC:  5.8 
cardio-vascular  (8333/1448) 
malformations,  rabbit 
VSD, DH,  C :  10.8 
max
hypoplastic  (490/45.2) 
kidney,  AUC:  3.4 
hypoplastic testes;  (4865/1448) 
decreased   
implantations,  LOAEL: 
increased  rat 
resorptions and  C :  4.7 
max
post implantation  (211/45.2) 
loss  AUC:  9.2 
(13,333/1448) 
rabbit 
LOAEL not 
identified 
a Nakatsuka and Fujii reported a NOAEL of 100 mg/kg in Sprague Dawley rats; the highest NOAEL of the 2 studies is reported here.  
b Extrapolated or actual reported value at 200 mg/kg oral dose in Sprague Dawley rats (Wientjes):  C  = 40 g/mL (visual inspection of Figure 1); AUC = 629 
max
 2430 gmin/mL (recalculated as 10.5  40.5 gh/mL).  C  data for aspirin is also available in Wistar rats administered 200 mg/kg (Higgs).   
max
c Extrapolated from reported value at 200 mg/kg oral dose in Sprague Dawley rats (Wientjes):  C  = 211 g/mL (Table 5); no AUC values were reported for 
max
salicylate.  C  data for salicylate is also available in Wistar rats administered 200 mg/kg (Higgs) and in Fischer rats administered 90 mg/kg (Kapetanovica).   
max
d Extrapolated from reported value at oral 90 mg/kg/day on D15 in Fischer rats (Kapetanovica):  AUC = 6000 gh/mL.  Note the AUC in Table 2 is reported as 
6.0 gh/mL, but this is incompatible with the plot in Figure 1a.  An AUC estimated from concentrations visually estimated from Figure 1a was 5319 
gh/mL (personal calculation); thus it is assumed that the reported value should actually be 6000 gh/mL. 
e Gupta reported a LOAEL of 250 mg/kg in Sprague Dawley rats; the lowest LOAEL of the 2 studies is reported here.  
46  Contains Nonbinding Recommendations 
 
 
f Data from Cappon is reported since the study design complied with ICH S5 standards.  Data are also available in which 200 mg/kg was reported as the NOAEL 
(McColl, Schardein), but these studies were pre-ICH S5.  McColl reported small auricles in hearts (18% vs 4.5% in controls) and increased presence of 13th 
rib (93% vs 56% in controls) at 200 mg/kg aspirin, but these are considered variations.  Schardein reported marked reduction in litter size at 200 mg/kg/day, 
but this dose was maternally toxic. 
g Extrapolated from reported values on D3 after 50 mg/kg/day oral dose in NZW rabbits (Marangos):  C  = 70 g/mL and AUC = 695 gh/mL.  Note that the 
max
extrapolation is 7-fold and that there are no data available on the linearity of the pharmacokinetics in rabbits. 
h Extrapolated to 6 daily doses every 4 hours from reported values after a single 1000 mg dose (Schurer):  C  = 10.89 g/mL, AUC = 12.38 gh/mL.  The C  
max max
after a single dose likely represents the C  at steady state since the half-life is short (approximately 0.5 hours) and no accumulation is expected using the 
max
equation:  accumulation = 1/(1  e-ktau), where k = 0.693/t12 with t12 = 0.5 hours and tau = 4 hours.  For AUC , the single-dose AUC at 1000 mg was 
(0-24h)
extrapolated to 650 mg and multiplied by 6 (the maximum recommended doses in 24 hours).  Data are also available following administration of 500 mg 
(Nagelschmitz). 
i Extrapolated to 6 daily doses every 4 hours from reported values after a single 1000 mg dose (Schurer):  C  = 53.5 g/mL, AUC = 371.32 gh/mL.  For C , 
max max
an accumulation factor of 1.3 was applied that was estimated from the equation:  accumulation = 1/(1  ektau), where k = 0.693/t12 with t12 = 2.0 hours and 
tau = 4 hours (i.e., 1/(1  e1.386) = 1/(1  0.25) = 1/0.75 = 1.3).  For AUC , the single-dose AUC at 1000 mg was extrapolated to 650 mg and multiplied by 
(0-24h)
6 (the maximum recommended doses in 24 hours).  Data are also available following administration of 500 mg (Nagelschmitz). 
j In the absence of PK data, C  ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 
max
 
References 
Cappon GD, Gupta U, Cook JC, Tassinari MS, Hurtt ME. Comparison of the developmental toxicity of aspirin in rabbits when 
administered throughout organogenesis or during sensitive windows of development. Birth Defects Res B Dev Reprod Toxicol. 
2003;68:38-46. 
Gupta U, Cook JC, Tassinari MS, Hurtt ME. Comparison of developmental toxicology of aspirin (acetylsalicylic acid) in rats using 
selected dosing paradigms. Birth Defects Res B Dev Reprod Toxicol. 2003;68:27-37. 
Kapetanovic IM, Bauer KS, Tessier DM, Lindeblad MO, Zakharov AD, Lubet R, et al. Comparison of pharmacokinetic and 
pharmacodynamic profiles of aspirin following oral gavage and diet dosing in rats. Chem Biol Interact. 2009;179:233-239. 
Marangos MN, Onyeji CO, Nicolau DP, Nightingale CH. Disposition kinetics of aspirin in female New Zealand white rabbits. Lab 
Anim Sci. 1995;45:67-69. 
Nakatsuka T, Fujii T. Comparative teratogenicity study of diflunisal (MK-647) and aspirin in the rat. Oyo Yakuri. 1979;17:551-557. 
Schurer M, Bias-Imhoff U, Schulz HU, Schwantes U, Riechers AM. Lack of influence of glycine on the single dose pharmacokinetics 
of acetylsalicylic acid in man. Int J Clin Pharmacol Ther. 1996;34:282-287. 
47  Contains Nonbinding Recommendations 
 
 
Wientjes MG, Levy G. Nonlinear pharmacokinetics of aspirin in rats. J Pharmacol Exp Ther. 1988;245:809-815. 
Additional References Evaluated  
Higgs GA, Salmon JA, Hendersonn B, Vane JR. Pharmacokinetics of aspirin and salicylate in relation to inhibition of arachidonate 
cyclooxygenase and antiinflammatory activity. Proc Natl Acad Sci. USA. 1986;84:1417-1420. 
McColl JD, Robinson S, Globus M. Effect of some therapeutic agents on the rabbit fetus. Toxicol Appl Pharmacol. 1967;10:244-252. 
Nagelschmitz J, Blunck M, Kraetzschmar J, Ludwig M, Wensing G, Hohlfeld T. Pharmacokinetics and pharmacodynamics of 
acetylsalicylic acid after intravenous and oral administration to healthy volunteers. Clin Pharmacol. 2014;6:51-59. 
Schardein JL, Blatz AT, Woosley ET, Kaump DH. Reproduction studies on sodium meclofenamate in comparison to aspirin and 
phenylbutazone. Toxicol Appl Pharmacol. 1969;15:46-55. 
 
48  Contains Nonbinding Recommendations 
 
 
ALL-TRANS-RETINOIC ACID (ATRA), TRETINOIN 
CAS No.:  302-79-4 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Marginsa  Notes 
NOAEL   LOAEL  Findings 
Dose  Dose  Dose  NOAEL/Human 
Dose 
Dose 
C   C   C   LOAEL/Human 
max max max
C  
C   max
AUC  AUC  max AUC 
AUC 
AUC 
5 mg/kg oral  10 mg/kg oral  cleft palate,   2 mg/kg oral  6 mg/kg  fetal  45  NOAEL:  tretinoin 
GD6-15  GD6-15  sporadic gross  GD6-18   oral GD6- resorptions  mg/m2/day in  induces its 
rat 
(Wistar)  (Wistar)  external and  18  and a  two divided  own 
[Tzimas, 
soft tissue  decrease in  doses  Cmax = 0.4  metabolism, 
[Seegmiller]  [Seegmiller]  1994]  [Tzimas, 
malformations,  live fetuses;  (0.15/0.394)  so PK 
1994]   
    skeletal    visceral  margins are 
AUC = 0.5 
C  = 0.15  C  = 0.30  alterations  C  = 0.10    ectopia, skin Cmax = 0.394  (0.25/0.537)  highly 
gm/amx Lb  gm/amx Lb  gm/amx Lc  C  =  erosions,  g/mLd  dependent 
max  
acaudia,  on day of 
0.30  AUC = 0.537 
AUC  =  AUC  =  AUC -  = 
(0-8h) (0-8h) (0 24h) g/mLc  torsion of  gh/mLd  rabbit  assessment 
0.25  0.50  0.207 
hindlimbs, 
gh/mLb  gh/mLb  gh/mLc  AUC(0-8h)  and  Cmax = 0.3 
= 0.622  (0.100/0.394) 
omphalocele 
gh/mLc 
AUC = 0.4 
(0.207/0.537) 
 
LOAEL: 
rat 
C  = 0.8 
max
(0.30/0.394) 
49  Contains Nonbinding Recommendations 
 
 
AUC = 0.9 
(0.50/0.537) 
 
rabbit 
C  = 0.8 
max
(0.300/0.394) 
AUC = 1.2 
(0.622/0.537) 
a Since tretinoin induces its own metabolism, which causes a significant decrease in plasma exposures with repeated dosing, single-dose PK data in animals and 
humans were used for calculating exposure margins. 
b Extrapolated or actual value after single 5 mg/kg oral dose on GD9 in Wistar rats (Tzimas 1997):  C  = 0.15 g/mL, AUC  = 0.25 gh/mL.  
max (0-8h)
Pharmacokinetic data are also available after a single dose of 6 mg/kg on GD12 (Collins, 1995):  C  = 0.320 g/mL from visual inspection of graph, AUC  
max (0-8h)
= 0.820 gh/mL; as well as after 6 daily doses (Collins 1994, 1995):  C  = 0.046 or 0.052 g/mL, and AUC  = 0.098 gh/mL or AUC  = 0.090 
max (0-24h) (0-10h)
gh/mL, respectively. 
c Extrapolated or actual value after single 6 mg/kg oral dose on GD12 in Swiss hare rabbits (Collins 1995):  C  = 0.300 g/mL from visual inspection of graph, 
max
AUC  =  0.622 gh/mL.  Pharmacokinetic data are also available following 6 daily doses in Swiss hare rabbits (Collins 1995):  C  = 0.110 g/mL, AUC
(0-8h) max (0-
 = 0.281 gh/mL; and from (Tzimas 1994):  C  = 0.105 g/mL, AUC  = 0.321 gh/mL. 
10h) max (0-24h)
d PK data after first dose (U.S. label). 
 
References 
Collins MD, Tzimas G, Burgin H, Hummler H, Nau H. Single versus multiple dose administration of all-trans-retinoic acid during 
organogenesis: differential metabolism and transplacental kinetics in rat and rabbit. Toxicol Appl Pharmacol. 1995;130:9-18. 
Seegmiller RE, Ford WH, Carter MW, Mitala JJ, Powers WJ Jr. A developmental toxicity study of tretinoin administered topically 
and orally to pregnant Wistar rats. J Am Acad Dermatol. 1997;36(3 Pt 2):S60-66. 
Tzimas G, Burgin H, Collins MD, Hummler H, Nau H. The high sensitivity of the rabbit to the teratogenic effects of 13-cis-retinoic 
acid (isotretinoin) is a consequence of prolonged exposure of the embryo to 13-cis-retinoic acid and 13-cis-4-oxo-retinoic acid, and 
not of isomerization to all-trans-retinoic acid. Arch Toxicol. 1994;68:119-128. 
50  Contains Nonbinding Recommendations 
 
 
Tzimas G, Thiel R, Chahoud I, Nau H. The area under the concentration-time curve of all-trans-retinoic acid is the most suitable 
pharmacokinetic correlate to the embryotoxicity of this retinoid in the rat. Toxicol Appl Pharmacol. 1997;143:436-444. 
U.S. label tretinoin. 
Additional References Evaluated 
Collins MD, Tzimas G, Hummler H, Burgin H, Nau H. Comparative teratology and transplacental pharmacokinetics of all-trans-
retinoic acid, 13-cis-retinoic acid, and retinyl palmitate following daily administrations in rats. Toxicol Appl Pharmacol. 
1994;127:132-144. [PK data after 6 daily doses] 
Kochhar DM, Christian MS. Tretinoin: a review of the nonclinical developmental toxicology experience. J Am Acad Dermatol. 
1997;36(3 Pt 2):S47-59. [review article of other papers already cited] 
Tembe EA, Honeywell R, Buss NE, Renwick AG. All-trans-retinoic acid in maternal plasma and teratogenicity in rats and rabbits. 
Toxicol Appl Pharmacol. 1996;141:456-472. [single-dose teratology and PK at 20 mg/kg] 
FDA, United States. Pharmtox review of NDA 021108/S000 (31 Aug 2000), page 16, 26. [p. 16:  same study as Seegmiller; p. 26:  
Review mentions only a modest increase in intrauterine death at 2.5 mg/kg in an oral rat developmental toxicity study, but there are 
no study details to allow confirmation.] 
U.S. label tretinoin. [Fetal resorptions and a decrease in live fetuses were stated as findings in all species studied, but the dose at which 
these occurred was not mentioned.] 
   
51  Contains Nonbinding Recommendations 
 
 
BOSENTAN 
CAS No.:  147536-97-8 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit  Rabbit  Notes 
Dose  Dose  Dose  LOAEL  Findings 
C   C   C   Dose 
max max max
AUC  AUC  AUC  C  
max
AUC 
60 mg/kg oral  300 mg/kg oral  Cesarean sections  1500 mg/kg/day  LOAEL not  none   
GD6-15 (FDA,  GD6-15 (FDA,  300 mg/kg:  agenesis of soft palate (1 oral (750 mg/kg  identified 
United States, p.  United States, p.  litter)  BID) GD7-18 
39, 155)  39, 155)  1500 mg/kg:  agenesis of soft palate  (FDA, United 
    (14 litters), shortened tongues,  States, p. 66) 
C  = 4.5  C  = 16.25  abnormal origin of the right   
max max
g/mLa  g/mLb  subclavian artery (1 litter);  C  = 1.435 
max
AUC = 13.2  AUC = 53.5  abnormalities of the skull (shortened  g/mLd 
gh/mLa  gh/mLb  and misshapen mandibles,  AUC = 27.7 
abnormally shaped palatine,  gh/mLd 
abnormally shaped tympanic annulus 
and hyoid bone, fusion of the 
pterygoid process with the tympanic 
annulus, bent internal pterygoid 
process) 
 
Spontaneous delivery fetuses (PPND 
groups) that died on study:c  
300 mg/kg:  agenesis of the soft 
palate, anophthalmia, and 
microphthalmia 
a Extrapolated from reported values in plasma after 10 doses of 200 mg/kg oral bosentan in pregnant rats (FDA, United States, p. 78):  C  = 15 g/mL, AUC = 
max
44 gh/mL. 
b Interpolated from reported values in plasma after 10 doses of 200 and 600 mg/kg oral bosentan in pregnant rats (FDA, United States, p. 78):  at 200 mg/kg, 
C  = 15 g/mL, AUC = 44 gh/mL; at 600 mg/kg, C  = 20 g/mL, AUC = 82 gh/mL. 
max max
52  Contains Nonbinding Recommendations 
 
 
c In a separate PPND study with higher levels of impurities and pup sacrifice on PND4, agenesis of the soft palate was also observed in 3 litters at 120 mg/kg 
(FDA, United States, p. 58). 
d Actual values in plasma after 12 doses of 1500 mg/kg/day oral bosentan administered as 2 divided doses (750 mg/kg each) 5 to 6 hours apart in pregnant 
Himalayan rabbits (FDA, United States, p. 78):  C  = 1.435 g/mL, AUC= 27.70 gh/mL. 
max  
 
References 
FDA, United States. Pharmacology Review NDA 021290 (30 Aug 2001). 
 
53  Contains Nonbinding Recommendations 
 
 
BUSULFAN 
CAS No.:  55-98-1 
 
Rat  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins   Notes 
NOAEL  Dose  NOAEL  LOAEL  Findings  Dosea  NOAEL/Human 
Dose   C   Dose   Dose  C   LOAEL/Human 
max max
C AUC  C   C   AUC 
max  max max
AUC  AUC  AUC 
NOAEL  3 mg/kg oral  3 mg/kg:  1.3 mg/kg  3.6 mg/kg  increased  4  8 mg daily  NOAEL:  human dose 
not  single dose  fused carpal  oral GD7 oral GD7- resorptions  oral  rat  is daily but 
identified GD12   bones  14  14  and  (0.06  0.13  NOAEL not  MEFL 
(18 mg/m2)    (15.6  (43.2  decreased  mg/kg, 2.4  4.7 identified  NOAEL was 
[Dodo]  10 mg/kg:   mg/m2)  mg/m2)  live young,   mg/m2)  rabbitd  single dose, 
  low incidence  [Somers]  [Somers]  abnormalities   C  = 10  margins 
max
C  = 0.84  of limb and      in liver and  for 8 mg dose  (1.3/0.13)  likely even 
max
g/mLb  rib  no rabbit  no rabbit  gall bladder  C  = 0.128  AUC = 3.3  lower if rats 
max
AUC = 2.70  malformations PK data  PK data  g/mLc  (15.6/4.7)  dosed through 
gh/mLb    found   found  AUC = 0.529    organogenesis 
30 mg/kg:   gh/mLc  LOAEL: 
high  rat 
incidence of  C  = 6.6 
max
limb and rib  (0.84/0.128) 
malformations  AUC = 5.1 
(2.7/0.529) 
rabbitd 
C  = 27.7 
max
(3.6/0.13) 
AUC = 9.2 
(43.2/4.7) 
a Note that Busulfex is a concentrated busulfan intravenous formulation with dimethylformamide indicated for bone marrow ablation.  Myleran is the original 
busulfan oral drug product indicated for treatment of chronic myelogenous leukemia.  The doses used below are for remission induction in chronic 
myelogenous leukemia. 
54  Contains Nonbinding Recommendations 
 
 
b Extrapolated from reported values after 1 mg/kg busulfan oral dose to fasted rats (strain not specified) (FDA, United States):  C  = 0.28 g/mL, AUC = 0.9 
max
gh/mL. 
c Extrapolated from the average of dose-normalized (to 2 mg) values across the range 2 to 6 mg (C  = 0.03 g/mL, AUC = 0.130 gh/mL) and dose-
max
normalized values (to 4 mg) from 4 and 8 mg in a separate study (C  = 0.068 g/mL, AUC = 0.269 gh/mL) (US label, Ehrsson). 
max
d In the absence of rabbit PK data, C  ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 
max
 
References 
Dodo T, Uchida K, Hirose T, Fukuta T, Kojima C, Shiraishi I, et al. Increases in discontinuous rib cartilage and fused carpal bone in 
rat fetuses exposed to the teratogens, busulfan, acetazolamide, vitamin A, and ketoconazole. Hum Exp Toxicol. 2010;29:439-450. 
Ehrsson H, Hassan M, Ehrnebo M, Beran M. Busulfan kinetics. Clin Pharmacol Ther. 1983;34:86-89. 
FDA, United States.  Pharmtox review NDA 020954 (04Feb1999), page 11.  
Somers GF. The evaluation of drugs for foetal toxicity and teratogenicity in the rabbit. Excerpta Medica International Congress. 
1969;181:227-234. [Proc Eur Soc Study Drug Toxic. 1969;10:227-34] 
Additional References Evaluated 
Bishop JB and Wassom JS. Toxicological review of busulfan (Myleran). Mutat Res. 1986;168:15-45. 
 
55  Contains Nonbinding Recommendations 
 
 
CARBAMAZEPINE 
CAS No.:  298-46-4 
 
Rat  Rat  Rabbit  Rabbit  Human  Margins 
NOAEL   LOAEL   NOAEL   LOAEL  Dose  NOAEL/Human 
Rabbit 
Dose  Dose  Rat Findings  Dose  Dose  C   LOAEL/Human Notes 
max
Findings 
C   C   C   C   AUC 
max max max max
AUC  AUC  AUC  AUC 
200 mg/kg  400 mg/kg  400 mg/kg GD4- NOAEL  225 mg/kg  No  Up to 800  NOAEL:  Human 
oral GD7-18 oral GD4-14 14 [FDA, United  was not  GD5-12  malformations  mg twice  Rat  exposure is 
[Vorhees]a  [FDA,  States 1967]  identified  [FDA, United up to 450  daily (1600  C  = 2.8  invariant, 
max
  United  abortions  [FDA,  States 1967]  mg/kg GD5- mg/day)  (33/11.7)  independent 
  States 1967,    United    12    AUC = 2.4  of dose. 
C  = 33  Vorhees]  600 mg/kg GD7- States  C  = 29    C  = 11.7  (547/232) 
max max max
g/mLb     18 SD rats  1967]  g/mLc  Decreased  g/mLd   
AUC  = C  = 65  [Vorhees]    AUC  =  numbers of  AUC  =  Rabbit  
(0-24 h) max (0-24h) (0-24h)
547  g/mLb  increased  267 gh/mLc fetuses,  232  No NOAEL 
gh/mLb  AUC  =  resorptions,  increased  gh/mLd  identified 
(0-24h)
1094  increased kinked  resorptions at     
gh/mLb  tails   225  450  LOAEL: 
  mg/kg  Rat 
650 mg/kg [U.S.  C  = 5.6 
max
label]  (65/11.7) 
offspring showed  AUC = 4.7 
low incidence of  (1094/232) 
cleft palate,   
talipes, or  Rabbit  
anophthalmos  C  = 2.5 
max
(29/11.7) 
AUC = 1.2 
(267/232) 
a Data from Vorhees was used for establishing the NOAEL because the data were much more detailed than provided in the FDA, United States review, which 
suggested a NOAEL of 300 mg/kg. 
56  Contains Nonbinding Recommendations 
 
 
b Extrapolated or actual data after 200 mg/kg oral single dose in Sprague Dawley male rats (Shi):  C  = 32.7 g/mL, AUC  = 32.8 mgmin/mL (547 
max (0-24h)
gh/mL).   
c Extrapolated from reported value after 80 mg/kg oral single dose in Angora grey rabbits (Kourmaravelou):  C  = 10.4 g/mL, AUC  = 94.8 gh/mL.  
max (0-24h)
Data are also available from Abushammala at a dose of ~20.6 mg/kg.  The data from Kourmaravelou were used because the dose was closer to the LOAEL, 
which provided a smaller extrapolation range (<3-fold). 
d From actual data for 1600 mg dose of conventional tablet carbamazepine (FDA, United States 1996).  C  = 11.66 g/mL, AUC = 232.27 gh/mL. 
max
 
References 
FDA, United States. Pharmtox review of Tegretol NDA 016608 Part 02 (19 December 1967), page 5. 
FDA, United States. Approval package of Carbatrol NDA 020712 Part 02 (23 December 1996), page 33. 
Koumaravelou K, Adithan C, Shashindran CH, Asad M, Abraham BK. Effect of honey on carbamazepine kinetics in rabbits. Indian J 
Exp Biol. 2002;40:560-563. 
Shi L, Dang XL, Liu XY, Wei HM, Yang MM, Zhang Y. Effect of Sophora flavescens on the pharmacokinetics of carbamazepine in 
rats. Arch Pharm Res. 2014;37:1617-1623. 
US Label Tegretol. 
Vorhees CV, Acuff KD, Weisenburger WP, Minck DR. Teratogenicity of carbamazepine in rats. Teratology. 1990;41:311-317. 
Additional References Evaluated 
Abushammala I. The effect of pioglitazone on pharmacokinetics of carbamazepine in healthy rabbits. Saudi Pharm J. 2015;23:177-
181.  
El-Sayed MG, Aly AE, Kadri M, Moustafa AM. Comparative study on the teratogenicity of some antiepileptics in the rat. East Afr 
Med J. 1983;60:407-415. 
Tolbert D, Cloyd J, Biton V, Bekersky I, Walzer M, Wesche D, et al. Bioequivalence of oral and intravenous carbamazepine 
formulations in adult patients with epilepsy. Epilepsia. 2015;56:915-923. (PK data for oral carbamazepine was similar to cited data, 
AUC is invariant across dose levels.)   
57  Contains Nonbinding Recommendations 
 
 
CISPLATIN 
CAS No.:  15663-27-1 
 
Rat NOAEL   Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit LOAEL  Rabbit Findings  Notes 
Dose  Dose  Dose  Dose 
C   C   C   C  
max max max max
AUC  AUC  AUC  AUC 
0.3 mg/kg IP on GD6,  1 mg/kg IP on  increased fetal  NOAEL not  LOAEL not  No data found   
8,11, or 14 in Wistar  GD8 or 11 in  mortality,  identified  identified 
rats (Keller)  Wistar rats  decreased live 
  (Keller)  fetuses per dam 
C  = 0.32 g/mLa   
max
AUC = 0.25 gh/mLa  C  = 1.08 
max
g/mLa 
AUC = 0.85 
gh/mLa 
a Extrapolated from values in plasma (unbound) after an intraperitoneal 5 mg/kg cisplatin single dose in male Donryu rats (Tamura):  C  = 5.4 g/mL, AUC
max (0-
 = 254 gmin/mL (4.23 gh/mL). 
inf)
 
References 
Keller KA, Aggarwal SK. Embryotoxicity of cisplatin in rats and mice. Toxicol Appl Pharmacol. 1983;69:245-256.   
Tamura T, Imai J, Matsukawa Y, Horikiri Y, Suzuki T, Yoshino H, et al. Pharmacokinetic behaviour of cisplatin in peritoneal fluid 
after intraperitoneal  administration of cisplatin-loaded microspheres. J Pharm Pharmacol. 2001;53:1331-1339.  
Additional References Evaluated 
Chen Y, Brott D, Luo W, Gangl E, Kamendi H, Barthlow H, et al. Assessment of cisplatin-induced kidney injury using an integrated 
rodent platform. Toxicol Appl Pharmacol. 2013;268:352-361. 
Darwish MA, Abo-Youssef AM, Khalaf MM, Abo-Saif AA, Saleh IG, Abdelghany TM. Resveratrol influences platinum 
pharmacokinetics: A novel mechanism in protection against cisplatin-induced nephrotoxicity. Toxicol Lett. 2018;290:73-82.  
Okada A, Fukushima K, Fujita M, Nakanishi M, Hamori M, Nishimura A, et al. Alterations in cisplatin pharmacokinetics and its 
acute/sub-chronic kidney injury over multiple cycles of cisplatin treatment in rats. Biol Pharm Bull. 2017;40:1948-1955.  
58  Contains Nonbinding Recommendations 
 
 
Sekiya S, Iwasawa H, Takamizawa H. Comparison of the intraperitoneal and intravenous routes of cisplatin administration in an 
advanced ovarian cancer model of the rat. Am J Obstet Gynecol. 1985;153:106-111.  [No C  or AUC values were reported.  
max
Substantial differences in PK were noted between the intravenous and intraperitoneal routes.] 
Toro-Cordova A, Flores-Cruz M, Santoyo-Salazar J, Carrillo-Nava E, Jurado R, Figueroa-Rodriguez PA, et al. Liposomes loaded with 
cisplatin and magnetic nanoparticles: physicochemical characterization, pharmacokinetics, and in vitro efficacy. Molecules. 
2018;23(9). pii: E2272. doi: 10.3390/molecules23092272.  [PK following 6 mg/kg intravenous cisplatin:  C  = 21.3 g/mL, AUC  
max (0-t)
= 7.49 gh/mLkg, which is 2.25 gh/mL in 300 g rats.] 
   
59  Contains Nonbinding Recommendations 
 
 
Summary of Cisplatin PK data evaluated 
Note:  There was no obvious choice for the best PK data to use.  Chen required a 15-fold extrapolation, Darwish was unclear whether 
the data were total Pt or unbound drug, and Tamura used a different strain of rat (Donryu) than used for the EFD toxicity study 
(Wistar).  There are substantial differences in PK between the intravenous and intraperitoneal routes (Sekiya, et al., 1985), so 
intravenous data were not used. 
Reference  Route  Dose  C  (g/mL)  AUC (gh/mL)  Notes 
max
(mg/kg)  Reported  Normalized  Reported  Normalized 
to 1.0 mg/kg   to 1.0 mg/kg  
Chen  IP  15  10.36  0.69  81.74 (0-inf)  5.45  unbound drug 
(diethyldithiocarbamate 
(DDTC)-derivatized) 
Darwish  IP  6  5.66  0.94  9.77  1.63  unclear whether unbound or 
total drug 
Tamuraa  IP  5  5.4  1.08  4.23   0.85  unbound drug (ultrafilterable) 
Okada  IV  5  7.3  1.5  3.0 (0-2h)  0.6  unbound drug (DDTC-
derivatized) 
Toro- IV  6  21.3  3.55  2.25b (0-t)  0.375  unbound (ultrafilterable, DDTC-
Cordova  derivatized) 
All studies used male Wistar rats except for Tamura et al., which used male Donryu rats. 
a PK parameters were derived from Figure 4 using scanning software (CurveUnscan).  
b Reported as AUC  = 7.49 gh/mLkg, which is 2.25 gh/mL in 300 g rats. 
(0-t)
 
   
60  Contains Nonbinding Recommendations 
 
 
CYCLOPHOSPHAMIDE 
CAS No.:  50-18-0 
 
Rat  Rat  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins   Notes 
NOAEL  LOAEL  NOAEL  LOAEL  Findings  Dose  NOAEL/Human 
Dose  Dose  Dose  Dose  C   LOAEL/Human 
max
C   C   C   C   AUC 
max max max max
AUC  AUC  AUC  AUC 
NOAEL  2.5 mg/kg  2.5 mg/kg   NOAEL  30 mg/kg IV  embryo-fetal  1600 mg/m2 (40  NOAEL:   MW CP = 
not  IP GD9  GD9 [Chaube] not  single doses  resorptions,  mg/kg) IV  rat:  261.086 
identified [Chaube]  embryo lethal  identified  on GD6-14  omphalocele, (highest dose, q  NOAEL not   MW PM = 
(<2.5      (<30  [Mirkes,  cleft lip/  3  4 weeks)f  identified, but  221.018 
mg/kg)  Cytoxan  5 mg/kg  mg/kg)  Fritz]  palate,    LOAEL margins   Cytoxan is a 
[Chaube] Cmax = 4.1  GD11 [von      forelimb  Cytoxan  were <0.1  prodrug, 
g/mLa   Kreybig,  Cytoxan  skeletal  Cmax = 106  rabbit  MEFL has 
AUC = 3.65  Mirkes]  Cmax = 151  defects   g/mLg  NOAEL not  been attributed 
gh/mLa  encephalocele,  g/mLc  AUC = 798  identified, but  to both 
  exencephaly,  AUC(0-8h) =  gh/mLg  LOAEL margins  phosphoramide 
PM  microcephaly,  24.1    were <1.5  mustard (PM) 
Cmax = 0.55  limb defects  gh/mLd  PM    and acrolein 
g/mLb  (i.e.,  PM  Cmax = 14.4  LOAEL:  metabolites 
AUC  =  syndactyly  C  = 0.07  g/mLh  rat 
(0-24h) max
2.13  and  g/mLe  AUC = 352  C :  0.04 
max
gh/mLb  ectrodactyly),  AUC  =  gh/mLh  (4.1/106) 
(0-8h)
defective  0.297   AUC:  0.005 
facial  gh/mLe  (3.65/798) 
development  rabbit 
(cleft palate)  C  =  1.4 
max
(151/106) 
AUC =  0.03 
(24.1/798) 
 
PM margins 
61  Contains Nonbinding Recommendations 
 
 
rat 
C  0.04 
max =
(0.55/14.4) 
AUC = 0.006 
(2.13/352) 
rabbit 
C  = 0.005 
max
(0.07/14.4) 
AUC = 0.0008 
(0.297/352) 
a Extrapolated from reported value after 20 mg/kg intravenous single dose in Sprague Dawley rats (Hong):  C  = 125.3 M (32.7 g/mL), AUC/D = 265.3 min/L 
0
(in rats with mean BW = 0.330 kg the administered dose = 6.6 mg/rat; thus AUC = 265.3 min/L x 6.6 mg = 1751 mgmin/L = 29.2 gh/mL). 
b Extrapolated from reported value after 20 mg/kg intravenous single dose in Sprague Dawley rats (Hong):  C  = 20 M (4.4 g/mL) from visual inspection of 
max
graph, AUC  = 76.9 Mh (17.0 gh/mL) from calculation based on concentration values estimated by visual inspection of graph. 
(0-24h)
c Extrapolated from reported value after 45 mg/kg cytoxan intravenous single dose in 2 New Zealand White rabbits (Holm):  C  = 227 g/mL from visual 
max
inspection of graphs (mean of 2 rabbits).  Values for the R and S isomers were added together; parent cytoxan is a racemic mixture.  Data are also available 
after a 20 mg/kg intravenous single dose in New Zealand White rabbits (Anthony), but the reported C  value (2.2 M [0.574 g/mL] from visual inspection 
max
of graph) is inconsistent with the reported AUC and thus was not used.   
d Extrapolated from reported value after 20 mg/kg cytoxan intravenous single dose in New Zealand White rabbits (Anthony):  AUC  = 3683 molmin/L 
(0-8h)
(16.0 gh/mL).  Data are also available after a 45 mg/kg intravenous single dose in 2 New Zealand White rabbits (Holm), but the reported AUC values for 
total racemate (3189 and 1259 gmin/mL [53.15 and 20.98 gh/mL]) in 2 rabbits differed by 2.5-fold and t  was 90 minutes so these values were not 
last
used.   
e Extrapolated from reported value after 20 mg/kg cytoxan intravenous single dose in NZW rabbits (Anthony):  C  = 0.22 M (0.049 g/mL) from visual 
max
inspection of graph, AUC  = 53.7 molmin/L (0.198 gh/mL).  
(0-8h)
f From SmPC.   
g Extrapolated from reported value after 1000 mg/m2 intravenous single dose cytoxan (Chan):  C  = 254.4 M (66.4 g/mL), AUC  = 1910 Mh (499 
0 (0-inf)
gh/mL). 
h Extrapolated from reported value after 1000 mg/m2 intravenous single-dose cytoxan (Chan):  C  = 40.5 M (9.0 g/mL), AUC  = 996.3 Mh (220 
0 (0-inf)
gh/mL). 
 
 
62  Contains Nonbinding Recommendations 
 
 
References 
Anthony LB, Long QC, Struck RF, Hande KR. The effect of cimetidine on cyclophosphamide metabolism in rabbits. Cancer 
Chemother Pharmacol. 1990;27:125-130. 
Chan KK, Hong PS, Tutsch K, Trump DL. Clinical pharmacokinetics of cyclophosphamide and metabolites with and without SR-
2508. Cancer Res. 1994;54:6421-6429.  
Chaube S, Kury G, Murphy ML: Teratogenic effects of cyclophosphamide (NSC-26271) in the rat.  Cancer Chemother Rep 
1967;51:363-376. 
Fritz H, Hess R. Effects of cyclophosphamide on embryonic development in the rabbit. Agents Actions. 1971;2:83-86. 
Holm KA, Kindberg CG, Stobaugh JF, Slavik M, Riley CM. Stereoselective pharmacokinetics and metabolism of the enantiomers of 
cyclophosphamide. Preliminary results in humans and rabbits. Biochem Pharmacol. 1990;39:1375-1384.  
Hong PS, Srigritsanapol A, Chan KK. Pharmacokinetics of 4-hydroxycyclophosphamide and metabolites in the rat. Drug Metab 
Dispos. 1991;19:1-7. 
Mirkes PE. Cyclophosphamide teratogenesis: a review. Teratog Carcinog Mutagen. 1985;5:75-88. 
von Kreybig T. Die teratogene wirkung cyclophosphamid wahrend der embryonalen entwicklungsphase bei der ratte. Naunyn-
Schniedeb Arch Exp Pathol Pharmakol. 1965;252:173-195. 
Additional References Evaluated 
Claussen U, Hettwer H, Voelcker G, Krengel HG, Servos G. The embryotoxicity of cyclophosphamide in rabbits during the 
histiotrophic phase of nutrition. Teratog Carcinog Mutagen. 1985;5:89-100. 
U.S. label cyclophosphamide. 
 
   
63  Contains Nonbinding Recommendations 
 
 
CYTARABINE 
CAS No.:  147-94-4 
 
Rat NOAEL   Rat LOAEL  Rat  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose   Dose   Findings  NOAEL  LOAEL Findings Dose   NOAEL/Human 
C   C   Dose    Dose   C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC 
10 mg/kg IP  20 mg/kg IP  20 mg/kg    no  no  no rabbit  100 mg/m2 IV  NOAEL:   half-life is 
single dose  single dose  cleft palate,  rabbit  rabbit  data  every 12 hours  rat  short, rapidly 
GD10,11, or 12  GD11 or 12  micrognathia, data  data  found  (days 1 to 7)  Cmax: 2.1  deaminated to 
[Chaube]  [Chaube]  deformed  foundb  foundb  many regimens  (5.8/2.8)  inactive 
    rear    are used including AUC: 2.4  uridine 
Cmax = ~5.8  Cmax = ~11.6  appendages,  CIV  (15.9/6.6)  arabinoside by 
g/mLa  g/mLa   paws and      cytidine 
AUC(0-inf) =  AUC(0-inf) =  tail; skeletal  Cmax = ~2.8  LOAEL:  deaminase 
~15.9 gh/mLa  ~31.7 gh/mLa defects  g/mLc  rat   active moiety 
  including  AUC = 6.6  Cmax: 4.1  is Ara-CTP 
distortion and  gh/mLc  (11.6/2.8)  which inhibits 
fusion of the  AUC: 4.8  DNA 
bones of the  (31.7/6.6)  polymerase 
skull and   MW = 
appendages,  243.217 
embryo-fetal 
mortality 
a Extrapolated or reported value after 20 mg/kg intraperitoneal [14C]cytarabine single dose in male Sprague Dawley rats (Parker):  C  = ~11.6 g/mL from 
max
visual inspection of graph, AUC  = ~31.7 gh/mL from calculation based on concentration values estimated by visual inspection of graph.  Note that the 
(0-inf)
reported plasma concentrations represent total radioactivity and that at 4 hours only 71% of the total plasma radioactivity was attributed to intact cytarabine 
(Parker).  The AUC value used, and the calculated margins, thus represent an upper bound, and the true AUC for intact cytarabine would certainly be lower.  
Also note that teratology was performed in Wistar rats.  PK data are also available in male Sprague Dawley rats administered 5 mg/kg intravenous cytarabine 
single dose (Zhang), in male Sprague Dawley rats administered 2.64 g/kg intravenous [3H]cytarabine (Simard), and in Wistar rats administered 5.4 mg/kg 
intramuscular cytarabine in solution with chitosan-beta-glycerophosphate (Mulik). 
64  Contains Nonbinding Recommendations 
 
 
b  Rabbit PK data are available in male New Zealand white rabbits administered single doses 50 mg/kg intravenous cytarabine (Zimmerman):  C  = 400 M 
max
(97 g/mL) from visual inspection of graph, AUC estimated from CL = 8.16 mL/(minkg) and dose = 50 mg/kg, AUC = dose/CL = (50/8.16)(1 h/60 min) = 
102 gh/mL. 
c  Extrapolated to 100 mg/m2 BID dose from reported value after single 100 mg intravenous dose (1.67 mg/kg, 60 mg/m2) (Wan):  C  = ~7.0 mol/L (1.7 
max
g/mL) from visual inspection of graph, AUC = dose/CL = 100 mg/845 mL/min = 1.97 gh/mL (which gives AUC = 3.29 gh/mL at 100 mg/m2 and 6.6 
gh/mL for 100 mg/m2 BID).  
 
65  Contains Nonbinding Recommendations 
 
 
Mouse NOAEL   Mouse LOAEL  Mouse Findings  Margins  Notes 
Dose  Dose  NOAEL/Human 
C   C   LOAEL/Human 
max max
AUC  AUC 
0.5 mg/kg IP GD6- 2 mg/kg IP GD6- cleft palate, renoureteral  NOAEL:  This table is included because:  (a) 
15 Swiss mice  15 Swiss mice  alterations, polydactyly,  mice  it shows that with the mouse 
[Ortega]  [Ortega]   oligodactyly   C :   0.16  teratology data, which was 
max
    (0.46/2.8)f  included in the U.S. label, exposure 
C  = ~0.50 g/mLd C  = ~2 g/mLd  AUC:  0.06  margins at the NOAEL were <1; 
max max
AUC = ~0.46  AUC= ~1.83  (0.39/6.6)f  (b) rat exposure margins at the 
gh/mLd  gh/mLd  LOAEL:  NOAEL were much higher; (c) 
    mice  rabbit data are not available, so it 
C  = ~0.41 g/mLe C  = ~1.62  C :   0.65  provides data in a 2nd species. 
max max max
AUC = 0.315  g/mLe  (1.81/2.8)f 
gh/mLe  AUC = 1.26  AUC:  0.23 
gh/mLe  (1.55/6.6)f 
d Extrapolated from reported value after administration of a 30 mg/kg intraperitoneal single dose cytarabine to Swiss mice (Dedrick):  C  = ~30 g/mL from 
max
visual inspection of graph, AUC  = ~27.5 gh/mL from calculation based on concentration values estimated by visual inspection of graph.  Note large 
(0-24h)
extrapolation range.  
e Extrapolated from reported value after administration of a 2.466 mmol/kg (600 mg/kg) intravenous single dose cytarabine to mice (Bayne): C  = 2 mol/mL 
max
(486 g/mL) from visual inspection of graph, AUC = 1.553 molh/mL (378 gh/mL).  Note large extrapolation range. 
f Mouse values were taken as the average of the 2 sources, which gave similar values despite the 20-fold difference in administered dose, suggesting PK was 
linear. 
 
References 
Bayne WF, Mayer LD, Swenson CE. Pharmacokinetics of CPX-351 (cytarabine/daunorubicin HCl) liposome injection in the mouse. J 
Pharm Sci. 2009;98:2540-2548.  
Chaube S, Kreis W, Uchida K, Murphy ML. The teratogenic effect of 1-beta-D-arabinofuranosylcytosine in the rat. Protection by 
deoxycytidine. Biochem Pharmacol. 1968;17:1213-1216. 
Dedrick RL, Forrester DD, Cannon JN, el-Dareer SM, Mellett LB. Pharmacokinetics of 1-beta-D-arabinofuranosylcytosine (ARA-C) 
deamination in several species. Biochem Pharmacol. 1973;22:2405-2417.  
66  Contains Nonbinding Recommendations 
 
 
Mulik R, Kulkarni V, Murthy RS. Chitosan-based thermosensitive hydrogel containing liposomes for sustained delivery of cytarabine. 
Drug Dev Ind Pharm. 2009;35(1):49-56.  
Parker RJ, Priester ER, Sieber SM. Comparison of lymphatic uptake, metabolism, excretion, and biodistribution of free and liposome-
entrapped [14C]cytosine-beta-D-arabinofuranoside following intraperitoneal administration to rats. Drug Metab Dispos. 1982;10:40-
46.  
Ortega A, Puig M, Domingo JL. Maternal and developmental toxicity of low doses of cytosine arabinoside in mice. Teratology. 
1991;44:379-384.  
Simard P, Hoarau D, Khalid MN, Roux E, Leroux JC. Preparation and in vivo evaluation of PEGylated spherulite formulations. 
Biochim Biophys Acta. 2005;1715(1):37-48. 
Wan SH, Huffman DH, Azarnoff DL, Hoogstraten B, Larsen WE. Pharmacokinetics of 1-beta-D-arabinofuranosylcytosine in humans. 
Cancer Res. 1974;34:392-397. 
Zhang B, Lu Y, Chen J, Wu W. Effects of interior gelation on pharmacokinetics and biodistribution of liposomes encapsulating an 
anti-cancer drug cytarabine. J Biomed Nanotechnol. 2010;6:704-709. 
Zimmerman CL. The disposition of cytosine arabinoside and its metabolite after single doses to rabbits. Biopharm Drug Dispos. 
1990;11:121-129. 
Additional References Evaluated 
Goto T, Endo A. Dose- and stage-related sex difference in the incidence of cytosine arabinoside induced digit anomalies in the mouse 
fetus. Teratology. 1987;35:35-40.  [Single-dose data only.] 
Kochhar DM, Penner JD, McDay JA. Limb development in mouse embryos. II. Reduction defects, cytotoxicity and inhibition of DNA 
synthesis produced by cytosine arabinoside. Teratology. 1978;18:71-92. [Single-dose data only.] 
Percy DH. Teratogenic effects of the pyrimidine analogues 5-iododeoxyuridine and cytosine arabinoside in late fetal mice and rats. 
Teratology. 1975;11:103-117.  [Rats were dosed subcutaneously on GD18-21 and offspring sacrificed on PND10 and 20.  NOAEL 
was 12.5 mg/kg and LOAEL was 25 mg/kg.] 
Scott WJ, Ritter EJ, Wilson JG. Studies on induction of polydactyly in rats with cytosine arabinoside. Dev Biol. 1975;45:103-111. 
[100 mg/kg was the only dose level.] 
 
67  Contains Nonbinding Recommendations 
 
 
DABRAFENIB 
CAS No.:  1195765-45-7 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit LOAEL Rabbit  Notes 
Dose  Dose  Dose  Dose  Findings 
C   C   C   C  
max max max max
AUC  AUC  AUC  AUC 
20 mg/kg oral  300 mg/kg oral  cardiac interventricular  No rabbit data  No rabbit data  None   
pcD1-17a  pcD1-17a  septal defects; decrease in  found  found 
(FDA, United  (FDA, United  the number of corpora 
States, p. 115)   States, p. 115)  lutea, number of implants, 
    and the number of live 
C  = 1.17 g/mLb  C  = 2.17 g/mLc  fetuses  
max max
AUC  = 4.10  AUC  = 22.6 
(0-t) (0-t)
gh/mLb  gh/mLc 
a From a combined female fertility and embryo-fetal development toxicity study in which females were dosed from 2 weeks prior to mating to post-coitum D17.  
Cesarean sections were performed on post-coitum D21. 
b Actual values in plasma after 20 mg/kg oral dabrefenib for 24 days in rats (FDA, United States, p. 119):  C  = 1.17 g/mL, AUC  = 4.10 gh/mL. 
max (0-t)
c Actual values in plasma after 300 mg/kg oral dabrefenib for 24 days in rats (FDA, United States, p. 119:  C  = 2.17 g/mL, AUC  = 22.6 gh/mL. 
max (0-t)
 
References 
FDA, United States.  Pharmacology Review NDA 202806 (25 Apr 2013). 
   
68  Contains Nonbinding Recommendations 
 
 
DASATINIB 
CAS No.:  302962-49-8 
 
Rat  Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit LOAEL  Rabbit Findings  Notes 
NOAEL  Dose  Dose  Dose 
Dose  C   C   C  
max max max
C   AUC  AUC  AUC 
max
AUC 
NOAEL not 2.5 mg/kg oral GD6- increased post- 6 mg/kg oral GD7- LOAEL for MEFL None:  findings in   
identified  15 (FDA, United  implantation loss and  19 (FDA, United  not identified  the definitive study 
  States, p. 225)  resorptions,  States, p. 236)  were limited to an 
  decreased litter size;    increase in skeletal 
C  = 0.021 g/mLa  bent scapula or  C  =  0.227 g/mL  variations (delays in 
max max
AUC  = 0.105  humerus  AUC  = 0.834  ossifications); 
(0-8h) (0-inf)
gh/mLa  gh/mL  embryolethality 
observed in the DRF 
at 10 mg/kg was 
associated with 
severe maternal 
toxicity  
a Actual values in plasma after 10 days (GD15) of 2.5 mg/kg oral dasatinib in pregnant Sprague Dawley rats (FDA, United States, p. 227):  C  = 0.021 g/mL, 
max
AUC  = 0.105 gh/mL. 
(0-8h)
b Actual values in plasma after 13 days (GD19) of 6 mg/kg oral dasatinib in pregnant NZW rabbits (FDA, United States, p. 238):  C  = 0.227 g/mL, AUC = 
max
0.834 gh/mL. 
References 
FDA, United States. Pharmacology Review NDA 21986/22072 (28 Jun 2006). 
Additional References Evaluated 
Kassem MG, Ezzeldin E, Korashy HM, Mostafa GA. High-performance liquid chromatographic method for the determination of 
dasatinib in rabbit plasma using fluorescence detection and its application to a pharmacokinetic study. J Chromatogr B Analyt Technol 
Biomed Life Sci. 2013;939:73-79.  [PK at 2.5 mg/kg was substantially different than reported in FDA, United States review:  C  = 
max
0.459 g/mL, AUC = 3.289 gh/mL.] 
69  Contains Nonbinding Recommendations 
 
 
FLUCONAZOLE 
CAS No.:  86386-73-4 
 
Rat NOAEL  Rat LOAEL Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose  Dose  NOAEL  LOAEL   Findings  Dose  NOAEL/Human 
C   C   Dose  Dose  C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC   
50 mg/kg oral  80 mg/kg  at 80 mg/kg:  25 mg/kg  75 mg/kg oral  abortions  400 mg  NOAEL:  
[U.S. label]  oral  embryolethality, oral  [U.S. Label,  (at    rat  
  [U.S. label]  cleft palate,  [U.S. Label,  FDA, United  maternally C  = 9.07  C  = 3.7 
max max
C  = 33.8    abnormal  FDA,  States1990a]  toxic  g/mLe  (33.8/9.07) 
max
g/mLa  C  = 54  craniofacial  United    dose)  AUC =  AUC = 2.8 
max (0-24h)
AUC  =  g/mLa  ossification  States  C  = 81  134.8 gh/mLe  (380/134.8) 
(0-inf) max
380 gh/mLb  AUC  =  adactylia,  1990a]  g/mLc  rabbit 
(0-inf)
  608  brachygnathia    AUC = 1563  C  = 3.0 (27/9.07) 
max
gh/mLb  [U.S. Label,  C  = 27  gh/mLd  AUC = 3.9 
max
  FDA, United  g/mLc    (521/134.8) 
States 1990a].  AUC = 521   
  gh/mLd  LOAEL: 
  rat  
C  = 6.0 (54/9.07) 
max
AUC = 4.5 
(608/134.8) 
rabbit 
C  = 8.9 (81/9.07) 
max
AUC = 11.6 
(1563/134.8) 
a Extrapolated from reported value after 20 mg/kg fluconazole oral single dose in rats (FDA, United States 1990a, p. 7):  C  = 13.5 g/mL. 
max
b Extrapolated from reported value after 20 mg/kg fluconazole oral single dose in rats (Humphrey):  AUC  = 152 gh/mL. 
(0-inf)
c:  Extrapolated from reported value after 10 mg/kg fluconazole oral single dose in rabbits (FDA, United States 1990a, p. 7):  C  = 10.8 g/mL. 
max
70  Contains Nonbinding Recommendations 
 
 
d Calculated using plasma clearance value for rabbits (0.8 mL/minkg, FDA, United States 1990a, p 8):  AUC = Dose/CL = (25 mg/kg)/(0.8 mL/minkg)(1 h/60 
min) = 521 gh/mL. 
e Actual value after 400 mg/day fluconazole oral single dose (FDA, United States 1990b, p. 7, 50-52):  C  = 9.07 g/mL, AUC  = 134.8 gh/mL.  Data are 
max (0-24h)
also available after 14 days of repeated administration, which shows significant drug accumulation.  C  = 18.89 g/mL, AUC  = 349.9 gh/mL.  Since 
max (0-24h)
PK was not available for repeated administration in animals, the single-dose human PK data were used for margin calculations.  
 
References 
Humphrey MJ, Jevons S, Tarbit MH. Pharmacokinetic evaluation of UK-49,858, a metabolically stable triazole antifungal drug, in 
animals and humans. Antimicrob Agents Chemother. 1985;28:648-653. 
FDA, United States. Pharmacology Review NDA 019949 (26 Jan 1990a), p. 7, 13. 
FDA, United States. Clinical Pharmacology Review NDA 019949 (17 Apr 1990b), p. 7, 50  52. 
U.S. label Diflucan. 
Additional References Evaluated 
Brammer KW, Farrow PR, Faulkner JK. Pharmacokinetics and tissue penetration of fluconazole in humans. Rev Infect Dis. 1990;12 
Suppl 3:S318-326. 
Pittrow L, Penk A. Plasma and tissue concentrations of fluconazole and their correlation to breakpoints. Mycoses. 1997;40:25-32.  
 
71  Contains Nonbinding Recommendations 
 
 
5-FLUOROURACIL 
CAS No.:  51-21-8 
 
Rat  Rat LOAEL Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
NOAEL  Dose   NOAEL  LOAEL  Findings  Dose   NOAEL/Human 
Dose   C   Dose   Dose   C   LOAEL/Human 
max max
C   AUC  C   C   AUC 
max max max
AUC  AUC  AUC 
 
10 mg/kg  15 mg/kg  Wilson:  NOAEL  40 mg/kg SC  limb  500 mg/m2  NOAEL:   note: half-
single dose  single dose  15 mg/kg:   not  GD12  anomalies (400  600  rat  life is very 
IP GD9  IP GD9  malformations, identified  [DeSesso]  85% of  mg/m2) in a  Cmax = 0.09  short (most 
[Wilson]  [Wilson]  embryo-fetal  [DeSesso]   term  variety of  (2.6/29)  patients 
    lethality  Cmax = 111  fetuses  dosing  AUC = 0.3  have 
Cmax = 2.6  Cmax = 3.87  Kuwagata:   g/mLb  regimens,  (3.89/11.5)  undetectable 
g/mLa  g/mLa  17 mg/kg:  AUC = 11  including doses rabbit  5-FU levels 
AUC = 3.89  AUC = 5.83  micro- gh/mLb   up to 3000  no NOAEL  in plasma 
gh/mLa  gh/mLa  /anophthalmos,  mg/m2 CIV for  identified  90 min after 
craniofacial  46 hoursc    IV) and PK 
defect,    LOAEL:  is nonlinear 
hydrocephaly,  Cmax = 29  rat   5FU is a 
brain hernia  g/mLd  Cmax = 0.1  prodrug: 
AUC = 11.5  (3.87/29)  thymidylate 
gh/mLd  AUC = 0.5  synthetase 
(5.83/11.5)  inhibitor is 
rabbit  5FdUMP 
Cmax = 3.8 (111/29)  MW = 
AUC = 1.0  130.077 
(11/11.5) 
a Extrapolated from reported value after 30 mg/kg 5FU intraperitoneal single dose in Sprague Dawley rats (Zhang):  C  = 7.74 g/mL, AUC = 11.66 gh/mL. 
max
b Extrapolated from reported value after 20 mg/kg 5FU intravenous single dose in rabbits (Kar):  C  = 0.427 mol/mL (55.5 g/mL), AUC = 2.535 
max
molmin/mL (5.5 gh/mL). 
72  Contains Nonbinding Recommendations 
 
 
c The dose of 500 mg/m2 IV bolus was used for comparison although higher doses (e.g., ~1500 mg/m2/day CIV) are used.  Very low margins were calculated, 
and using higher human doses would make them even lower. 
d Extrapolated from reported value after 14.7 mg/kg (544 mg/m2) 5FU oral single dose (Schaaf):  C  = 32 g/mL from visual inspection of graph,  AUC = 
max
12.55 gh/mL.  Data are also available after a 370 mg/m2 dose (Bocci):  C  = 48.41 g/mL, AUC = 13.61 gh/mL. 
max
 
References 
DeSesso JM, Scialli AR, Goeringer GC. Teratology. 1995;51:172 (abstract) 
Kar R, Cohen RA, Terem TM, Nahabedian MY, Wile AG. Pharmacokinetics of 5-fluorouracil in rabbits in experimental regional 
chemotherapy. Cancer Res. 1986;46:4491-4495.  
Kuwagata M, Takashima H, Nagao T. A comparison of the in vivo and in vitro response of rat embryos to 5-fluorouracil. J Vet Med 
Sci. 1998;60:93-99. 
Schaaf LJ, Dobbs BR, Edwards IR, Perrier DG. Nonlinear pharmacokinetic characteristics of 5-fluorouracil (5-FU) in colorectal 
cancer patients. Eur J Clin Pharmacol. 1987;32:411-418. 
Wilson JG. Teratogenic interaction of chemical agents in the rat. J Pharmacol Exp Therapeut. 1964;144:429-436. 
Zhang C, Li G, Wang Y, Cui F, Zhang J, Huang Q. Preparation and characterization of 5-fluorouracil-loaded PLLA-PEG/PEG 
nanoparticles by a novel supercritical CO  technique. Int J Pharm. 2012;436:272-281. 
2
Additional References Evaluated 
Bocci G, Danesi R, Di Paolo AD, Innocenti F, Allegrini G, Falcone A, et al. Comparative pharmacokinetic analysis of 5-fluorouracil 
and its major metabolite 5-fluoro-5,6-dihydrouracil after conventional and reduced test dose in cancer patients. Clin Cancer Res. 
2000;6:3032-3037. 
Chaube S, Murphy ML. The teratogenic effects of the recent drugs active in cancer chemotherapy. In: Woolham, DHM, editor. 
Advances in Teratology, Volume 3. New York: Academic Press. 1968. pp. 180-237. [no incidence was provided, but confirms 
malformations in rats as detailed by Kuwagata] 
Huang Y, Wei Y, Yang H, Pi C, Liu H, Ye Y, Zhao L. A 5-fluorouracil-loaded floating gastroretentive hollow microsphere: 
development, pharmacokinetic in rabbits, and biodistribution in tumor-bearing mice. Drug Des Devel Ther. 2016;10:997-1008. [no 
systemic PK, only oral after 50 mg/kg dose; C  = 2.55g/mL, AUC = 5.82 gh/mL] 
max
Shuey DL, Lau C, Logsdon TR, Zucker RM, Elstein KH, Narotsky MG, et al. Biologically based dose-response modeling in 
developmental toxicology: biochemical and cellular sequelae of 5-fluorouracil exposure in the developing rat. Toxicol Appl 
73  Contains Nonbinding Recommendations 
 
 
Pharmacol. 1994a;126:129-144. [Malformations were seen at 35 and 40 mg/kg administered SC on GD14; MEFL effects were seen at 
lower doses in other studies.] 
Shuey DL, Buckalew AR, Wilke TS, Rogers JM, Abbott BD. Early events following maternal exposure to 5-fluorouracil lead to 
dysmorphology in cultured embryonic tissues. Teratology. 1994b;50:379-386. [10  40 mg/kg SC on GD14, all malformations studied 
in explants] 
U.S. Adrucil label. [confirms malformations in rats as detailed by Kuwagata] 
Zhao B, Zhao XL. [Pharmacokinetic studies on 5-fluorouracil and its metabolite in rabbits by high pressure liquid chromatography]. 
Zhongguo Yao Li Xue Bao (Acta Pharmacol Sin). 1988;9:275-278. Chinese. [PK after 170 mg/kg IV dose, which would require 
greater extrapolation than data from Kar] 
 
74  Contains Nonbinding Recommendations 
 
 
HYDROXYUREA 
CAS No.:  127-07-1 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose   Dose   NOAEL  LOAEL Findingsa  Dose  NOAEL/Human 
C    C    Dose   Dose   C    LOAEL/Human 
max max max
AUC  AUC  C    C    AUC 
max max
  AUC  AUC 
100 mg/kg IP  137 mg/kg IP Embryo-fetal  NOAEL  30  650 mg/kg  oral for oncology  NOAEL:   PK is 
GD9-12  GD9-12  lethality,  not  mg/kg  SC GD12  indications:   rat  nonlinear 
[Wilson]  [Wilson]  ocular and  identified [U.S.  [DeSesso  80 mg/kg Q3D,  Cmax = 0.9  with short 
    cerebral    label]  1990]:  cleft  20  30  (47.3/52)  half-life (15 
Cmax = 47.3  Cmax = 80.6  malformations PK not    lip, cleft  mg/kg/day   Cmax dose = 2.9  min in rats, 2 
g/mLb  g/mLb  available    palate,  oral for sickle cell  (100/35)d   4 h 
AUC not  AUC not  PK not  reduction  anemia  AUC = 0.5   in humans) 
available  available  available deformities  15  35  (600/1295)e   MW = 
of limbs and  mg/kg/day (555   rabbit  76.05g/mol 
tail  1295 mg/m2)   NOAEL not   PK after IP 
    identified 
and IV is 
750 mg/kg  C  = 52 g/mLc   
max similar 
SC GD12  AUC  = 184  LOAEL: 
(0-inf) (Wilson) 
[DeSesso  gh/mLc  rat 
 bioavailability 
1977]:  skull  C  = 1.6 
max is 70  80% in 
and facial  (80.6/52) 
rats and 
anomalies as  C  dose = 3.9 
max humans, 
well as  (137/35)d 
respectively 
severe  AUC = 0.6 
(Beckloff) 
reduction  (822/1295)e 
 no robust data 
deformities   
for adverse 
of all limbs  rabbitf 
human 
C  = 0.9 
max pregnancy 
(30/35) 
outcomes   
75  Contains Nonbinding Recommendations 
 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose   Dose   NOAEL  LOAEL Findingsa  Dose  NOAEL/Human 
C    C    Dose   Dose   C    LOAEL/Human 
max max max
AUC  AUC  C    C    AUC 
max max
  AUC  AUC 
AUC = 0.3 
(360/1295) 
a U.S. label states that Hydroxyurea is embryotoxic and causes fetal malformations (partially ossified cranial bones, absence of eye sockets, hydrocephaly, 
bipartite sternebrae, missing lumbar vertebrae) at 180 mg/kg/day in rats and 30 mg/kg/day in rabbits, but it is not clear which effects are in which species.  
Thus, 30 mg/kg is accepted as the LOAEL, but the findings are listed from publications with rabbits with SC doses of 650 and 750 mg/kg. 
b Actual values after 100 and 137 mg/kg hydroxyurea IP doses in pregnant Wistar rats (Wilson):  C  = 47.3 at 100 mg/kg and 80.6 g/mL at 137 mg/kg. 
max
c Extrapolated from reported value after 1000 mg (16.7 mg/kg) hydroxyurea oral single dose (MHRA):  C  = 24.6 g/mL, AUC  = 87.79 gh/mL.  The 
max (0-inf)
dose for margin calculations was chosen to be 35 mg/kg/day.  Although higher intermittent doses are used for oncology indications, the dose for sickle cell 
anemia is believed to be more relevant for assessing risk of developmental toxicity.  As summarized in the table below, other human PK data are also 
available. 
d 
Although rat C  data are available, this was after IP administration whereas the human data is after oral administration.  Thus, in the absence of more direct 
max
PK comparisons, the estimated ratio based on mg/kg dose is also provided. 
e In the absence of rat AUC data, AUC ratio was based on mg/m2 dose ratio. 
f In the absence of rabbit PK data, C  ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 
max
 
References 
DeSesso JM, Jordan RL. Drug-induced limb dysplasias in fetal rabbits. Teratology. 1977;15:199-211. 
DeSesso JM, Goeringer GC. Ethoxyquin and nordihydroguaiaretic acid reduce hydroxyurea developmental toxicity. Reprod Toxicol. 
1990;4:267-275.  
MHRA Public Assessment Report PL 10880/128-9, page 48. 
U.S. label Hydrea and Droxea. 
Wilson JG, Scott WJ, Ritter EJ, Fradkin R. Comparative distribution and embryotoxicity of hydroxyurea in pregnant rats and rhesus 
monkeys. Teratology. 1975;11:169-178. 
 
76  Contains Nonbinding Recommendations 
 
 
 
 
 
Additional References Evaluated 
Beckloff GL, Lerner HJ, Frost D, Russo-Alesi FM, Gitomer S. Hydroxyurea (NSC-32065) in biologic fluids: dose-concentration 
relationship. Cancer Chemother Rep. 1965;48:57-58. [PK data in cancer subjects, no AUC] 
Charache S, Dover GJ, Moore RD, Eckert S, Ballas SK, Koshy M, et al. Hydroxyurea: effects on hemoglobin F production in patients 
with sickle cell anemia. Blood. 1992;79:2555-2565. [PK data in sickle cell anemia subjects] 
Chaube S, Murphy ML. The effects of hydroxyurea and related compounds on the rat fetus. Cancer Res. 1966;26:1448-1457. [effects 
of single and repeated IP doses 125 mg/kg] 
Gwilt PR, Tracewell WG. Pharmacokinetics and pharmacodynamics of hydroxyurea. Clin Pharmacokinet. 1998;34:347-358. [review 
article of PK publications] 
Millicovsky G, DeSesso JM. Cardiovascular alterations in rabbit embryos in situ after a teratogenic dose of hydroxyurea: an in vivo 
microscopic study. Teratology. 1980;22:115-124. [effects on ex vivo embryos after 500 and 750 mg/kg to does on GD12] 
Philips FS, Sternberg SS, Schwartz HS, Cronin AP, Sodergren JE, Vidal PM. Hydroxyurea. I. Acute cell death in proliferating tissues 
in rats. Cancer Res. 1967;27:61-75. [C  after 46, 184, and 1840 mg/kg IV dose, nonlinear PK] 
max
Tracewell WG, Vaughan WP, Gwilt PR. Nonlinear disposition of hydroxyurea. J Pharm Sci. 1994;83:1060-1061. [formal PK analysis 
of Philips data] 
Villani P, Maserati R, Regazzi MB, Giacchino R, Lori F. Pharmacokinetics of hydroxyurea in patients infected with human 
immunodeficiency virus type I. J Clin Pharmacol. 1996;36:117-121. [PK in HIV subjects] 
 
Human Pharmacokinetic Data 
Reference  Population  Dose  Route  C  (g/mL)  AUC (gh/mL)  Notes 
max
Charache  sickle cell  25 mg/kg   oral  19  AUC( 1216  AUC units were published as g 
0-6) = 
anemia  mL/min, value seems wrong since 
(Cmax x 6 h = 114 gh/mL) 
77  Contains Nonbinding Recommendations 
 
 
Villani  HIV  mean 7.6 mg/kg  oral  0.135 nmol/L  AUC  = 540   
(0-12h)
BID  = 0.135  molh/L = 41.1 
mol/mL =  gh/mL;  
10.3 g/mL  AUC  = 82.1 
(0-24h)
gh/mL 
MHRA  not stated  BE  1000 mg (16.6  oral  24.6  AUC  = 87.79  use these values 
(0-inf)
review  study  mg/kg)  gh/mL 
Beckloff  cancer  20 mg/kg   oral  20.7     
80 mg/kg   oral  128.1     
 
78  Contains Nonbinding Recommendations 
 
 
IBRUTINIB 
CAS No.:  936563-96-1 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit LOAELa  Rabbit  Notes 
Dose  Dose  Dose  Dose  Findingsb 
C   C   C   C  
max max max max
AUC  AUC  AUC  AUC 
40 mg/kg oral GD6- 80 mg/kg oral  malformations including  30 mg/kg oral  100 mg/kg oral  increased pre-   
17 (FDA, United  GD6-17 (FDA,  dextrocardia,  GD7-19 (FDA,  GD7-19 (FDA,  and post-
States, p. 126)  United States, p.  retroesophageal aortic  United States, p.  United States, p.  implantation loss 
  126)  arch, persistent truncus  135)  135)c  (increased early 
C  = 1.31 g/mLc    arteriosus, right-sided      resorptions), 
max
AUC  = 5.348  C  = 2.627  aortic arch, and  C  = 0.311  C  = 1.83  decreased viable 
(0-24h) max max max
gh/mLc  g/mLd  interrupted aortic arch;  g/mLe  g/mLf  fetuses, abortions 
AUC  =  increased post- AUC = 1.31  AUC = 21.00 
(0-24h)
13.729 gh/mLd  implantation loss  gh/mLe  gh/mLf 
(increased early 
resorptions), decreased 
viable fetuses 
a The LOAEL for MEFL was a maternally toxic dose as indicated by increased mortality and abortions, clinical signs, and reductions in body weight and food 
consumption. 
b This was a dose-range-finding study with limited numbers of animals (n=6) and fetal evaluations limited to external morphology.  It is thus unknown if there 
were visceral or skeletal alterations. 
c Actual values in plasma after 11 doses of 40 mg/kg oral ibrutinib in pregnant rats (FDA, United States, p. 130):  C  = 1.31 g/mL, AUC  = 
max (0-24h)
5.348 gh/mL. 
d Actual values in plasma after 11 doses of 100 mg/kg oral ibrutinib in pregnant rats (FDA, United States, p. 130):  C  = 2.627 g/mL, AUC  = 13.729 
max (0-24h)
gh/mL. 
e Actual values in plasma after 13 doses of 30 mg/kg oral ibrutinib in pregnant rabbits (FDA, United States, p. 136):  C  = 0.311 g/mL, AUC = 1.31 gh/mL. 
max
f 
Actual values in plasma after 13 doses of 100 mg/kg oral ibrutinib in pregnant rabbits (FDA, United States, p. 136):  C  = 1.83 g/mL, AUC = 21.00 
max
gh/mL. 
 
79  Contains Nonbinding Recommendations 
 
 
References 
FDA, United States. Pharmacology Review NDA 020552 (21 Aug 2013). 
 
80  Contains Nonbinding Recommendations 
 
 
IBUPROFEN 
CAS No.:  15687-27-1 
 
Rat NOAEL  Rat LOAEL  Rat  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose  Dose  Findings  NOAELc  LOAEL  Findings  Dose  NOAEL/Human 
C   C   Dose  Dose  C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC 
180 mg/kg oral  oral GD1-20:  GD1-20:  60 mg/kg oral  No  None  Maximum dose is 800  NOAEL:   
GD1-20  No LOAEL  None  GD1-29  LOAEL  mg QID, 3200 mg/day  rat 
[Adams]  idenitifed    [Adams]  identified  (13.3 mg/kg/dose, 53  C  = 3.4 
max
  [Adams]   GD9-10:  C  = 26.6  mg/kg/day)   (205/59.7) 
max
C  = 205    ventricular g/mLd  [U.S. label]  AUC = 0.7 
max
g/mLa  oral GD9-10:   septal  AUC  =    (597/839) 
(0-inf)
AUC = 597  300 mg/kg   defects  80.5 gh/mLd  C  = 59 g/mLe   
max
gh/mLa  [Cappon    AUC = 839 gh/mLe  rabbitc 
2003]b    60 mg/kg 
  500 mg/kg oral  NOAEL 
at 300 mg/kg   GD9-11  C  = 0.5 
max
C  = 341  [Cappon 2003]b  (26.6/59) 
max
g/mLa  C  = 222  AUC = 0.1 
max
AUC = 995  g/mLd  (80.5/839) 
gh/mLa  AUC  = 671  500 mg/kg 
(0-inf)
gh/mLd  NOAEL 
C  = 3.8 
max
(222/59) 
AUC = 0.8 
(671/839) 
 
LOAEL: 
rat 
C  = 5.8 
max
(341/59) 
81  Contains Nonbinding Recommendations 
 
 
Rat NOAEL  Rat LOAEL  Rat  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose  Dose  Findings  NOAELc  LOAEL  Findings  Dose  NOAEL/Human 
C   C   Dose  Dose  C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC 
AUC = 1.2 
(995/839) 
 
rabbit  no 
LOAEL 
a Extrapolated from reported value after 25 mg/kg ibuprofen (suspension) single oral dose in Sprague Dawley rats (You):  C  = 28.4 g/mL,  AUC = 
max (0-inf)
4971.3 gmin/mL (82.9 gh/mL).  Note that different data (5- to 7-fold lower values) are available from the same laboratory at 25 mg/kg where the only 
difference appears to be that ibuprofen was administered in hard gelatin capsules versus a suspension (Newa):  C  = 5.32 g/mL, AUC = 12.41 gh/mL. 
max
b To enhance detection of VSD and midline defects (seen in humans and with other NSAIDS), exposure was limited to the sensitive period of cardiovascular 
development and midline closure (i.e., GD9-10 in rats and GD9-11 in rabbits).  By limiting the exposure period, maternal GI toxicity was reduced, allowing 
for the administration of higher doses. 
c Two values are included for the rabbit NOAEL since neither study design was ideal for assessing the risk of developmental toxicity according to current 
conventions.  The study by Adams dosed rabbits on GD1-29 instead of the conventional GD7-19, whereas the study by Cappon dosed rabbits only on GD9-11 
to enhance detection of VSD and midline defects. 
d Extrapolated from reported value after 56 mg/kg ibuprofen single oral dose in male New Zealand White rabbits (Kondal):  C  = 24.85 g/mL,  AUC  = 
max (0-inf)
75.14 gh/mL. 
e Extrapolated from reported value after 14.8 mg/kg (mean) ibuprofen single oral dose (Konstan):  C  = 65.5 g/mL,  AUC  = 14.0 mgmin/mL (233 
max (0-inf)
gh/mL).  Note the C  was multiplied by 0.9 (13.3/14.8) to give the extrapolated C .  The C  after a single dose likely represents the C  at steady state 
max max max max
since the half-life is short (approximately 1.8 to 2 hours (U.S. label)) and little accumulation is expected using the equation:  accumulation = 1/(1  ektau), 
where k = 0.693/t12 with t12 = 2 hours and tau = 6 hours (yielding an accumulation factor of 1.1).  The AUC was multiplied by 4 to get the daily AUC for QID 
dosing (at 59.2 mg/kg/day), and then by 0.9 to give the extrapolated AUC for 53 mg/kg/day. 
 
References 
Adams SS, Bough RG, Cliffe EE, et al. Absorption, distribution and toxicity of ibuprofen. Toxicol Appl Pharmacol. 1969;15:310-30. 
Cappon GD, Cook JC, Hurtt ME. Relationship between cyclooxygenase 1 and 2 selective inhibitors and fetal development when 
administered to rats and rabbits during the sensitive periods for heart development and midline closure. Birth Defects Res B Dev 
Reprod Toxicol. 2003;68:47-56. 
82  Contains Nonbinding Recommendations 
 
 
Kondal A1, Garg SK. Influence of acidic beverage (Coca-Cola) on pharmacokinetics of ibuprofen in healthy rabbits. Indian J Exp 
Biol. 2003;41:1322-1324. 
Konstan MW, Krenicky JE, Finney MR, Kirchner HL, Hilliard KA, Hilliard JB, et al. Effect of ibuprofen on neutrophil migration in 
vivo in cystic fibrosis and healthy subjects J Pharmacol Exp Ther. 2003;306:1086-1091. 
Newa M, Bhandari KH, Kim JO, Im JS, Kim JA, Yoo BK, et al.. Enhancement of solubility, dissolution and bioavailability of 
ibuprofen in solid dispersion systems. Chem Pharm Bull (Tokyo). 2008;56:569-574. 
U.S. Motrin label. 
You X, Xing Q, Tuo J, Song W, Zeng Y, Hu H. Optimizing surfactant content to improve oral bioavailability of ibuprofen in 
microemulsions: just enough or more than enough? Int J Pharm. 2014;471:276-284.  
Additonal References Evaluated 
Cappon GD, Fleeman TL, Cook JC, Hurtt ME. Combined treatment potentiates the developmental toxicity of ibuprofen and 
acetazolamide in rats. Drug Chem Toxicol. 2005;28:409-421. [confirmed VSD findings in Cappon 2003] 
Cook JC, Jacobson CF, Gao F, Tassinari MS, Hurtt ME, DeSesso JM. Analysis of the nonsteroidal anti-inflammatory drug literature 
for potential developmental toxicity in rats and rabbits. Birth Defects Res B Dev Reprod Toxicol. 2003;68:5-26. [review article: 
captured data from Adams] 
Malm H, Borisch C.  Analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), muscle relaxants, and antigout medications. In: 
Schaefer C, Peters P, Miller RK, editors.  Drugs during pregnancy and lactation:  treatment options and risk assessment (Third 
Edition).  Boston: Academic Press; 2015. p. 27-58. [mainly human data] 
 
83  Contains Nonbinding Recommendations 
 
 
IMATINIB 
CAS No.:  152459-95-5 (220127-57-1 as mesilate) 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit LOAEL  Rabbit  Notes 
Dose  Dose  Dose  Dose  Findings 
C   C   C   C  
max max max max
AUC  AUC  AUC  AUC 
30 mg/kg oral  100 mg/kg oral  exencephaly and/or  60 mg/kg oral GD7-19  LOAEL not  None   
GD6-15 (FDA,  GD6-15 (FDA,  protruding tongue,  (FDA, United States,  identified 
United States, p.  United States, p.  encephalocele, absent  p. 72)   
69)  69)  frontal or parietal   
    bones;  C  = 53.06 g/mLc 
max
C  = 3.57  C  = 12.14  increased post- AUC = 699.8 
max max
g/mLa  g/mLb  implantation loss,  gh/mLc 
AUC = 39.28  AUC = 142.55  decreased live fetuses 
gh/mLa  gh/mLb 
a Interpolated from reported values in plasma after 15 and 50 mg/kg imatinib oral single dose in female rats (FDA, United States, p. 24):  at 15 mg/kg, C  = 
max
1.69 g/mL, AUC  = 15.40 gh/mL; at 50 mg/kg, C  = 6.07 g/mL, AUC  = 71.276 gh/mL. 
(0-24h) max (0-24h)
b Extrapolated from reported value in plasma after 50 mg/kg imatinib oral single dose in female rats (FDA, United States, p. 24):  C  = 6.07 g/mL, AUC  
max (0-24h)
= 71.276 gh/mL. 
c Reported value after 60 mg/kg oral imatinib single dose in rabbits species (FDA, United States, p. 26):  C  = 53.06 g/mL, AUC  = 699.8 gh/mL. 
max (0-24h)
References 
FDA, United States. Pharmacology Review NDA 021335 (04 May 2001). 
84  Contains Nonbinding Recommendations 
 
 
ISOTRETINOIN (13-CIS-RETINOIC ACID) 
CAS No.:  4759-48-2 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL  Rat LOAEL  
NOAEL   LOAEL   Dose  NOAEL/Human 
Dose  Dose  Rabbit 
Rat Findings  Dose  Dose  C   LOAEL/Human  Notes 
max
C   C   Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
50 mg/kg  100 mg/kg  LOAEL:  3 mg/kg oral  15 mg/kg oral increased  0.5 mg/kg BID  NOAEL:   
oral dose on  oral dose on  microtia and  GD8-11  GD8-11  resorptions,  (1 mg/kg/day)  rat 
GD10  GD10  talipes.  [Eckhoff]  [Eckhoff]  malformations   C  = 2.8 
max
[Tembe]  [Tembe]  Higher doses:      including eye  C  = 0.32  (0.9/0.32) 
max
    microcephaly,  C  = 0.95  C  = 3.1  defects, tail  g/mLd  AUC = 0.6 
max max
C  = 0.9  C  = 1.8  anotia,  g/mLb  g/mLc  defects,  AUC = 7.52  (4.8/7.52) 
max max
g/mLa  g/mLa  exophthalmos, AUC = 12.2  AUC = 49.1  cardiomegaly,  gh/mLd  rabbit 
AUC  =  AUC  =  protruding  gh/mLb  gh/mLc  skin tag on    C  = 3.0 
(0-10h) (0-10h) max
4.8 gh/mLa  9.6 gh/mLa  tongue, cleft  face  (0.95/0.32) 
lip,  AUC = 1.6 
mandibular  (12.2/7.52) 
hypoplasia,   
cleft palate,  LOAEL: 
overdeveloped  rat 
papillae, anal  C  = 5.6 
max
atresia, spina  (1.8/0.32) 
bifida,  AUC = 1.3 
deformed tail,  (9.6/7.52) 
and acaudate;   
increased  rabbit 
resorptions  C  = 9.7 
max
(3.1/0.32) 
AUC = 6.5 
(49.1/7.52) 
a Extrapolated from reported value after 500 mg/kg isotretinoin oral single dose in Wistar rats (Tembe):  C  = 9.07 g/mL,  AUC  = 47.9 gh/mL. 
max (0-10h)
85  Contains Nonbinding Recommendations 
 
 
b Actual values after 3 mg/kg isotretinoin oral single dose in New Zealand White rabbits (Eckhoff):  C  = 0.952 g/mL,  AUC = 12.2 gh/mL. 
max
c Actual values after 15 mg/kg isotretinoin oral single dose in New Zealand White rabbits (Eckhoff):  C  = 3.099 g/mL,  AUC  = 49.1 gh/mL. 
max (0-10h)
d Extrapolated from reported value after 80 mg (1.33 mg/kg) isotretinoin oral single dose with food (U.S. label):  C  = 0.86 g/mL,  AUC  =  10.0 
max (0-10h)
gh/mL.  The C  extrapolation was based on a 0.5 mg/kg dose, whereas the AUC extrapolation was based on the daily dose of 1 mg/kg/day.  PK data are 
max
also available while fasting, but the higher values from the fed state were used for margin calculations:  C  = 0.3 g/mL, AUC = 3.7 gh/mL. 
max
References 
Eckhoff C, Chari S, Kromka M, Staudner H, Juhasz L, Rudiger H, et al. Teratogenicity and transplacental pharmacokinetics of 13-cis-
retinoic acid in rabbits. Toxicol Appl Pharmacol. 1994;125:34-41. 
Tembe EA, Honeywell R, Buss NE, Renwick AG. All-trans-retinoic acid in maternal plasma and teratogenicity in rats and rabbits. 
Toxicol Appl Pharmacol. 1996;141:456-472. 
U.S. label isotretinoin. 
86  Contains Nonbinding Recommendations 
 
 
METHOTREXATE 
CAS No.:  59-05-2 
 
Rat  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  NOAEL  Notes 
NOAEL  Dose  NOAEL  LOAEL  Findings  Dosec  Margins 
Dose  C   Dose  (Dose  C  
max max
C   AUC  C   C   AUC 
max max max
AUC  AUC  AUC 
NOAEL  0.1 mg/kg IP  resorbed  NOAEL  0.3 mg/kg  hydrocephalus,  psoriasis:  10  25  NOAEL:  Note:  animal 
not  GD9 [Jordan,  litters,  not  IV GD10  microphthalmia, mg Q7D (5.9  14.7  rat  MEFL data is 
identified  Woo]  malformations identified  [Jordan]  cleft lip and  mg/m2) oral or IVc  NOAEL not  after single 
      palate,    identified   dose, so 
C  = 0.21  C  = 1.58 micrognathia,  ALL:  induction   rabbit  margins 
max max
g/mLa  g/mLb  dysplastic sacral 3.3 mg/m2 daily;  NOAEL not  would likely 
AUC = 0.067  AUC =  and caudal  maintenance  15  identified   be even lower 
gh/mLa  0.61  vertebrate,  mg/m2 oral    if dosed 
gh/mLb  phocomelia,  twice/week  LOAEL:  throughout 
hemimelia,  choriocarcinoma:   rat  organogenesis 
syndactyly, and  15  30 mg oral  C  = 0.1 
max
ectrodactyly;  QDx5 (8.8  17.6  (0.21/2.14) 
embryolethality, mg/m2)   AUC = 0.02 
resorptions  lymphoma:  10  25  (0.067/3.28) 
mg QDx4-8 oral  rabbit 
(5.9  14.7 mg/m2);  C  = 0.7 
max
0.625  2.5 mg/kg  (1.58/2.14) 
(23  92.5 mg/m2)  AUC = 0.2 
mycosis fungoides:   (0.61/3.28) 
5  50 mg Q7D oral 
(2.9  29 mg/m2) 
RA: 7.5 mg Q7D 
oral (4.4 mg/m2) 
 
C  = 2.14 g/mLd 
max
87  Contains Nonbinding Recommendations 
 
 
AUC = 3.28 
gh/mLd 
a Extrapolated from reported value after 0.31 mg/kg methotrexate intravenous single dose in Wistar rats (Scheufler 1982):  C  = 0.64 g/mL, AUC  = 0.207 
0 (0.1-4h)
gh/mL.  Other PK data are also available as shown in the table below.  The data from Scheufler 1982 were chosen for margin calculations because it 
required the least degree of extrapolation in the same strain as the teratology study. 
b Extrapolated from reported value after 1.33 mg/kg methotrexate intravenous single dose in male rabbits (Iven):  C  = 7 g/mL, AUC= 2.72 gh/mL.  Data 
max  
are also available after a 10 mg/kg methotrexate intravenous single dose in female New Zealand White rabbits (Stagni):  C  = 74 g/mL, AUC= 31.4 
max  
gh/mL.  The data from Iven were chosen for margin calculations because it required the least degree of extrapolation to the dose in the teratology study. 
c As noted, there is a wide variety of doses, schedules, and routes used in a variety of indications (U.S. label).  An intravenous dose of 25 mg (14.7 mg/m2) in 
psoriasis was chosen for PK margin comparisons since this was the highest dose in a non-oncology indication and would also provide a higher exposure than 
a 50 mg (29 mg/m2) oral dose (mycosis fungoides) since oral bioavailability is only ~40%. 
d Extrapolated to 14.7 mg/m2 from reported value after 30 mg/m2 methotrexate intravenous single dose (Campbell):  C  = 4.37 g/mL from visual inspection of 
max
graph, AUC  = 6.69 gh/mL.  Oral data are also available (Campbell):  C  = 0.50 g /mL from visual inspection of graph, AUC  = 2.34gh/mL. 
(0-inf) max (0-inf)
 
 
References 
Campbell MA, Perrier DG, Dorr RT, Alberts DS, Finley PR. Methotrexate: bioavailability and pharmacokinetics. Cancer Treat Rep. 
1985;69:833-838. 
Iven H, Brasch H, Engster J. Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate in rabbits. Cancer Chemother Pharmacol. 
1985;15:115-120. 
Jordan RL, Wilson JG, Schumacher HJ. Embryotoxicity of the folate antagonist methotrexate in rats and rabbits. Teratology. 
1977;15:73-79. 
Scheufler E. Evidence of nonlinear pharmacokinetics of methotrexate in the rat. Pharmacology. 1982;25:51-56. 
U.S. label methotrexate. 
Woo DC, McClain RM, Hoar RM. Potentiation of methotrexate embryolethality by aspirin in rats. Teratology. 1978;17:37-41. 
 
Additional References Evaluated 
Berry CL. Transient inhibition of DNA synthesis by methotrexate, in the rat embryo and foetus. J Embryol Exp Morphol. 
1971;26:469-4274. [increased resoprtions at 1 mg/kg] 
88  Contains Nonbinding Recommendations 
 
 
Hyoun SC, Obican SG, Scialli AR. Teratogen update: methotrexate. Birth Defects Res A Clin Mol Teratol. 2012;94:187-207. [review 
article] 
Kim MM, Lee SH, Lee MG, Hwang SJ, Kim CK. Pharmacokinetics of methotrexate after intravenous and intramuscular injection of 
methotrexate-bearing positively charged liposomes to rats. Biopharm Drug Dispos. 1995;16:279-93. [PK in Sprague Dawley rats at 4 
mg/kg dose] 
Scheufler E, Zetler G, Iven H. Pharmacokinetics and organ distribution of methotrexate in the rat. Pharmacology. 1981;23:75-81. [PK 
only at 31 mg/kg dose] 
Stagni G, Shukla C. Pharmacokinetics of methotrexate in rabbit skin and plasma after iv-bolus and iontophoretic administrations. J 
Control Release. 2003;93:283-292. [PK only at 10 mg/kg dose] 
Wilson JG, Scott WJ, Ritter EJ, Fradkin R. Comparative distribution and embryotoxicity of methotrexate in pregnant rats and rhesus 
monkeys. Teratology. 1979;19:71-79. [no AUC data, only concentrations at 0.25 hours] 
 
Rat Pharmacokinetic Data 
Reference Dose  Route Strain  C    AUC  Notes 
max
(mg/kg)  (g/mL)  (gh/mL) 
Wilson  0.3  IV  Wistar  0.40   C  was estimated from graph since 1st timepoint was 0.25 
0
hours 
Scheufler  31  IV  Wistar  177  AUC  = 38.4  C  is C  
(0-inf) max 0
1981 
Scheufler  0.31  IV  Wistar  0.64 AUC  =   
(0.1-4h)
1982  0.207 
Kim  4.0  IV  Sprague  40  AUC  = 2.88  C  was from visual inspection of graph, AUC was 173 
(0-inf) max
Dawley  gmin/mL 
 
89  Contains Nonbinding Recommendations 
 
 
PHENYTOIN 
CAS No.:  57-41-0 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose  Dose  NOAEL  LOAEL  Findings  Dose  NOAEL/Human 
C   C   Dose  Dose  C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC 
150 mg/kg  300 mg/kg  external findings  50 mg/kg  75 mg/kg  open eyes,  up to 625  NOAEL:   
oral GD6-15  oral GD6-15  (protruding  oral GD7- oral GD7- cleft palate,  mg/day oral  rat 
[Kim]  [Kim]  tongue,  18  18  and limb  solutione  C  = 0.9 
max
    meningoencepha- [McClain]  [McClain]  abnormalities    (13.4/14.5) 
C  = 13.4  C  = 26.8  locele, domed      that included  C  = 14.5  AUC = 0.7 
max max max
g/mLa   g/mLa  head, anasarca,  C = 27  C = 34  shortened and  g/mLf  (205/291) 
max  max 
AUC = 205  AUC = 410  and limb  g/mLb   g/mLd  curved long  AUC = 291  rabbit 
gh/mLa  gh/mLa  hyperflexion),  AUC  = AUC  = bones, pes  gh/mLg  C = 1.9 
(0-24h) (0-24h) max 
skeletal  193  290  cavus,  (27/14.5) 
malformation  gh/mLc  gh/mLc  syndactyly  AUC = 0.7 
(short rib)  (193/291) 
 
LOAEL: 
rat  
C  = 1.8 
max
(26.8/14.5) 
AUC = 1.4 
(410/291) 
rabbit  
C  = 2.3 
max
(34/14.5) 
AUC = 1.0 
(290/291) 
 
90  Contains Nonbinding Recommendations 
 
 
a Actual or extrapolated from reported value after 150 mg/kg phenytoin oral dose on GD8 in Sprague Dawley rats (Rowland):  C  = 13.4 g/mL, AUC  = 
max (0-inf)
205 gh/mL.  PK data are also available on GD17:  C  = 30.2 g/mL, AUC  = 906 gh/mL. 
max (0-inf)
b Actual value after 50 mg/kg phenytoin oral single dose in female New Zealand White rabbits (McClain):  C  = 27 g/mL.  PK data are also available after 30 
max
mg/kg phenytoin oral dose in male New Zealand White rabbits (Medhi):  C  = 12.8 g/mL.  The value from McClain was used because it was from 
max
females, required no extrapolation, and was generated in conjunction with the developmental toxicity study. 
c Extrapolated from reported value after 30 mg/kg phenytoin oral dose in male New Zealand White rabbits (Medhi):  AUC = 116 gh/mL, from calculation 
based on concentration values estimated by visual inspection of graph since published value was inconsistent with other data in the paper. 
d Interpolated from actual values after 50 or 100 mg/kg phenytoin oral single dose in female New Zealand White rabbits (McClain):  C  = 27 g/mL and 41 
max
g/mL at 50 and 100 mg/kg, respectively.   
e Phenytoin is available as an oral solution with an MRHD of 625 mg/day (dosing interval not clear) and as extended-release capsules with an MRHD up to 600 
mg/day (in 3 divided doses).  For exposure comparisons, a dose of 250 mg (10 mL) as a single dose was used for C  and a dose of 625 mg/day oral solution 
max
was used for AUC since exposure was higher for the solution than for extended-release capsules (FDA, United States 1986). 
f Extrapolated to a 250 mg dose from reported value after 125 mg phenytoin oral solution single dose (FDA, United States 2002):  C  = 2.268 g/mL, AUC  
max (0-inf)
= 58.2 gh/mL.  PK data are also available for a 100 mg oral solution dose and for extended-release capsules (FDA, United States 1986).  For C , an 
max
accumulation factor of 3.2 was applied that was estimated from the equation:  accumulation = 1/(1  ektau), where k = 0.693/t12 with t12 = 14.924 hours and 
tau = 8 hours (i.e., 1/(1  e0.372) = 1/(1  0.690) = 1/0.31 = 3.2). 
g Extrapolated to 625 mg/day from reported value after 125 mg phenytoin oral solution single dose (FDA, United States 2002):  AUC  = 58.2 gh/mL. 
(0-inf)
 
References 
ANDA #40-420 Bioequivalence Review, Phenytoin FDA, United States Approval package, Clinical Pharmacology and 
Biopharmaceutics Review 040420/S-000. 
FDA, United States Approval Package (Bioequivalence Review) for ANDA 088771 (22 Oct 1986), p. 32.  
FDA, United States Approval Package (Bioequivalence Review) for ANDA 040420 (19 Apr 2002), p. 40. 
Kim SH, Lee IC, Baek HS, Lim JH, Moon C, Shin DH, Kim SH, Park SC, Kim JC. Dose-response effects of diphenylhydantoin on 
pregnant dams and embryo-fetal development in rats. Birth Defects Res B Dev Reprod Toxicol. 2012;95:337-45. 
McClain RM, Langhoff L. Teratogenicity of diphenylhydantoin in the New Zealand white rabbit. Teratology. 1980;21:371-379. 
Medhi B, Prakash A, Joshi R, Byrav DS. Effect of esomeprazole on pharmacokinetics of phenytoin in rabbits. Indian J Physiol 
Pharmacol. 2012;56:382-387.  
91  Contains Nonbinding Recommendations 
 
 
Rowland JR, Binkerd PE, Hendrickx AG. Developmental toxicity and pharmacokinetics of oral and intravenous phenytoin in the rat. 
Reprod Toxicol. 1990;4:191-202.  
U.S. label Dilantin oral solution. 
U.S. label Dilantin extended release capsules. 
 
POMALIDOMIDE 
CAS No.:  19171-19-8 
 
Rat  Rat LOAEL Rat Findings  Rabbit  Rabbit  Rabbit Findings  Human  Margins  Notes 
NOAEL  Dose   NOAEL  LOAEL Dose   Dose   NOAEL/Human 
Dose   C   Dose   C   C   LOAEL/Human 
max max max
C   AUC  C   AUC  AUC 
max max
AUC  AUC 
NOAEL  25 mg/kg  absence of  NOAEL  10 mg/kg  interventricular septal  4 mg per day  NOAEL:    
not  oral GD6-17  urinary  not  GD7-19  defects; misaligned,  x21 (2.4  rat 
identified [FDA,  bladder and  identified [FDA, United  fused, or small caudal  mg/m2/day)  NOAEL not 
United States thyroid gland,  States 2013a]  vertebrae    identified  
2013a]  fusion, and    C  = 0.079  rabbit 
max
  misalignment  C  = 0.072  g/mLc  NOAEL not 
max
C  = 2.7  of lumbar and  g/mLb  AUC  =  identified  
max (0-24h)
g/mLa   thoracic  AUC = 0.418  0.402   
AUC  =  vertebral  gh/mLb  gh/mLd  LOAEL: 
(0-24)
34.3  elements  rat 
gh/mLa  (vertebral,  C  = 34 
max
central and/or  (2.7/0.079) 
neural arches)  AUC = 85 
  (34.3/0.402) 
resorptions;   
increased post- rabbit 
implantation  C  = 0.9 
max
(0.072/0.079) 
92  Contains Nonbinding Recommendations 
 
 
loss, decreased  AUC = 1.0 
viable fetuses  (0.418/0.402) 
a Actual value on GD17 after 25 mg/kg pomalidomide oral dose in pregnant Sprague Dawley rats (FDA, United States 2013a, p. 152):  C  = 2.729 g/mL, 
max
AUC  = 34.34 gh/mL. 
(0-24h)
b Actual value on GD17 after 10 mg/kg pomalidomide oral dose in pregnant New Zealand White rabbits (FDA, United States 2013a, p. 163):  C  = 0.072 
max
g/mL, AUC = 0.418 gh/mL. 

c Actual value after 4 mg pomalidomide oral dose for 8 days in multiple myeloma subjects (FDA, United States 2013b, p. 24):  C  = 0.079 g/mL. 
max
d Actual value after 4 mg pomalidomide oral dose for 4 weeks (FDA, United States 2013a, p. 180):  AUC  = 0.402 gh/mL. 
(0-24h)
 
References 
FDA, United States Pharmtox Review for Pomalyst, NDA 204026 (08 Feb 2013a), pp. 149-156, 158-170, 178-180. 
FDA, United States ClinPharm Review for Pomalyst, NDA 204026 (08 Feb 2013b), p. 25. 
Additional References Evaluated 
Gay F, Mina R, Troia R, Bringhen S. Pharmacokinetic evaluation of pomalidomide for the treatment of myeloma. Expert Opin Drug 
Metab Toxicol. 2013;9:1517-1527. [review article, data from Hoffman] 
Hoffmann M, Kasserra C, Reyes J, Schafer P, Kosek J, Capone L, et al. Absorption, metabolism and excretion of [14C]pomalidomide 
in humans following oral administration. Cancer Chemother Pharmacol. 2013;71:489-501. [PK in healthy volunteers, used data for 
patients from FDA, United States reviews] 
   
93  Contains Nonbinding Recommendations 
 
 
RIBAVIRIN 
CAS No.:  36791-04-5 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit NOAEL Rabbit  Rabbit  Notes 
Dose  Dose  Dose  LOAEL  Findings 
C   C   C   Dose 
max max max
AUC  AUC  AUC  C  
max
AUC 
0.3 mg/kg oral  1.0 mg/kg oral  hydrocephaly, retinal 0.3 mg/kg oral  1.0 mg/kg oral  anomalous  Ribavirin undergoes 
GD6-15 (FDA,  GD6-15 (FDA,  folds, diaphragmic  GD6-18 (FDA,  GD6-18 (FDA,  cervicothoracic significant 1st pass 
United States, p.  United States, p.  hernia, displaced  United States, p.  United States,  arteries  metabolism. As a 
64)  64)  adrenal, displaced  68)  p. 68)  prodrug, it is rapidly 
    oesophagus, vascular     anabolized to ribavirin 
C  = 3.8  C  = 12.7  defects; extra  No rabbit PK  No rabbit PK  monophosphate and 
max max
ng/mLa  ng/mLa  vertabra, scoliosis,  data found  data found  ribavirin triphosphate, 
AUC = 8.28  AUC = 27.6  fused ribs and  which play a role in its 
ngh/mLa  ngh/mLa  vertebrae, split  antiviral activity (Dixit).  
sternum,  It is also deribosylated 
ectrodactyly,  to triazole carboxamide 
malrotated hind  (Lin).  The contribution 
limbs; increased  of each of these 
post-implantation  metabolites to the 
loss  developmental effects 
in rats is unknown. 
a Extrapolated from reported value in plasma after 10 mg/kg ribavirin oral single dose in female Sprague Dawley rats (FDA, United States, p. 76):  C  = 0.127 
max
g/mL, AUC = 0.276 gh/mL. Note 10-fold extrapolation. 
References 
FDA, United States. Pharmacology Review NDA 020903 (18 May 1998). 
Additional References Evaluated 
Dixit NM, Perelson AS. The metabolism, pharmacokinetics and mechanisms of antiviral activity of ribavirin against hepatitis C virus. 
Cell Mol Life Sci. 2006;63:832-842. 
94  Contains Nonbinding Recommendations 
 
 
Liao S, Jin X, Li J, Zhang T, Zhang W, Shi W, et al. Effects of silymarin, glycyrrhizin, and oxymatrine on the pharmacokinetics of 
ribavirin and its major metabolite in rats. Phytother Res. 2016;30:618-626. [at 30 mg/kg in fasted male Sprague Dawley rats:  C  = 
max
1.36 g/mL, AUC  = 14.7 gh/mL] 
(0-inf)
Lin CC, Yeh LT, Luu T, Lourenco D, Lau JY. Pharmacokinetics and metabolism of [14C]ribavirin in rats and cynomolgus monkeys. 
Antimicrob Agents Chemother. 2003;47:1395-1398. [at 30 mg/kg in fasted male Sprague Dawley rats:  C  = 0.433 g/mL, AUC
max (0-
 = 3.04 gh/mL] 
inf)
95  Contains Nonbinding Recommendations 
 
 
TACROLIMUS 
CAS No.:  104987-11-3 
 
Rat NOAEL   Rat LOAEL  Rat Findings  Rabbit NOAEL  Rabbit LOAEL  Rabbit Findings  Notes 
Dose  Dose  Dose  Dose 
C   C   C   C  
max max max max
AUC  AUC  AUC  AUC 
1.0 mg/kg oral  3.2 mg/kg oral  slight increase  0.32 mg/kg oral  1.0 mg/kg oral  ventricular   Maternal 
GD7-17 (FDA,  GD7-17 (FDA,  in post- GD6-18 (FDA,  GD6-18 (FDA,  hypoplasia,  toxicity seen in 
United States, p.  United States, p.  implantation  United States, p.  United States, p.  interventricular septal  both rats and 
18)  18)  loss (late  19)  19)  defect, bulbous aortic  rabbits at 
    resorptions)      arch and stenosis of  LOAEL 
Cmax = 2.9  Cmax = 20  Cmax = 0.93  Cmax =  2.9  arch and ductus   Ratio of blood: 
ng/mLa  ng/mLb  ng/mLc  ng/mLc  arteriosus,  plasma is 4:1 
AUC(0-inf) = 10.9  AUC(0-inf) = 68.9  AUC = 17.6  AUC = 55  omphalocele,   Metabolites are 
ngh/mLa  ngh/mLb  gh/mLc  ngh/mLc  gallbladder agenesis,  3-fold parent 
skeletal   99% protein 
malformations; 
bound 
increased post-
implantation loss, 
decreased litter size 
a Actual values in plasma after 1.0 mg/kg tacrolimus oral single dose in male rats (FDA, United States, p. 25):  C  = 2.9 ng/mL, AUC  = 10.9 ngh/mL. 
max (0-inf)
b Actual values in plasma after 3.2 mg/kg tacrolimus oral single dose in male rats (FDA, United States, p. 25):  C  = 20 ng/mL, AUC  = 68.9 ngh/mL. 
max (0-inf)
c Extrapolated from reported value after 2 mg/kg tacrolimus oral single dose in NZW rabbits (Piekoszewski):  C  = 5.79 ng/mL, AUC = 110 ngh/mL. 
max
References 
FDA, United States. Pharmacology Review NDA 50-708/50-709 (08 Apr 1994). 
Piekoszewski W, Chow FS, Jusko WJ. Disposition of tacrolimus (FK 506) in rabbits. Role of red blood cell binding in hepatic 
clearance. Drug Metab Dispos. 1993;21:690-698. 
 
96  Contains Nonbinding Recommendations 
 
 
Additional References Evaluated 
Iwasaki K, Shiraga T, Nagase K, Hirano K, Nozaki K, Noda K. Pharmacokinetic study of FK 506 in the rat. Transplant Proc. 
1991;23:2757-2759. 
97  Contains Nonbinding Recommendations 
 
 
THALIDOMIDE 
CAS No.:  50-35-1 
 
Rat NOAEL  Rat LOAEL  Rat  Rabbit  Rabbit  Rabbit Findings  Human  Margins  Notes 
Dose   Dose   Findingsa NOAEL  LOAEL  Dose   NOAEL/Human 
C   C   Dose   Dose   C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC 
10 mg/kgb  50 mg/kgb   decreased 20 mg/kg oral  60 mg/kg oral   resorptions  50 mg oralf  NOAEL:   
[Janer]  [Newman,  implanta- GD7-19  GD7-19   rotated or flexed    rat 
  Schardein]  tion sites  [Christian]  [Christian]  limbs (4/38  Cmax = 0.62  Cmax = 1.6 
        fetuses at 60  g/mLg  (0.97/0.62) 
Cmax =  Cmax =      mg/kg and 15/25  AUC(0-inf) =  AUC = 2.2 
0.97g/mLc  4.87g/mLc  at GD19  at GD19  fetuses at 180  4.9 gh/mLg  (10.75/4.9) 
AUC(0-24h) =  AUC(0-24h) =  Cmax = 0.82  Cmax = 2.16  mg/kg)  rabbit 
10.75  53.75  g/mLd  g/mLe   hydrocephaly  Cmax = 1.3 
gh/mLc  gh/mLc  AUC(0-24h) =  AUC(0-24h) =  (n=2/38)   (0.82/0.62) 
4.18  14.4 g.h/mLe  increased post- AUC = 0.9 
gh/mLd  (4.18/4.9) 
implantation loss, 
 
including dead 
LOAEL: 
fetuses, and 
rat 
numerous 
C  = 7.9 
external and  max
(4.87/0.62) 
visceral 
AUC = 11.0 
malformations at 
(53.75/4.9) 
180 mg/kg 
rabbit 
C  = 3.5 
max
(2.16/0.62) 
AUC = 2.9 
(14.4/4.9) 
98  Contains Nonbinding Recommendations 
 
 
a Numerous developmental toxicity studies in rats have been reported in the literature with a variety of divergent results in different strains (Newman, Neubert, 
Janer, Schardein).  Many of these older studies do not meet todays standards for design.  Although malformations cannot be reproducibly induced, 
embryolethality appears to be a common effect at doses 100 mg/kg (Newman).   
b Based on literature reviews by Newman and Schardein, a dose of 50 mg/kg was chosen as the LOAEL.  Based on review by Janer, 10 mg/kg appeared to be the 
highest dose with no evidence of developmental toxicity. 
c Extrapolated or actual value after 50 mg/kg thalidomide oral dose for 8 days in female Fischer rats (FDA, United States p. 86):  C  = 4.87 g/mL, AUC  
max (0-24h)
= 53.75 gh/mL.  PK data are also available after 30 mg/k oral single dose in female Fischer rats (FDA, United States, p. 22, 91):  C  = 10.4 g/mL, 
max
AUC  = 63.99 gh/mL; and after a 100 mg/kg oral single dose in male Sprague Dawley rats (FDA, United States, p. 73):  C  = 21.60 g/mL, AUC
(0-18h) max (0-
 = 348.5 gh/mL. 
48h)
d Actual value after 20 mg/kg thalidomide oral doses in pregnant New Zealand White rabbits (Christian).  GD7:  C  = 1.77 g/mL, AUC  = 13.4 gh/mL; 
max (0-24h)
GD19:  C  = 0.824 g/mL, AUC  = 4.18 gh/mL. 
max (0-24h)
e Actual value after 60 mg/kg thalidomide oral doses in pregnant New Zealand White rabbits (Christian).  GD7:  C  = 6.39 g/mL, AUC  = 78.7 gh/mL; 
max (0-24h)
GD19:  C  = 2.16 g/mL, AUC  = 14.4 gh/mL.   
max (0-24h)
f Currently approved doses range from 100 to 400 mg/day.  A dose of 50 mg was used for PK comparisons because that was the lowest dose used to treat 
insomnia when thalidomide was first developed.  Also, one 50 mg tablet of thalidomide during the time-sensitive window is sufficient to cause birth defects 
in 50% of pregnancies (Vargesson). 
g Actual value after 50 mg single dose to healthy volunteers (Teo, U.S. label):  C  = 0.62 g/mL, AUC = 4.90 gh/mL.  
max
 
References 
Christian MS, Laskin OL, Sharper V, Hoberman A, Stirling DI, Latriano L.  Evaluation of the developmental toxicity of lenalidomide 
in rabbits. Birth Defects Res B Dev Reprod Toxicol. 2007;80:188-207. 
FDA, United States.  Pharmtox review NDA 020785 (11 May 1998).   
Janer G, Slob W, Hakkert BC, Vermeire T, Piersma AH.  A retrospective analysis of developmental toxicity studies in rat and rabbit: 
what is the added value of the rabbit as an additional test species? Regul Toxicol Pharmacol. 2008;50:206-217. 
Neubert R, Neubert D. Peculiarities and possible mode of actions of thalidomide. In: Kavlock RJ, Daston GP, editors.  Handbook of 
experimental pharmacology 124: Drug toxicity in embryonic development II. New York: Springer-Verlag; 1997. p. 41-119. 
Newman LM, Johnson EM, Staples RE.  Assessment of the effectiveness of animal developmental toxicity testing for human safety. 
Reprod Toxicol. 1993;7:359-390.  
99  Contains Nonbinding Recommendations 
 
 
Schardein JL, Macina OT.  Human developmental toxicants: aspects of toxicology and chemistry. Boca Raton: CRC Press; 2007. p. 
127-141. 
Teo SK, Colburn WA, Tracewell WG, Kook KA, Stirling DI, Jaworsky MS, Scheffler MA, Thomas SD, Laskin OL.  Clinical 
pharmacokinetics of thalidomide. Clin Pharmacokinet. 2004;43:311-327. 
Vargesson N.  Thalidomide embryopathy: an enigmatic challenge. ISRN Development Biol. 2013;2013:Article ID 241016.  
http://dx.doi.org/10.1155/2013/241016.  
U.S. label Thalomid. 
 
Additional References Evaluated 
Brock N, [Experimental contribution to the testing of teratogenic drug effects in the laboratory rat].  Naunyn-Schmiedebergs Archiv 
fur experimentelle Pathologie und Pharmakologie. 1964;249:117-145 [500 mg/kg only dose tested]. 
EMA Assessment Report for Thalidomide Pharmion. EMEA/176582/2008, p. 13. [same PK values as FDA, United States review, 
AUC  = 55.25 gh/mL at 50 mg/kg on D8] 
(0-inf)
Eriksson T, Riesbeck K, Ostraat O, Ekberg H, Bjorkman S.  Drug exposure and flow cytometry analyses in a thalidomide treatment 
schedule that prolongs rat cardiac graft survival. Transplant Proc. 1992;24:2560-2561.  [no PK parameters published] 
FDA, United States.  Pharmtox review NDA 021430 (23 Nov 2005).  [review for multiple myeloma, no new PK or teratology data 
from NDA 020785] 
FDA, United States.  Pharmtox review NDA 204026 (08 Feb 2013).  [Thalidomide was used as a positive control in the rabbit 
developmental toxicity study ata dose of 180 mg/kg.] 
100  Contains Nonbinding Recommendations 
 
 
TOPIRAMATE 
CAS No.:  97240-79-4 
 
Rat  Rat  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
NOAEL   LOAEL  NOAEL   LOAEL  Findings  Dose  NOAEL/Human 
Dose  Dose  Dose  Dose  C   LOAEL/Human 
max
C   C   C   C   AUC 
max max max max
AUC  AUC  AUC  AUC 
100 mg/kg  400  ectrodactyly,  20 mg/kg  35 mg/kg  Embryo- 400 mg/day in NOAEL:   In rats: Although 
oral GD6-15  mg/kg  hydronephrosis oral GD6-18 oral GD6-18 fetal  two divided  rat  reduced fetal 
[U.S. label,  oral GD6- [U.S. label,  [U.S. label,  mortality  doses  Cmax = 3.6  BW and 
FDA, United 15  FDA,  FDA,  at 35    (49/13.5)   increased 
States  [U.S.  United  United  mg/kg   Cmax = 13.5  AUC = 3.9  incidence of 
1996a]  label,  States  States  g/mLe  (893/229)  structural 
  FDA,  1996a]  1996a]  AUC = 229  rabbit  variations were 
Cmax = 49  United      gh/mLe  Cmax = 1.0  observed at 20 
g/mLa  States  Cmax = 13  Cmax = 23  (13/13.5)  mg/kg, the 
AUC = 893  1996a]  g/mLd  g/mLd  AUC = 0.3  NOAEL for 
gh/mLb    AUC = 67  AUC = 117  (67/229)  MEFL is 
Cmax =  gh/mLd   gh/mLd    assumed to be 
168.6  LOAEL:  100 mg/kg  
g/mLc  rat   In rats: Clinical 
AUC =  Cmax = 12.5  signs of maternal 
3573  (169/13.5)   toxicity were 
gh/mLb  AUC = 15.6  seen at 400 
(3573/229)  mg/kg and 
  maternal BW 
rabbit  gain was reduced 
Cmax = 1.7  at 100 mg/kg 
(23/13.5)   In rabbits: 
AUC = 0.5  maternal toxicity 
(117/229)  (decreased BW 
gain, clinical 
101  Contains Nonbinding Recommendations 
 
 
signs, and/or 
mortality) was 
seen at 35 
mg/kg 
a Extrapolated from reported value after 200 mg/kg topiramate for GD12-15 (4 days) in pregnant female Sprague Dawley rats (FDA, United States, p. 48):  C  
1.5h
= 97.3 g/mL. 
b Extrapolated from reported value after 30 mg/kg topiramate for 8 days in female Sprague Dawley rats (FDA, United States, p. 12):  C  = 22.2 g/mL, AUC = 
max
268.2 gh/mL. 
c Actual value after 400 mg/kg topiramate for GD12-15 (4 days) in pregnant female Sprague Dawley rats (FDA, United States, p. 48):  C  = 168.6 g/mL. 
1.5h
d Extrapolated from reported value after 60 mg/kg topiramate for 14 days in female New Zealand White rabbits (FDA, United States, p. 13):  C  =39.1 g/mL, 
max
AUC = 201 gh/mL. 
e Extrapolated from reported value after 100 mg/kg topiramate BID oral for 14 days (FDA, United States 1996b):  C  = 6.76 g/mL, AUC  = 57.2 
max (0-24h)
gh/mL.  PK data at a number of other doses and schedules and in combination with other drugs are also available (FDA, United States 1995b, Bialer). 
 
References 
Bialer M, Doose DR, Murthy B, Curtin C, Wang SS, Twyman RE, et al. Pharmacokinetic interactions of topiramate. Clin 
Pharmacokinet. 2004;43:763-780. 
FDA, United States. Pharmtox Review NDA 020505 (24 Dec 1996a). 
FDA, United States. Clinical Pharmacology Review NDA 020505 (24 Dec 1996b), p. 39. 
   
102  Contains Nonbinding Recommendations 
 
 
TRIMETHADIONE 
CAS No.:  127-48-0 
 
Rat NOAEL  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
Dose  Dose  NOAEL  LOAEL Findings Dose  NOAEL/Human 
C   C   Dose  Dose  C   LOAEL/Human 
max max max
AUC  AUC  C   C   AUC 
max max
AUC  AUC 
60 mg/kg oral  240 mg/kg  240 mg/kg  No rabbit data  No  No  600 mg QID  Trimethadione  Dimethadione 
GD6-15  oral GD6-18  GD6-15  found  rabbit  rabbit  (10 mg/kg x4)  NOAEL:  is the only 
[Buttar 1976]  [Buttar 1976]  [Buttar]:    data  data  [highest dose,  rat  metabolite, 
    adverse fetal    found  found  U.S. label]  C  = 1.4  has much 
max
Trimethadione Trimethadione effects on  Trimethadione    (58.9/42.75)  higher 
C  =  58.9  C  = 235  survival and  AUC = 10.78  Trimethadione AUC = 0.2  exposures 
max max
g/mLa  g/mLa  litter size  gh/mLc  C  = 42.75  (203/1000)  than 
max
AUC  =  AUC  =    g/mLd  rabbit  trimethadione, 
(0-inf) (0-inf)
203 gh/mLa  814 gh/mLa  250 mg/kg  AUC  =  NOAEL not  and is a 
(0-inf)
    GD7-18  1000  identified  confirmed 
Dimethadione  Dimethadione  [Vorhees]:  gh/mLd    teratogen 
C  =  C  =  391  embryolethality,    LOAEL:  (Buttar 1978).  
max max
97.7g/mLb  g/mLb  malformations  Dimethadione  rat  Thus, margins 
AUC  =  AUC  =  (primarily  C  = 1251  C  = 5.5  for 
(0-inf) (0-inf) max max
4872  19,488  cardiac, with a  g/mLe  (235/42.75)  dimethadione 
gh/mLb  gh/mLb  lower incidence  AUC  =  AUC = 0.8  are also listed. 
(0-inf)
of esophageal  36,670  (814/1000) 
and kidney  gh/mLe  rabbit 
defects)  LOAEL not 
identified 
 
Dimethadione 
NOAEL: 
rat 
103  Contains Nonbinding Recommendations 
 
 
C  = 0.1 
max
(97.7/1251) 
AUC = 0.1 
(4872/36670) 
 
LOAEL: 
rat 
C  = 0.3 
max
(391/1251) 
AUC = 0.5 
(19488/36670) 
a Extrapolated from reported value after 100 mg/kg trimethadione oral single dose in male Wistar rats (Tanaka 1981):  C  = 98.1 g/mL, AUC  = 339 
max (0-inf)
gh/mL.  
b Extrapolated from reported value after 100 mg/kg trimethadione oral single dose in male Wistar rats (Tanaka 1981):  dimethadione C  = 162.8 g/mL, 
max
AUC  = 8120 gh/mL. 
(0-inf)
c Actual value after 4 mg/kg trimethadione intravenous single dose in Japanese White rabbits (Tanaka 1999):  AUC  = 10.78 gh/mL calculated from Cl = 
(0-inf)
0.371 L/(kgh). 
d Extrapolated from reported value after 4 mg/kg trimethadione oral single dose (Kobayashi):  C  = 6.0 g/mL, AUC  = 100.1 gh/mL.  For C , an 
max (0-inf) max
accumulation factor of 2.85 was applied that was estimated from the equation:  accumulation = 1/(1  ektau), where k = 0.693/t12 with t12 = 9.6 hours and tau 
= 6 hours (i.e., 1/(1  e0433) = 1/(1  0.649) = 1/0.351 = 2.85). 
e Extrapolated from reported value after 4 mg/kg trimethadione oral single dose (Kobayashi):  dimethadione C  = 12.83 g/mL, AUC  = 3667 gh/mL.  
max (0-inf)
For C , an accumulation factor of 39 was applied that was estimated from the equation:  accumulation = 1/(1  ektau), where k = 0.693/t12 with t12 = 160 
max
hours and tau = 6 hours (i.e., 1/(1  e0.026) = 1/(1  0.974) = 1/0.026 = 39). 
 
References 
Buttar HS, Dupui I, Khera KS. Fetotoxicity of trimethadione and paramethadione in rats. Toxicol Appl Pharmacol. 1976;37:126. 
[abstract] 
Buttar HS, Dupuis I, Khera KS. Dimethadione-induced fetotoxicity in rats. Toxicology. 1978;9:155-164. 
Tanaka E, Kinoshita H, Yamamoto T, Kuroiwa Y, Takabatake E. Pharmacokinetic studies of trimethadione and its metabolite in rats 
with chemical-induced liver injury. J Pharmacobiodyn. 1981;4:576-583.  
104  Contains Nonbinding Recommendations 
 
 
Tanaka E, Ishikawa A, Horie T. In vivo and in vitro trimethadione oxidation activity of the liver from various animal species including 
mouse, hamster, rat, rabbit, dog, monkey and human. Hum Exp Toxicol. 1999;18:12-16. 
Vorhees CV. Fetal anticonvulsant syndrome in rats: dose- and period-response relationships of prenatal diphenylhydantoin, 
trimethadione and phenobarbital exposure on the structural and functional development of the offspring. J Pharmacol Exp Ther. 
1983;227:274-287. 
U.S. label trimethadione. 
Additional References Evaluated 
Midha KK.  Metabolism and disposition of trimethadione in pregnant rats. Epilepsia. 1979;20:417-423. [Only useful data are 
concentrations at 6 hours after last dose following dosing 60 and 240 mg/kg GD6-15: at 60 mg/kg , C  = 11.3 g/mL] 
6h
Schardein JL, Schwetz BA, Kenel MF. Species sensitivities and prediction of teratogenic potential. Environ Health Perspect. 
1985;61:55-67.  [Claimed rats are an insensitive species for detecting trimethadione teratogenesis.] 
Tanaka E, Yoshida T, Kuroiwa Y. Dose-independent pharmacokinetics of trimethadione and its metabolite in rats. J Pharm Sci. 
1985;74:340-341. [PK values after 4 mg/kg trimethadione oral single dose in male Wistar rats: trimethadione C  = 3.0 g/mL, 
max
AUC  = 8.21 gh/mL, and dimethadione C  = 10.2 g/mL, AUC  = 465.8 gh/mL.  The values after 100 mg/kg  (Tanaka 
(0-inf) max (0-inf)
1981) were used instead.] 
Taylor JD, Bertcher EL. The determination and distribution of trimethadione (tridione) in animal tissues. J Pharmacol Exp Ther. 
1952;106:277-285. [levels in rabbit brain after 1000 mg/kg IP] 
105  Contains Nonbinding Recommendations 
 
 
VALPROIC ACID 
CAS No.:  99-66-1 (sodium valproate: 1069-66-5) 
 
Rat Findings  Rabbit  Rabbit  Rabbit Findings Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL   LOAEL  Dose  NOAEL/Human 
Dose  Dose 
Dose  Dose  C    LOAEL/Human  Notes 
max
C   C  
max max
C   C    AUC   
max max
AUC  AUC 
AUC  AUC 
65 mg/kg oral  200 mg/kg  hydronephrosis, 150 mg/kg oral  350 mg/kg resorptions;  60  NOAEL:   
GD6-15, SD  oral, SD rats,  cardiovascular  GD6-18   oral GD6- external  mg/kg/day  rat 
rats [FDA,  GD7-18  defects  [FDA, United  18   abnormalities  oral in 2  C  = 0.4 
max
United States,  [Voorhees],    States, 1977]  [FDA,  (cleft palate,  divided  (73.8/205) 
1995]  GD8-17    United  umbilical  doses (30  AUC = 0.06 
  [Binkerd];  C  = 410  States,  hernia, bilateral  mg/kg/dose)  (230/4180) 
max
  [U.S.  g/mLb  1977]  talipes,  [highest  rabbit 
  Depacon  AUC = 690    exencephaly,  approved  C  = 2.0 
max
C  = 73.8  label]  gh/mLb  C  = 957 hypoplastic  dose, U.S.  (410/205) 
max max
g/mLa      g/mLb  ears,  Depakote  AUC = 0.2 
AUC = 230  C  = 227  AUC =  gastrochisis,  and  (690/4180) 
max
gh/mLa  g/mLa  1610  bilateral  Depakene   
AUC = 707  gh/mLb  talipes);  labels]  LOAEL: 
gh/mLa   visceral    rat 
malformations    C  = 1.1 
max
(intraventricular  C  = 205  (227/205) 
max
septal defects,  g/mLc  AUC = 0.2 
misshapen  AUC  =  (707/4180) 
(0-inf)
ventricle, renal  4180  rabbit 
agenesis);  gh/mLd  C  = 4.7 
max
skeletal  (957/205) 
malformations  AUC = 0.4 
(supernumerary  (1610/4180) 
ribs, fused ribs) 
106  Contains Nonbinding Recommendations 
 
 
a Extrapolated or actual value after 200 mg/kg valproic acid oral dose on GD17 in pregnant Sprague Dawley rats (Binkerd):  C  = 227 g/mL, AUC = 707 
max
gh/mL.  PK data are also available on GD8:  C  = 341g/mL, AUC = 1019 gh/mL 
max
b Extrapolated from reported value after 70 mg/kg valproic acid oral single dose in male New Zealand White rabbits (Bourin):  C  = 191.3 g/mL, AUC  
max (0-inf)
= 322 gh/mL. Rabbit PK data are also available after 50 mg/kg oral (FDA, United States), 20 mg/kg oral (van Jaarsveld), 43 mg/kg intravenous 
(Nakashima), and 75 mg/kg intravenous (Yokogawa). 
c Extrapolated from reported value after 1000 mg valproic acid oral BID for 5 days (Nitsche):  C  = 114 g/mL. 
max
d Extrapolated from reported value after 1000 mg valproic acid oral single dose (Nitsche):  AUC  = 1161 gh/mL. 
(0-inf)
 
References 
Binkerd PE, Rowland JM, Nau H, Hendrickx AG. Evaluation of valproic acid (VPA) developmental toxicity and pharmacokinetics in 
Sprague-Dawley rats. Fundam Appl Toxicol. 1988;11:485-493.  
Bourin M, Guenzet J, Thomare P, Kergueris MF, Ortega A, Larousse C. Effects of administration route on valproate pharmacokinetics 
in the rabbit. Fundam Clin Pharmacol. 1991;5:331-339. 
FDA, United States Approval Package, NDA 018081 (S-001, S-025) and 018082 (S-008) (1995), Part 2. p. 7-8, 10, 12, 28. 
FDA, United States Pharmtox reviews IND 011152 (March 1977), p. 31-32, 34. 
Nitsche V, Mascher H. The pharmacokinetics of valproic acid after oral and parenteral administration in healthy volunteers. Epilepsia. 
1982;23:153-162 
Ong LL, Schardein JL, Petrere JA, Sakowski R, Jordan H, Humphrey RR, et al. Teratogenesis of calcium valproate in rats. Fundam 
Appl Toxicol. 198;3:121-126. 
Vorhees CV. Teratogenicity and developmental toxicity of valproic acid in rats. Teratology. 1987;35(2):195-202.   
U.S. Depacon (valproate injection) label. 
U.S. Depakene (valproate capsule) label. 
U.S. Depakote (valproex tablets) label. 
 
Additional References Evaluated 
FDA, United States Pharmtox reviews IND 011152 (1977), p. 48.  [after 50 mg/kg [14C]valproic acid oral single dose in rabbits (FDA, 
United States):  C  = 86 g/mL] 
max
107  Contains Nonbinding Recommendations 
 
 
Katayama H, Mizukami K, Yasuda M, Hatae T. Effects of carnitine on valproic acid pharmacokinetics in rats. J Pharm Sci. 
2016;105:3199-3204. [PK data in male Wistar rats after 32 mg/kg oral: C  = 40.7 g/mL, AUC  = 3458 gmin/mL (57.6 
max (0-inf)
gh/mL)] 
Nakashima M, Takeuchi N, Hamada M, Matsuyama K, Ichikawa M, Goto S. In vivo microdialysis for pharmacokinetic investigations: 
a plasma protein binding study of valproate in rabbits. Biol Pharm Bull. 1994;17:1630-1634.  [PK after 43 mg/kg intravenous valproic 
acid in anesthetized male Japanese Albino rabbits: C  = 157 g/mL, AUC  = 308 gh/mL] 
0 (0-inf)
Rha JH, Jang IJ, Lee KH, Chong WS, Shin SG, Lee N, Myung HJ. Pharmacokinetic comparison of two valproic acid formulations--a 
plain and a controlled release enteric-coated tablets. J Korean Med Sci. 1993 Aug;8(4):251-256. 
van Jaarsveld MF, Walubo A, du Plessis JB. Interaction between valproic acid and acyclovir after intravenous and oral administration 
in a rabbit model. Basic Clin Pharmacol Toxicol. 2007;101:434-440. [PK after 20 mg/kg valproic acid oral single dose in New 
Zealand White rabbts:  C  = 64.2 g/mL, AUC  = 227 gh/mL]. 
max (0-inf)
Yokogawa K, Iwashita S, Kubota A, Sasaki Y, Ishizaki J, Kawahara M, Matsushita R, Kimura K, Ichimura F, Miyamoto K. Effect of 
meropenem on disposition kinetics of valproate and its metabolites in rabbits. Pharm Res. 2001;18:1320-1326.  [PK after 75 mg/kg 
intravenous dose in male albino rabbits:  C  = 238 g/mL, AUC  = 17.5 mgmin/L (292 gh/mL)] 
max (0-6h)
Zaccara G, Messori A, Moroni F. Clinical pharmacokinetics of valproic acid--1988. Clin Pharmacokinet. 1988;15:367-389. 
108  Contains Nonbinding Recommendations 
 
 
VISMODEGIB 
CAS No.:  879085-55-9 
 
Rat  Rat LOAEL  Rat Findings  Rabbit  Rabbit  Rabbit  Human  Margins  Notes 
NOAEL  Dose  NOAEL  LOAEL  Findings Dose  NOAEL/Human 
Dose  C   Dose  Dose  C   LOAEL/Human 
max max
C   AUC  C   C   AUC 
max max max
AUC  AUC  AUC 
NOAEL  10 mg/kg GD6-17  malformations no rabbit  no rabbit  no rabbit  150 mg oral  NOAEL:  MW = 
not  oral [FDA, United  included  data found  data found  data    rat:    421.3 
identified States, 2011]  absent and/or  found  C  = 13.0  NOAEL not 
max
  fused digits  g/mLb  identified 
C  = 7.22 g/mLa  on the hind  AUC  = 306  rabbit: 
max (0-24h)
AUC  = 50.5  limb, open  gh/mLb  no data found  
(0-24h)
gh/mLa  perineum,   
multiple  LOAEL: 
craniofacial  rat 
anomalies  C  = 0.6 
max
(7.22/13) 
AUC = 0.2 
(50.5/306) 
rabbit 
no data found  
a Reported value after 10 daily oral doses of 10 mg/kg vismodegib in female pregnant Wistar rats (FDA, United States, 2011):  C  = 7.22 g/mL, AUC  = 
max (0-24h)
50.5 gh/mL. 
b Reported value after 14 daily oral doses of 150 mg vismodegib (FDA, United States, 2012):  C  = 30.9 M (13.0 g/mL), AUC  = 727 molh/L (306 
max (0-24h)
gh/mL).  
 
References 
FDA, United States.  Pharmacology Review NDA 203388 (08 Sep 2011), p. 66-69. 
FDA, United States.  Clinical Pharmacology Review NDA 203388 (13 Jan 2012), p. 48.   
109  Contains Nonbinding Recommendations 
 
 
1.3.2  Negative Control Reference Compounds  
 
CETIRIZINE 
CAS No.:  83881-51-0 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human  Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
NOAEL  225 mg/kg oral  225 mg/kg:   NOAEL  Not  No MEFL  10 mg MRHD  NOAEL:  None 
(MEFL)  GD6-15   pre- and  (MEFL)  established  observed    rat (75   
75 mg/kg oral  (FDA, United  post- 135 mg/kg  Exposure values  mg/kg/day) 
GD6-15   States 1989)  implantation oral   after single dose:  C : 136 
max
(FDA, United    loss in  GD6-18   C  = 0.33  (45/0.33) 
max
States 1989)  C  = 128  presence of  (FDA,  g/mLd  AUC: 111 
max
  g/mLa  maternal  United  AUC : 3.0  (334/3.02) 
(0-24h)
C  = 45  AUC = 1010  toxicity  States 1989)  ghr/mLd  Rabbit (135 
max
g/mLa  gh/mLb  (death,    mg/kg/day) 
AUC = 301    clinical  C  = 137  C :  415 
max max
gh/mLb  signs)  g/mLc  (137/0.33) 
    AUC = 642  AUC: 213 
Exposure data    gh/mLc  (642/3.02) 
at lower doses     
8 mg/kg oral   Exposure  LOAEL: 
GD6-15   data at lower  Rat (225 
(FDA, United  doses  mg/kg/day) 
States 1989)  15 mg/kg  C : 388 
max
  oral   (128/0.33) 
C  = 4.6  GD6-18   AUC: 334 
max
g/mLa  (FDA,  (1010/3.02) 
AUC = 32  United  rabbit 
gh/mLb  States 1989)  Not applicable 
110  Contains Nonbinding Recommendations 
 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human  Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
   
25 mg/kg oral   C  = 15 
max
GD6-15   g/mLc 
(FDA, United  AUC = 71 
States 1989)  gh/mLc 
   
C  = 12  45 mg/kg 
max
g/mLa  oral  
AUC = 41  GD6-18  
gh/mLb  (FDA, 
United 
States 1989) 
C  =  51 
max
g/mLc 
AUC = 116 
gh/mLc 
 
a From reported C  values in a 4-week, repeated-dose toxicity study in rats at steady state (day 23) at doses of 25, 75, and 225 mg/kg/day. C  for 8 mg/kg/day 
max max
was linearly extrapolated from these data (FDA, United States 1993, page 4). 
b From reported AUC values in a 4-week, repeated-dose toxicity study in rats at steady state (day 23) at doses of 25 mg/kg/day and 225 mg/kg/day. AUC for 8 
and 75 mg/kg/day were linearly extrapolated from these data (FDA, United States 1993, page 4). 
c From reported C  and AUC values in pregnant rabbits exposed from GD6-18 at steady state (GD18) at doses of 25, 45 and 90 mg/kg/day. C  and AUC for 
max max
15 and 135 mg/kg/day were linearly extrapolated from these data (FDA, United States 1993, page 5). 
d Single administration of 10 mg cetirizine with water (FDA, United States, 2003). 
 
 
111  Contains Nonbinding Recommendations 
 
 
References 
FDA, United States. Pharmacology review of NDA 019835 (11 Apr 1989) part 01, pages 10-11 (rat and rabbit EFD overview). 
FDA, United States. Pharmacology review of NDA 019835 (11 Apr 1989) part 02, pages 10-30 (rat and rabbit EFD summary). 
FDA, United States. Pharmacology review of NDA 019835 (18 Oct 1993), pages 4 (rat PK data) and 5 (rabbit PK data). 
FDA, United States. Clinical Pharmacology and Biopharmaceutics Review of NDA 021621/S-000 (31 Oct 2003) (Clinical AUC, 
single dose, page 11). 
U.S. Label Zyrtec. 
European Union (EU) SmPC Zyrtec. 
 
 
 
SAXAGLIPTIN 
CAS No.:  361442-04-8 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
NOAEL  Not  No MEFL  NOAEL  Not  No MEFL  5 mg MRHD  NOAEL:  BMS-
(MEFL)  established  observed  (MEFL)  established  observed    rat (900  510849 is a 
900 mg/kg oral      200  mg/kg  Exposure  mg/kg/day)  major 
GD6-15     oral   values after    active 
(FDA, United  GD7-19   single dose:  Saxagliptin  metabolite 
States 2009)  (FDA,    C : 10,375  of 
max
  United  Saxagliptin  (249/0.024)  saxagliptin. 
Saxagliptin  States 2009)  C  = 0.024  AUC: 8,294   (U.S. Label 
max
C  = 249    g/mLd  (647/0.078)  and 
max
g/mLa  Saxagliptin    European 
112  Contains Nonbinding Recommendations 
 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
AUC  = 647  C  = 43  AUC :  BMS-510849  public 
0-24 max (0-24h)
gh/mLa  g/mLc  0.078  C : 449  assessment 
max
  AUC  =  ghr/mLd  (21.1/0.047)  reports (EU 
0-24
BMS-510849  111    AUC: 673  EPAR) 
C  = 21.1  gh/mLa  BMS-510849  (144/0.214)  Onglyza) 
max
g/mLb    C  = 0.047   
max
AUC  = 144  BMS-510849  g/mLd  Rabbit (200 
0-24
gh/mLa  C  = 125  AUC :  mg/kg/day) 
max (0-24h)
  g/mLc  0.214   
  AUC  =  ghr/mLd  Saxagliptin 
0-24
Exposure data  434  C : 1,792 
max
at lower doses  gh/mLa  (43/0.024) 
64 mg/kg oral     AUC: 1,423 
GD6-15     (111/0.078) 
  Exposure   
Saxagliptin  data at lower  BMS-510849 
C  = 17.7  doses  C : 2,659 
max max
g/mLa  8 mg/kg oral   (125/0.047) 
AUC  = 23.6  GD7-19   AUC: 2,028 
0-24
gh/mLa    (434/0.214) 
  Saxagliptin   
BMS-510849  C  = 2  LOAEL: 
max
C  = 1.5  g/mLc  rat 
max
g/mLb  AUC  =  Not applicable 
0-24
AUC  = 6.3  2.5  rabbit 
0-24
gh/mLa  gh/mLa  Not applicable 
   
240 mg/kg oral   BMS-510849 
113  Contains Nonbinding Recommendations 
 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
GD6-15   C  = 5 
max
  g/mLc 
Saxagliptin  AUC  = 
0-24
C  = 66.3  7.4 
max
g/mLa  gh/mLa 
AUC  = 121   
0-24
gh/mLa  40 mg/kg 
  oral  
BMS-510849  GD7-19  
C  = 5.6   
max
g/mLb  Saxagliptin 
AUC  = 28.9  C  = 9 
0-24 max
gh/mLa  g/mLc 
AUC  = 
0-24
12.3 
gh/mLa 
 
BMS-510849 
C  = 25 
max
g/mLc 
AUC  = 
0-24
47.9 
gh/mLa 
a From reported AUC values in pregnant rats (GD15) and pregnant rabbits (GD19) at steady state at doses of 64, 240, and 900 mg/kg/day saxagliptin for rat and 
8, 40, and 200 mg/kg/day saxagliptin for rabbit (FDA, United States, 2009, part 02, page 84). 
b From reported C  values in a 4-week repeated-dose toxicity study in female rats at steady state (day 28) at doses of 150, 300, and 225 mg/kg/day, 
max
corresponding to 50, 78, and 139 g/mL for saxagliptin and 4.6, 7.9, and 11 g/mL for the active metabolite. Saxagliptin Cmax values were linearly 
extrapolated from these data (FDA, United States, 2009, part 04, page 56). 
114  Contains Nonbinding Recommendations 
 
 
c From reported C  values in a rabbit EFD study at steady state (GD19) at 40 mg/kg/day saxagliptin (C  8.5 g/mL). Saxagliptin C  values were linearly 
max max max
extrapolated from these data. 
d Single administration of 5 mg saxagliptin (U.S. Label Onglyza, page 12). 
 
 
 
115  Contains Nonbinding Recommendations 
 
 
References 
FDA, United States. Pharmacology Review of NDA 022350/S-000 (3 March 2009) Part 02, page 84 (rat and rabbit AUC data 
Saxagliptin and active metabolite). 
FDA, United States. Pharmacology Review of NDA 022350/S-000 (3 March 2009) Part 03, pages 57-59 (rat and rabbit EFD studies). 
FDA, United States. Pharmacology Review of NDA 022350/S-000 (3 March 2009) Part 04, page 56 (rat Cmax data Saxagliptin and 
active metabolite). 
FDA, United States. Pharmacology Review of NDA 200678Orig1s000 (10 January 2010) for Saxagliptin + metformin, page 44 table 
30 (rabbit Cmax data Saxagliptin and active metabolite). 
U.S. Label Onglyza. 
EU EPAR Onglyza. 
114  Contains Nonbinding Recommendations 
 
 
VILDAGLIPTIN 
CAS No.:  274901-16-5 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
NOAEL  Not established  No MEFL  NOAEL  Not  No MEFL  50 mg BID  NOAEL:   
(MEFL)    observed  (MEFL)  established  observed  MRHD  rat (750 
750 mg/kg    150 mg/kg  (100 mg/day)  mg/kg/day) 
oral     oral      
GD6-17   GD7-20   Exposure values  AUC: 117 
(TGA,  (TGA,  after 50 mg BID  (241/2.06) 
Australia  Australia     
2010)  2010)  AUC : 2.06  Rabbit (150 
(0-24h)
    ghr/mLb  mg/kg/day) 
AUC  =  AUC  =     
0-24 0-24
241 gh/mLa  80 gh/mLa    AUC: 39 
    (80/2.06) 
Exposure data  Exposure   
at lower doses  data at lower  LOAEL: 
75 mg/kg oral   doses  Not applicable 
GD6-17   15 mg/kg 
  oral  
AUC  = 23  GD7-20  
0-24
gh/mLa   
  AUC  = 6 
0-24
225 mg/kg  gh/mLa 
oral    
GD6-17   50 mg/kg 
  oral  
GD7-20  
115  Contains Nonbinding Recommendations 
 
 
Rabbit  Rabbit  Human  Margins 
Rat NOAEL   Rat LOAEL  
NOAEL  LOAEL  Dose  NOAEL/Human 
Dose  Dose  Rat  Rabbit 
Dose  Dose  C   LOAEL/Human Notes 
max
C   C   Findings  Findings 
max max
C   C   AUC 
max max
AUC  AUC 
AUC  AUC 
AUC  = 68   
0-24
gh/mLa  AUC  = 
0-24
19 gh/mLa 
a Calculated from exposure ratios compared to human exposure at MRHD (2.06 ghr/mL at 50 mg BID) of AUC data provided within the rat and rabbit EFD 
studies (TGA, Australia, 2010, page 19). 
b Human exposure data at 50 mg BID (TGA, Australia, 2010, page 14). 
 
 
 
References 
TGA, Australia. Australian Public Assessment Report for Vildagliptin (April 2010) pages 19 (EFD studies); 14, 24 (exposure data); 
and 72 (pregnancy). 
EU EPAR Galvus. 
EU SmPC. Galvus. 
 
 
116