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 Additional copies are available from: Office of Communications, Division of Drug Information Center for Drug Evaluation and Research Food and Drug Administration 10001 New Hampshire Ave., Hillandale Bldg., 4th Floor Silver Spring, MD 20993-0002 Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 Email: druginfo@fda.hhs.gov https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs and/or Office of Communication, Outreach and Development Center for Biologics Evaluation and Research Food and Drug Administration 10903 New Hampshire Ave., Bldg. 71, Room 3128 Silver Spring, MD 20993-0002 Phone: 800-835-4709 or 240-402-8010 Email: ocod@fda.hhs.gov 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 Industry 1 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 pharmaceutical’s 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 pharmaceutical’s 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 FDA’s 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 (Cmax) at the MRHD can be an appropriate endpoint for high dose selection. Doses providing an exposure in pregnant animals > 25˗fold 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, Cmax, minimum plasma concentration (Cmin), body surface 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. Cmax: Maximum plasma concentration. Cmin: Minimum plasma concentration. 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 experience and availability parturition (e.g., • Extensive historical data nonsteroidal anti- inflammatory drugs in late pregnancy) • Less sensitive than humans to fertility perturbations • Limited application for foreign proteins o Limited or no pharmacologic activity o Potential impact of 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 (NHP) physiologically similar to variability across groups humans than other species • Low fecundity • More likely than rodents to o Single offspring 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 assay’s predictions, endpoint(s) assessed, and the applicability domain, should be supported empirically. To qualify 6 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 method’s 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 Cmax LOAEL/Human Notes Cmax Findings Cmax Cmax Cmax AUC 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) Cmax = 1.9 metabolite (Kistler) (Kistler) dilated (Kistler) (Kistler) (1.5/0.79) (free acid) of renal 2 mg/kg: cleft Exposure AUC = 1.8 etretinate Cmax = 1.5 Cmax = 3.0 pelvis no PK no PK palate, skull and tail values at (6.6/3.6) (ethyl ester) µg/mLa µg/mLa data data malformations, steady state: rabbitc AUC = 6.6 AUC = 13.2 30 mg/kg: available available ectrodactyly of the Cmax = 0.79 Cmax = 0.2 µg∙h/mLa µg∙h/mLa cleft fore- and hindfeet µg/mL b (0.2/0.83) palate; and malformations AUC(0-24h): 3.6 AUC = 0.08 malformed of the long bones µg∙h/mLb (2.4/29.4) humeri, radii and LOAEL: ulnae rat Cmax = 3.8 (3.0/0.79) AUC = 3.7 (13.2/3.6) rabbitc Cmax = 0.7 (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 Cmax LOAEL/Human Notes Cmax Findings Cmax Cmax Cmax AUC AUC AUC AUC AUC AUC = 0.2 (7.2/29.4) a Extrapolated from reported values at 5 mg/kg (Brouwer): Cmax = ~1.0 µg/mL from visual inspection of graph, AUC = 4.4 µg∙h/mL. b Steady state values after 21 daily doses administered with food (FDA, United States): Cmax = 0.786 µg/mL, AUC(0-24h) = 3.569 µg∙h/mL. c In the absence of rabbit PK data, Cmax ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 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 Cmax LOAEL/Human Cmax Cmax Cmax Cmax AUC 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 Cmax = 3.5 (salicylic acid) 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 Cmax= 7.08 25.3/48.3) parent and is h Cmax = ~25 Cmax = ~40 severe defects of aspirin: µg/mL rabbitj pharmacologically µg/mLb µg/mLb vertebral and aspirin AUC(0-24h) = Cmax = 32.4 active. Since AUC = 6.6 AUC = 10.5 costal bones; PK data 48.3 µg∙h/mLh (350/10.8) aspirin – 25.3 – 40.5 increased in rabbits AUC = 1.8 concentrations µg∙h/mL b µg∙h/mL b resorptions is not salicylic acid (4200/2294) were often BLQ, available Cmax = 45.2 salicylate salicylate salicylate Gupta (250 µg/mLi LOAEL: exposure data are Cmax = 132 Cmax = 211 mg/kg): salicylate AUC = 1448 rat also reported. µg/mLc µg/mLc ablepharia, Cmax = µg∙h/mLi Cmax = 5.6 AUC = AUC = cranio- 490 (40/7.08) salicylic acid 8333 13,333 rachischisis, µg/mLg AUC = 0.2 – 0.8 MW = 138.12 µg∙h/mLd µg∙h/mLd exencephaly, AUC = (10.5/48.3 to g/mol various low 4865 40.5/48.3) occurrence head µg∙h/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, Cmax: 2.9 various low (132/45.2) occurrence AUC: 5.8 cardio-vascular (8333/1448) malformations, rabbit VSD, DH, Cmax: 10.8 hypoplastic (490/45.2) kidney, AUC: 3.4 hypoplastic testes; (4865/1448) decreased implantations, LOAEL: increased rat resorptions and Cmax: 4.7 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): Cmax = 40 µg/mL (visual inspection of Figure 1); AUC = 629 – 2430 µg∙min/mL (recalculated as 10.5 – 40.5 µg∙h/mL). Cmax data for aspirin is also available in Wistar rats administered 200 mg/kg (Higgs). c Extrapolated from reported value at 200 mg/kg oral dose in Sprague Dawley rats (Wientjes): Cmax = 211 µg/mL (Table 5); no AUC values were reported for salicylate. Cmax data for salicylate is also available in Wistar rats administered 200 mg/kg (Higgs) and in Fischer rats administered 90 mg/kg (Kapetanovica). d Extrapolated from reported value at oral 90 mg/kg/day on D15 in Fischer rats (Kapetanovica): AUC = 6000 µg∙h/mL. Note the AUC in Table 2 is reported as 6.0 µg∙h/mL, but this is incompatible with the plot in Figure 1a. An AUC estimated from concentrations visually estimated from Figure 1a was 5319 µg∙h/mL (personal calculation); thus it is assumed that the reported value should actually be 6000 µg∙h/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): Cmax = 70 µg/mL and AUC = 695 µg∙h/mL. Note that the 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): Cmax = 10.89 µg/mL, AUC = 12.38 µg∙h/mL. The Cmax after a single dose likely represents the Cmax at steady state since the half-life is short (approximately 0.5 hours) and no accumulation is expected using the equation: accumulation = 1/(1 – e-k∙tau), where k = 0.693/t½ with t½ = 0.5 hours and tau = 4 hours. For AUC(0-24h), the single-dose AUC at 1000 mg was 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): Cmax = 53.5 µg/mL, AUC = 371.32 µg∙h/mL. For Cmax, an accumulation factor of 1.3 was applied that was estimated from the equation: accumulation = 1/(1 – e–k∙tau), where k = 0.693/t½ with t½ = 2.0 hours and tau = 4 hours (i.e., 1/(1 – e–1.386) = 1/(1 – 0.25) = 1/0.75 = 1.3). For AUC(0-24h), the single-dose AUC at 1000 mg was 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). j In the absence of PK data, Cmax ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 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 Cmax Cmax Cmax LOAEL/Human Cmax Cmax AUC AUC 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 rat induces its (Wistar) (Wistar) external and 18 and a two divided own [Tzimas, Cmax = 0.4 [Seegmiller] [Seegmiller] soft tissue [Tzimas, decrease in doses metabolism, 1994] malformations, live fetuses; (0.15/0.394) so PK 1994] skeletal visceral AUC = 0.5 margins are alterations ectopia, skin Cmax = 0.394 highly Cmax = 0.15 Cmax = 0.30 Cmax = 0.10 (0.25/0.537) b b c erosions, µg/mLd dependent µg/mL µg/mL µg/mL Cmax = acaudia, on day of AUC(0-24h) = 0.30 c AUC = 0.537 AUC(0-8h) = AUC(0-8h) = torsion of assessment µg/mL µg·h/mLd rabbit 0.25 0.50 0.207 hindlimbs, µg·h/mLb µg·h/mLb µg·h/mLc AUC(0-8h) and Cmax = 0.3 = 0.622 omphalocele (0.100/0.394) µg·h/mLc AUC = 0.4 (0.207/0.537) LOAEL: rat Cmax = 0.8 (0.30/0.394) 49 Contains Nonbinding Recommendations AUC = 0.9 (0.50/0.537) rabbit Cmax = 0.8 (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): Cmax = 0.15 µg/mL, AUC(0-8h) = 0.25 µg·h/mL. Pharmacokinetic data are also available after a single dose of 6 mg/kg on GD12 (Collins, 1995): Cmax = 0.320 µg/mL from visual inspection of graph, AUC(0-8h) = 0.820 µg·h/mL; as well as after 6 daily doses (Collins 1994, 1995): Cmax = 0.046 or 0.052 µg/mL, and AUC(0-24h) = 0.098 µg·h/mL or AUC(0-10h) = 0.090 µg·h/mL, respectively. c Extrapolated or actual value after single 6 mg/kg oral dose on GD12 in Swiss hare rabbits (Collins 1995): Cmax = 0.300 µg/mL from visual inspection of graph, AUC(0-8h) = 0.622 µg·h/mL. Pharmacokinetic data are also available following 6 daily doses in Swiss hare rabbits (Collins 1995): Cmax = 0.110 µg/mL, AUC(0- 10h) = 0.281 µg·h/mL; and from (Tzimas 1994): Cmax = 0.105 µg/mL, AUC(0-24h) = 0.321 µg·h/mL. d PK data after first dose (U.S. label). References Collins MD, Tzimas G, Bürgin 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, Bürgin 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, Bürgin 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 Cmax Cmax Cmax Dose AUC AUC AUC Cmax 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) Cmax = 4.5 Cmax = 16.25 abnormal origin of the right μg/mLa µg/mLb subclavian artery (1 litter); Cmax = 1.435 AUC = 13.2 AUC = 53.5 abnormalities of the skull (shortened µg/mLd μg∙h/mLa µg∙h/mLb and misshapen mandibles, AUC = 27.7 abnormally shaped palatine, µg∙h/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): Cmax = 15 µg/mL, AUC = 44 µg·h/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, Cmax = 15 µg/mL, AUC = 44 µg·h/mL; at 600 mg/kg, Cmax = 20 µg/mL, AUC = 82 µg·h/mL. 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): Cmax = 1.435 µg/mL, AUC = 27.70 µg·h/mL. 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 Cmax Dose Dose Cmax LOAEL/Human Cmax AUC Cmax Cmax AUC 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 Cmax = 10 margins Cmax = 0.84 of limb and in liver and for 8 mg dose (1.3/0.13) likely even µg/mLb rib no rabbit no rabbit gall bladder Cmax = 0.128 AUC = 3.3 lower if rats AUC = 2.70 malformations PK data PK data µg/mLc (15.6/4.7) dosed through µg·h/mLb found found AUC = 0.529 organogenesis 30 mg/kg: µg∙h/mLc LOAEL: high rat incidence of Cmax = 6.6 limb and rib (0.84/0.128) malformations AUC = 5.1 (2.7/0.529) rabbitd Cmax = 27.7 (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): Cmax = 0.28 µg/mL, AUC = 0.9 µg·h/mL. c Extrapolated from the average of dose-normalized (to 2 mg) values across the range 2 to 6 mg (Cmax = 0.03 µg/mL, AUC = 0.130 µg∙h/mL) and dose- normalized values (to 4 mg) from 4 and 8 mg in a separate study (Cmax = 0.068 µg/mL, AUC = 0.269 µg∙h/mL) (US label, Ehrsson). d In the absence of rabbit PK data, Cmax ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 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 Cmax LOAEL/Human Notes Findings Cmax Cmax Cmax Cmax AUC AUC AUC AUC AUC 200 mg/kg 400 mg/kg400 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 Cmax = 2.8 invariant, United abortions [FDA, States 1967] mg/kg GD5- mg/day) (33/11.7) independent States 1967, United 12 AUC = 2.4 of dose. Cmax = 33 Vorhees] 600 mg/kg GD7- States Cmax = 29 Cmax = 11.7 (547/232) µg/mLb 18 SD rats 1967] µg/mLc Decreased µg/mLd AUC(0-24 h) = Cmax = 65 [Vorhees] AUC(0-24h) = numbers of AUC(0-24h) = Rabbit 547 µg/mLb increased 267 µg∙h/mLc fetuses, 232 No NOAEL µg∙h/mL b AUC(0-24h) = resorptions, increased µg∙h/mL d identified 1094 increased kinked resorptions at µg∙h/mLb tails 225 – 450 LOAEL: mg/kg Rat 650 mg/kg [U.S. Cmax = 5.6 label] (65/11.7) offspring showed AUC = 4.7 low incidence of (1094/232) cleft palate, talipes, or Rabbit anophthalmos Cmax = 2.5 (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): Cmax = 32.7 µg/mL, AUC(0-24h) = 32.8 mg·min/mL (547 µg∙h/mL). c Extrapolated from reported value after 80 mg/kg oral single dose in Angora grey rabbits (Kourmaravelou): Cmax = 10.4 µg/mL, AUC(0-24h) = 94.8 µg∙h/mL. 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). Cmax = 11.66 µg/mL, AUC = 232.27 µg∙h/mL. 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 Cmax Cmax Cmax Cmax 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 Cmax = 0.32 μg/mLa AUC = 0.25 μg∙h/mLa Cmax = 1.08 µg/mLa AUC = 0.85 µg∙h/mLa a Extrapolated from values in plasma (unbound) after an intraperitoneal 5 mg/kg cisplatin single dose in male Donryu rats (Tamura): Cmax = 5.4 µg/mL, AUC(0- inf) = 254 µg·min/mL (4.23 µg·h/mL). 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 Cmax or AUC values were reported. 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: Cmax = 21.3 µg/mL, AUC(0-t) = 7.49 µg·h/mL·kg, which is 2.25 µg·h/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 Cmax (μg/mL) AUC (μg∙h/mL) Notes (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(0-t) = 7.49 µg·h/mL·kg, which is 2.25 µg·h/mL in 300 g rats. 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 Cmax LOAEL/Human Cmax Cmax Cmax Cmax AUC 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, µg/mLa Kreybig, Cytoxan skeletal Cmax = 106 rabbit MEFL has AUC = 3.65 Mirkes] Cmax = 151 defects µg/mLg NOAEL not been attributed µg·h/mLa encephalocele, µg/mLc AUC = 798 identified, but to both exencephaly, AUC(0-8h) = µg·h/mLg LOAEL margins phosphoramide PM microcephaly, 24.1 were <1.5 mustard (PM) Cmax = 0.55 limb defects µg·h/mLd PM and acrolein µg/mLb (i.e., PM Cmax = 14.4 LOAEL: metabolites AUC(0-24h) = syndactyly Cmax = 0.07 µg/mLh rat 2.13 and µg/mLe AUC = 352 Cmax: 0.04 µg·h/mLb ectrodactyly), AUC(0-8h) = µg·h/mLh (4.1/106) defective 0.297 AUC: 0.005 facial µg·h/mLe (3.65/798) development rabbit (cleft palate) Cmax = 1.4 (151/106) AUC = 0.03 (24.1/798) PM margins 61 Contains Nonbinding Recommendations rat Cmax = 0.04 (0.55/14.4) AUC = 0.006 (2.13/352) rabbit Cmax = 0.005 (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): C0 = 125.3 µM (32.7 µg/mL), AUC/D = 265.3 min/L (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 mg∙min/L = 29.2 µg∙h/mL). b Extrapolated from reported value after 20 mg/kg intravenous single dose in Sprague Dawley rats (Hong): Cmax = 20 µM (4.4 µg/mL) from visual inspection of graph, AUC(0-24h) = 76.9 µM·h (17.0 µg·h/mL) from calculation based on concentration values estimated by visual inspection of graph. c Extrapolated from reported value after 45 mg/kg cytoxan intravenous single dose in 2 New Zealand White rabbits (Holm): Cmax = 227 µg/mL from visual 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 Cmax value (2.2 µM [0.574 µg/mL] from visual inspection 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(0-8h) = 3683 µmol·min/L (16.0 µg·h/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 µg∙min/mL [53.15 and 20.98 µg∙h/mL]) in 2 rabbits differed by 2.5-fold and tlast was ≤90 minutes so these values were not used. e Extrapolated from reported value after 20 mg/kg cytoxan intravenous single dose in NZW rabbits (Anthony): Cmax = 0.22 µM (0.049 µg/mL) from visual inspection of graph, AUC(0-8h) = 53.7 µmol·min/L (0.198 µg·h/mL). f From SmPC. g Extrapolated from reported value after 1000 mg/m2 intravenous single dose cytoxan (Chan): C0 = 254.4 µM (66.4 µg/mL), AUC(0-inf) = 1910 µM·h (499 µg·h/mL). h Extrapolated from reported value after 1000 mg/m2 intravenous single-dose cytoxan (Chan): C0 = 40.5 µM (9.0 µg/mL), AUC(0-inf) = 996.3 µM·h (220 µg·h/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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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 µg/mLa µg/mLa paws and cytidine AUC(0-inf) = AUC(0-inf) = tail; skeletal Cmax = ~2.8 LOAEL: deaminase ~15.9 µg·h/mLa ~31.7 µg·h/mLa defects µg/mLc rat • active moiety including AUC = 6.6 Cmax: 4.1 is Ara-CTP distortion and µg∙h/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): Cmax = ~11.6 µg/mL from visual inspection of graph, AUC(0-inf) = ~31.7 µg∙h/mL from calculation based on concentration values estimated by visual inspection of graph. Note that the 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 µg/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): Cmax = 400 µM (97 µg/mL) from visual inspection of graph, AUC estimated from CL = 8.16 mL/(min·kg) and dose = 50 mg/kg, AUC = dose/CL = (50/8.16)(1 h/60 min) = 102 µg·h/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): Cmax = ~7.0 µmol/L (1.7 µg/mL) from visual inspection of graph, AUC = dose/CL = 100 mg/845 mL/min = 1.97 µg·h/mL (which gives AUC = 3.29 µg∙h/mL at 100 mg/m2 and 6.6 µg∙h/mL for 100 mg/m2 BID). 65 Contains Nonbinding Recommendations Mouse NOAEL Mouse LOAEL Mouse Findings Margins Notes Dose Dose NOAEL/Human Cmax Cmax LOAEL/Human 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 Cmax: 0.16 teratology data, which was (0.46/2.8)f included in the U.S. label, exposure Cmax = ~0.50 µg/mLd Cmax = ~2 µg/mLd AUC: 0.06 margins at the NOAEL were <1; AUC = ~0.46 AUC= ~1.83 (0.39/6.6)f (b) rat exposure margins at the µg∙h/mLd µg∙h/mLd LOAEL: NOAEL were much higher; (c) mice rabbit data are not available, so it Cmax = ~0.41 µg/mLe Cmax = ~1.62 Cmax: 0.65 provides data in a 2nd species. AUC = 0.315 µg/mLe (1.81/2.8)f µg∙h/mL e AUC = 1.26 AUC: 0.23 µg∙h/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): Cmax = ~30 µg/mL from visual inspection of graph, AUC(0-24h) = ~27.5 µg∙h/mL from calculation based on concentration values estimated by visual inspection of graph. Note large 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): Cmax = 2 µmol/mL (486 µg/mL) from visual inspection of graph, AUC = 1.553 µmol∙h/mL (378 µg∙h/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 Cmax Cmax Cmax Cmax 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 Cmax = 1.17 μg/mLb Cmax = 2.17 µg/mLc fetuses AUC(0-t) = 4.10 AUC(0-t) = 22.6 μg∙h/mL b µg∙h/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): Cmax = 1.17 µg/mL, AUC(0-t) = 4.10 µg·h/mL. c Actual values in plasma after 300 mg/kg oral dabrefenib for 24 days in rats (FDA, United States, p. 119: Cmax = 2.17 µg/mL, AUC(0-t) = 22.6 µg·h/mL. 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 Cmax Cmax Cmax Cmax AUC AUC AUC 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 Cmax = 0.021 μg/mLa bent scapula or Cmax = 0.227 µg/mL variations (delays in AUC(0-8h) = 0.105 humerus AUC(0-inf) = 0.834 ossifications); μg∙h/mLa μg∙h/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): Cmax = 0.021 µg/mL, AUC(0-8h) = 0.105 µg·h/mL. b Actual values in plasma after 13 days (GD19) of 6 mg/kg oral dasatinib in pregnant NZW rabbits (FDA, United States, p. 238): Cmax = 0.227 µg/mL, AUC = 0.834 µg·h/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: Cmax = 0.459 μg/mL, AUC = 3.289 μg∙h/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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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 Cmax = 9.07 Cmax = 3.7 e Cmax = 33.8 abnormal FDA, States1990a] toxic µg/mL (33.8/9.07) µg/mLa Cmax = 54 craniofacial United dose) AUC(0-24h)= AUC = 2.8 AUC(0-inf) = µg/mLa ossification States Cmax = 81 134.8 µg∙h/mL (380/134.8) e 380 µg∙h/mL AUC(0-inf) = b adactylia, 1990a] µg/mLc rabbit 608 brachygnathia AUC = 1563 Cmax = 3.0 (27/9.07) µg∙h/mLb [U.S. Label, Cmax = 27 µg∙h/mLd AUC = 3.9 FDA, United µg/mLc (521/134.8) States 1990a]. AUC = 521 µg∙h/mLd LOAEL: rat Cmax = 6.0 (54/9.07) AUC = 4.5 (608/134.8) rabbit Cmax = 8.9 (81/9.07) 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): Cmax = 13.5 µg/mL. b Extrapolated from reported value after 20 mg/kg fluconazole oral single dose in rats (Humphrey): AUC(0-inf) = 152 µg·h/mL. c : Extrapolated from reported value after 10 mg/kg fluconazole oral single dose in rabbits (FDA, United States 1990a, p. 7): Cmax = 10.8 µg/mL. 70 Contains Nonbinding Recommendations d Calculated using plasma clearance value for rabbits (0.8 mL/min∙kg, FDA, United States 1990a, p 8): AUC = Dose/CL = (25 mg/kg)/(0.8 mL/min∙kg)(1 h/60 min) = 521 µg∙h/mL. e Actual value after 400 mg/day fluconazole oral single dose (FDA, United States 1990b, p. 7, 50-52): Cmax = 9.07 µg/mL, AUC(0-24h) = 134.8 µg∙h/mL. Data are also available after 14 days of repeated administration, which shows significant drug accumulation. Cmax = 18.89 µg/mL, AUC(0-24h) = 349.9 µg∙h/mL. Since 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 Cmax Dose Dose Cmax LOAEL/Human Cmax AUC Cmax Cmax AUC 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 malformations, IP GD9 identified [DeSesso] 85% of mg/m2) in a Cmax = 0.09 short (most [Wilson] embryo-fetal [Wilson] [DeSesso] term variety of (2.6/29) patients lethality Cmax = 111 fetuses dosing AUC = 0.3 have Cmax = 2.6 Cmax = 3.87 Kuwagata: µg/mLb regimens, (3.89/11.5) undetectable µg/mLa µg/mLa ≥17 mg/kg: AUC = 11 including doses rabbit 5-FU levels AUC = 3.89 AUC = 5.83 micro- µg·h/mLb up to 3000 no NOAEL in plasma µg·h/mLa µg·h/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 µg/mLd Cmax = 0.1 prodrug: AUC = 11.5 (3.87/29) thymidylate µg·h/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): Cmax = 7.74 µg/mL, AUC = 11.66 µg·h/mL. b Extrapolated from reported value after 20 mg/kg 5FU intravenous single dose in rabbits (Kar): Cmax = 0.427 µmol/mL (55.5 µg/mL), AUC = 2.535 µmol∙min/mL (5.5 µg·h/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): Cmax = 32 µg/mL from visual inspection of graph, AUC = 12.55 µg·h/mL. Data are also available after a 370 mg/m2 dose (Bocci): Cmax = 48.41 µg/mL, AUC = 13.61 µg·h/mL. 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 CO2 technique. Int J Pharm. 2012;436:272-281. 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; Cmax = 2.55µg/mL, AUC = 5.82 µg·h/mL] 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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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 µg/mLb µg/mLb available palate, oral for sickle cell (100/35)d –4h 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 Cmax = 52 µg/mLc similar SC GD12 AUC(0-inf) = 184 LOAEL: (Wilson) [DeSesso µg·h/mLc rat • bioavailability 1977]: skull Cmax = 1.6 is 70 – 80% in and facial (80.6/52) rats and anomalies as Cmax dose = 3.9 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 Cmax = 0.9 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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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): Cmax = 47.3 at 100 mg/kg and 80.6 µg/mL at 137 mg/kg. c Extrapolated from reported value after 1000 mg (16.7 mg/kg) hydroxyurea oral single dose (MHRA): Cmax = 24.6 µg/mL, AUC(0-inf) = 87.79 µg·h/mL. The 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 Cmax data are available, this was after IP administration whereas the human data is after oral administration. Thus, in the absence of more direct 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, Cmax ratio was based on mg/kg dose ratio and AUC was based on mg/m2 dose ratio. 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. [Cmax after 46, 184, and 1840 mg/kg IV dose, nonlinear PK] 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 Cmax (µg/mL) AUC (µg∙h/mL) Notes Charache sickle cell 25 mg/kg oral 19 AUC(0-6) = 1216 AUC units were published as “µg anemia mL/min”, value seems wrong since (Cmax x 6 h = 114 µg∙h/mL) 77 Contains Nonbinding Recommendations Villani HIV mean 7.6 mg/kg oral 0.135 nmol/L AUC(0-12h) = 540 BID = 0.135 µmol·h/L = 41.1 µmol/mL = µg·h/mL; 10.3 µg/mL AUC(0-24h) = 82.1 µg∙h/mL MHRA not stated – BE 1000 mg (16.6 oral 24.6 AUC(0-inf) = 87.79 use these values review study mg/kg) µg∙h/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 Cmax Cmax Cmax Cmax 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 Cmax = 1.31 μg/mLc arteriosus, right-sided resorptions), AUC(0-24h) = 5.348 Cmax = 2.627 aortic arch, and Cmax = 0.311 Cmax = 1.83 decreased viable μg∙h/mLc µg/mLd interrupted aortic arch; µg/mLe µg/mLf fetuses, abortions AUC(0-24h) = increased post- AUC = 1.31 AUC = 21.00 13.729 µg∙h/mLd implantation loss µg∙h/mLe µg∙h/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): Cmax = 1.31 µg/mL, AUC(0-24h) = 5.348 µg·h/mL. d Actual values in plasma after 11 doses of 100 mg/kg oral ibrutinib in pregnant rats (FDA, United States, p. 130): Cmax = 2.627 µg/mL, AUC(0-24h) = 13.729 µg·h/mL. e Actual values in plasma after 13 doses of 30 mg/kg oral ibrutinib in pregnant rabbits (FDA, United States, p. 136): Cmax = 0.311 µg/mL, AUC = 1.31 µg·h/mL. f Actual values in plasma after 13 doses of 100 mg/kg oral ibrutinib in pregnant rabbits (FDA, United States, p. 136): Cmax = 1.83 µg/mL, AUC = 21.00 µg·h/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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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 Cmax = 3.4 [Adams] GD9-10: Cmax = 26.6 mg/kg/day) (205/59.7) Cmax = 205 ventricular µg/mLd [U.S. label] AUC = 0.7 µg/mLa oral GD9-10: septal AUC(0-inf) = (597/839) AUC = 597 300 mg/kg defects 80.5 µg∙h/mLd Cmax = 59 µg/mLe µg∙h/mL a [Cappon AUC = 839 µg∙h/mLe rabbitc 2003]b 60 mg/kg 500 mg/kg oral NOAEL at 300 mg/kg GD9-11 Cmax = 0.5 Cmax = 341 [Cappon 2003]b (26.6/59) µg/mLa Cmax = 222 AUC = 0.1 AUC = 995 µg/mLd (80.5/839) µg∙h/mLa AUC(0-inf) = 671 500 mg/kg µg∙h/mLd NOAEL Cmax = 3.8 (222/59) AUC = 0.8 (671/839) LOAEL: rat Cmax = 5.8 (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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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): Cmax = 28.4 µg/mL, AUC(0-inf)= 4971.3 µg·min/mL (82.9 µg∙h/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): Cmax = 5.32 µg/mL, AUC = 12.41 µg·h/mL. 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): Cmax = 24.85 µg/mL, AUC(0-inf) = 75.14 µg·h/mL. e Extrapolated from reported value after 14.8 mg/kg (mean) ibuprofen single oral dose (Konstan): Cmax = 65.5 µg/mL, AUC(0-inf) = 14.0 mg·min/mL (233 µg∙h/mL). Note the Cmax was multiplied by 0.9 (13.3/14.8) to give the extrapolated Cmax. The Cmax after a single dose likely represents the Cmax at steady state 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 – e–k∙tau), where k = 0.693/t½ with t½ = 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 Cmax Cmax Cmax Cmax 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; Cmax = 53.06 µg/mLc Cmax = 3.57 Cmax = 12.14 increased post- AUC = 699.8 μg/mLa µg/mLb implantation loss, µg∙h/mLc AUC = 39.28 AUC = 142.55 decreased live fetuses μg∙h/mLa µg∙h/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, Cmax = 1.69 µg/mL, AUC(0-24h) = 15.40 µg·h/mL; at 50 mg/kg, Cmax = 6.07 µg/mL, AUC(0-24h) = 71.276 µg·h/mL. b Extrapolated from reported value in plasma after 50 mg/kg imatinib oral single dose in female rats (FDA, United States, p. 24): Cmax = 6.07 µg/mL, AUC(0-24h) = 71.276 µg·h/mL. c Reported value after 60 mg/kg oral imatinib single dose in rabbits species (FDA, United States, p. 26): Cmax = 53.06 µg/mL, AUC(0-24h) = 699.8 µg∙h/mL. 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 Cmax LOAEL/Human Notes Cmax Cmax Findings Cmax Cmax AUC 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 Cmax = 2.8 [Tembe] [Tembe] Higher doses: including eye Cmax = 0.32 (0.9/0.32) microcephaly, Cmax = 0.95 Cmax = 3.1 defects, tail µg/mLd AUC = 0.6 Cmax = 0.9 Cmax = 1.8 anotia, µg/mLb µg/mLc defects, AUC = 7.52 (4.8/7.52) a a µg/mL µg/mL exophthalmos, AUC = 12.2 AUC = 49.1 cardiomegaly, µg∙h/mLd rabbit AUC(0-10h) = AUC(0-10h) = protruding µg∙h/mLb µg∙h/mLc skin tag on Cmax = 3.0 4.8 µg∙h/mLa 9.6 µg∙h/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 Cmax = 5.6 atresia, spina (1.8/0.32) bifida, AUC = 1.3 deformed tail, (9.6/7.52) and acaudate; increased rabbit resorptions Cmax = 9.7 (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): Cmax = 9.07 µg/mL, AUC(0-10h) = 47.9 µg·h/mL. 85 Contains Nonbinding Recommendations b Actual values after 3 mg/kg isotretinoin oral single dose in New Zealand White rabbits (Eckhoff): Cmax = 0.952 µg/mL, AUC = 12.2 µg·h/mL. c Actual values after 15 mg/kg isotretinoin oral single dose in New Zealand White rabbits (Eckhoff): Cmax = 3.099 µg/mL, AUC(0-10h) = 49.1 µg·h/mL. d Extrapolated from reported value after 80 mg (1.33 mg/kg) isotretinoin oral single dose with food (U.S. label): Cmax = 0.86 µg/mL, AUC(0-10h) = 10.0 µg·h/mL. The Cmax 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 also available while fasting, but the higher values from the fed state were used for margin calculations: Cmax = 0.3 µg/mL, AUC = 3.7 µg∙h/mL. 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 Cmax Dose (Dose Cmax Cmax AUC Cmax Cmax AUC 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 Cmax = 0.21 Cmax = 1.58 micrognathia, ALL: induction – rabbit margins µg/mLa µg/mLb dysplastic sacral 3.3 mg/m2 daily; NOAEL not would likely AUC = 0.067 AUC = and caudal maintenance – 15 identified be even lower µg∙h/mLa 0.61 vertebrate, 2 mg/m oral if dosed µg∙h/mLb phocomelia, twice/week LOAEL: throughout hemimelia, choriocarcinoma: rat organogenesis syndactyly, and 15 – 30 mg oral Cmax = 0.1 ectrodactyly; QDx5 (8.8 – 17.6 (0.21/2.14) 2 embryolethality, mg/m ) AUC = 0.02 resorptions lymphoma: 10 – 25 (0.067/3.28) mg QDx4-8 oral rabbit (5.9 – 14.7 mg/m2); Cmax = 0.7 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) Cmax = 2.14 µg/mLd 87 Contains Nonbinding Recommendations AUC = 3.28 µg·h/mLd a Extrapolated from reported value after 0.31 mg/kg methotrexate intravenous single dose in Wistar rats (Scheufler 1982): C0 = 0.64 µg/mL, AUC(0.1-4h) = 0.207 µg·h/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): Cmax = 7 µg/mL, AUC = 2.72 µg·h/mL. Data are also available after a 10 mg/kg methotrexate intravenous single dose in female New Zealand White rabbits (Stagni): Cmax = 74 µg/mL, AUC = 31.4 µg·h/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): Cmax = 4.37 µg/mL from visual inspection of graph, AUC(0-inf) = 6.69 µg·h/mL. Oral data are also available (Campbell): Cmax = 0.50 µg /mL from visual inspection of graph, AUC(0-inf) = 2.34µg·h/mL. 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, Običan 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 Cmax AUC Notes (mg/kg) (µg/mL) (µg∙h/mL) Wilson 0.3 IV Wistar 0.40 — C0 was estimated from graph since 1st timepoint was 0.25 hours Scheufler 31 IV Wistar 177 AUC(0-inf) = 38.4 Cmax is C0 1981 Scheufler 0.31 IV Wistar 0.64 AUC(0.1-4h) = 1982 0.207 Kim 4.0 IV Sprague 40 AUC(0-inf) = 2.88 Cmax was from visual inspection of graph, AUC was 173 Dawley µg∙min/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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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 Cmax = 0.9 meningoencepha- [McClain] [McClain] abnormalities (13.4/14.5) Cmax = 13.4 Cmax = 26.8 locele, domed that included Cmax = 14.5 AUC = 0.7 µg/mLa µg/mLa head, anasarca, Cmax = 27 Cmax = 34 shortened and µg/mLf (205/291) AUC = 205 AUC = 410 and limb µg/mLb µg/mL d curved long AUC = 291 rabbit µg∙h/mLa µg∙h/mLa hyperflexion), AUC(0-24h) = AUC(0-24h) = bones, pes µg∙h/mLg Cmax = 1.9 skeletal 193 290 cavus, (27/14.5) malformation µg∙h/mLc µg∙h/mLc syndactyly AUC = 0.7 (short rib) (193/291) LOAEL: rat Cmax = 1.8 (26.8/14.5) AUC = 1.4 (410/291) rabbit Cmax = 2.3 (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): Cmax = 13.4 µg/mL, AUC(0-inf) = 205 µg∙h/mL. PK data are also available on GD17: Cmax = 30.2 µg/mL, AUC(0-inf) = 906 µg·h/mL. b Actual value after 50 mg/kg phenytoin oral single dose in female New Zealand White rabbits (McClain): Cmax = 27 µg/mL. PK data are also available after 30 mg/kg phenytoin oral dose in male New Zealand White rabbits (Medhi): Cmax = 12.8 µg/mL. The value from McClain was used because it was from 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 µg·h/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): Cmax = 27 µg/mL and 41 µg/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 Cmax and a dose of 625 mg/day oral solution 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): Cmax = 2.268 µg/mL, AUC(0-inf) = 58.2 µg·h/mL. PK data are also available for a 100 mg oral solution dose and for extended-release capsules (FDA, United States 1986). For Cmax, an accumulation factor of 3.2 was applied that was estimated from the equation: accumulation = 1/(1 – e–k∙tau), where k = 0.693/t½ with t½ = 14.924 hours and tau = 8 hours (i.e., 1/(1 – e–0.372) = 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(0-inf) = 58.2 µg·h/mL. 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 Cmax Dose Cmax Cmax LOAEL/Human Cmax AUC Cmax AUC AUC 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 Cmax = 0.079 rabbit misalignment Cmax = 0.072 µg/mLc NOAEL not Cmax = 2.7 of lumbar and µg/mLb AUC(0-24h) = identified µg/mLa thoracic AUCτ = 0.418 0.402 AUC(0-24) = vertebral µg·h/mLb µg·h/mLd LOAEL: 34.3 elements rat µg·h/mLa (vertebral, Cmax = 34 central and/or (2.7/0.079) neural arches) AUC = 85 (34.3/0.402) resorptions; increased post- rabbit implantation Cmax = 0.9 (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): Cmax = 2.729 µg/mL, AUC(0-24h) = 34.34 µg·h/mL. b Actual value on GD17 after 10 mg/kg pomalidomide oral dose in pregnant New Zealand White rabbits (FDA, United States 2013a, p. 163): Cmax = 0.072 µg/mL, AUCτ = 0.418 µg·h/mL. c Actual value after 4 mg pomalidomide oral dose for 8 days in multiple myeloma subjects (FDA, United States 2013b, p. 24): Cmax = 0.079 µg/mL. d Actual value after 4 mg pomalidomide oral dose for 4 weeks (FDA, United States 2013a, p. 180): AUC(0-24h) = 0.402 µg·h/mL. 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 DoseLOAEL Findings Cmax Cmax CmaxDose AUC AUC AUC Cmax 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 Cmax = 3.8 Cmax = 12.7 defects; extra No rabbit PK No rabbit PK monophosphate and 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 ng∙h/mLa ng∙h/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): Cmax = 0.127 µg/mL, AUC = 0.276 µg·h/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: Cmax = 1.36 μg/mL, AUC(0-inf) = 14.7 µg·h/mL] 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: Cmax = 0.433 μg/mL, AUC(0- inf) = 3.04 µg·h/mL] 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 Cmax Cmax Cmax Cmax 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 ng∙h/mLa ng∙h/mLb µg∙h/mLc ng∙h/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): Cmax = 2.9 ng/mL, AUC(0-inf) = 10.9 ng·h/mL. b Actual values in plasma after 3.2 mg/kg tacrolimus oral single dose in male rats (FDA, United States, p. 25): Cmax = 20 ng/mL, AUC(0-inf) = 68.9 ng·h/mL. c Extrapolated from reported value after 2 mg/kg tacrolimus oral single dose in NZW rabbits (Piekoszewski): Cmax = 5.79 ng/mL, AUC = 110 ng·h/mL. 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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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 µg/mLg (0.97/0.62) Cmax = Cmax = mg/kg and 15/25 AUC(0-inf) = AUC = 2.2 0.97µg/mLc 4.87µg/mLc at GD19 at GD19 fetuses at 180 4.9 µg·h/mLg (10.75/4.9) AUC(0-24h) = AUC(0-24h) = Cmax = 0.82 Cmax = 2.16 mg/kg) rabbit 10.75 53.75 µg/mLd µg/mLe • hydrocephaly Cmax = 1.3 µg·h/mLc µg·h/mLc AUC(0-24h) = AUC(0-24h) = (n=2/38) (0.82/0.62) 4.18 14.4 µg.h/mLe • increased post- AUC = 0.9 µg·h/mLd implantation loss, (4.18/4.9) including dead fetuses, and LOAEL: numerous rat external and Cmax = 7.9 visceral (4.87/0.62) malformations at AUC = 11.0 180 mg/kg (53.75/4.9) rabbit Cmax = 3.5 (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 today’s 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): Cmax = 4.87 µg/mL, AUC(0-24h) = 53.75 µg∙h/mL. PK data are also available after 30 mg/k oral single dose in female Fischer rats (FDA, United States, p. 22, 91): Cmax = 10.4 µg/mL, AUC(0-18h) = 63.99 µg∙h/mL; and after a 100 mg/kg oral single dose in male Sprague Dawley rats (FDA, United States, p. 73): Cmax = 21.60 µg/mL, AUC(0- 48h) = 348.5 µg∙h/mL. d Actual value after 20 mg/kg thalidomide oral doses in pregnant New Zealand White rabbits (Christian). GD7: Cmax = 1.77 µg/mL, AUC(0-24h) = 13.4 µg·h/mL; GD19: Cmax = 0.824 µg/mL, AUC(0-24h) = 4.18 µg·h/mL. e Actual value after 60 mg/kg thalidomide oral doses in pregnant New Zealand White rabbits (Christian). GD7: Cmax = 6.39 µg/mL, AUC(0-24h) = 78.7 µg·h/mL; GD19: Cmax = 2.16 µg/mL, AUC(0-24h) = 14.4 µg·h/mL. 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): Cmax = 0.62 g/mL, AUC = 4.90 µg∙h/mL. 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(0-inf) = 55.25 µg∙h/mL at 50 mg/kg on D8] Eriksson T, Riesbeck K, Ostraat O, Ekberg H, Björkman 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 Cmax LOAEL/Human Cmax Cmax Cmax Cmax AUC 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 e 1996a] label, States States µg/mL (893/229) structural FDA, 1996a] 1996a] AUC = 229 rabbit variations were e Cmax = 49 United µg·h/mL Cmax = 1.0 observed at 20 µg/mLa States Cmax = 13 Cmax = 23 (13/13.5) mg/kg, the AUC = 893 1996a] µg/mLd µg/mLd AUC = 0.3 NOAEL for µg∙h/mLb AUC = 67 AUC = 117 (67/229) MEFL is Cmax = µg∙h/mLd µg∙h/mLd assumed to be 168.6 LOAEL: 100 mg/kg µg/mLc rat • In rats: Clinical AUC = Cmax = 12.5 signs of maternal 3573 (169/13.5) toxicity were µg∙h/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): C1.5h = 97.3 µg/mL. b Extrapolated from reported value after 30 mg/kg topiramate for 8 days in female Sprague Dawley rats (FDA, United States, p. 12): Cmax = 22.2 µg/mL, AUC = 268.2 µg·h/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): C1.5h = 168.6 µg/mL. d Extrapolated from reported value after 60 mg/kg topiramate for 14 days in female New Zealand White rabbits (FDA, United States, p. 13): Cmax =39.1 µg/mL, AUC = 201 µg·h/mL. e Extrapolated from reported value after 100 mg/kg topiramate BID oral for 14 days (FDA, United States 1996b): Cmax = 6.76 µg/mL, AUC(0-24h) = 57.2 µg·h/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 Cmax Cmax Dose Dose Cmax LOAEL/Human AUC AUC Cmax Cmax AUC 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] Cmax = 1.4 has much Trimethadione Trimethadione effects on Trimethadione (58.9/42.75) higher Cmax = 58.9 Cmax = 235 survival and AUC = 10.78 Trimethadione AUC = 0.2 exposures µg/mLa µg/mLa litter size” µg∙h/mLc Cmax = 42.75 (203/1000) than AUC(0-inf) = AUC(0-inf) = µg/mLd rabbit trimethadione, 203 µg∙h/mLa 814 µg∙h/mLa 250 mg/kg AUC(0-inf) = NOAEL not and is a GD7-18 1000 identified confirmed Dimethadione Dimethadione [Vorhees]: µg∙h/mLd teratogen Cmax = Cmax = 391 embryolethality, LOAEL: (Buttar 1978). b b 97.7µg/mL µg/mL malformations Dimethadione rat Thus, margins AUC(0-inf) = AUC(0-inf) = (primarily Cmax = 1251 Cmax = 5.5 for 4872 19,488 cardiac, with a µg/mLe (235/42.75) dimethadione µg∙h/mLb µg∙h/mLb lower incidence AUC(0-inf) = AUC = 0.8 are also listed. of esophageal 36,670 (814/1000) and kidney µg∙h/mLe rabbit defects) LOAEL not identified Dimethadione NOAEL: rat 103 Contains Nonbinding Recommendations Cmax = 0.1 (97.7/1251) AUC = 0.1 (4872/36670) LOAEL: rat Cmax = 0.3 (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): Cmax = 98.1 µg/mL, AUC(0-inf) = 339 µg·h/mL. b Extrapolated from reported value after 100 mg/kg trimethadione oral single dose in male Wistar rats (Tanaka 1981): dimethadione Cmax = 162.8 µg/mL, AUC(0-inf) = 8120 µg∙h/mL. c Actual value after 4 mg/kg trimethadione intravenous single dose in Japanese White rabbits (Tanaka 1999): AUC(0-inf) = 10.78 µg∙h/mL calculated from Cl = 0.371 L/(kg∙h). d Extrapolated from reported value after 4 mg/kg trimethadione oral single dose (Kobayashi): Cmax = 6.0 µg/mL, AUC(0-inf) = 100.1 µg·h/mL. For Cmax, an accumulation factor of 2.85 was applied that was estimated from the equation: accumulation = 1/(1 – e–k∙tau), where k = 0.693/t½ with t½ = 9.6 hours and tau = 6 hours (i.e., 1/(1 – e–0433) = 1/(1 – 0.649) = 1/0.351 = 2.85). e Extrapolated from reported value after 4 mg/kg trimethadione oral single dose (Kobayashi): dimethadione Cmax = 12.83 µg/mL, AUC(0-inf) = 3667 µg·h/mL. For Cmax, an accumulation factor of 39 was applied that was estimated from the equation: accumulation = 1/(1 – e–k∙tau), where k = 0.693/t½ with t½ = 160 hours and tau = 6 hours (i.e., 1/(1 – e–0.026) = 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 , C6h = 11.3 µg/mL] 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 Cmax = 3.0 µg/mL, AUC(0-inf) = 8.21 µg·h/mL, and dimethadione Cmax = 10.2 µg/mL, AUC(0-inf) = 465.8 µg∙h/mL. The values after 100 mg/kg (Tanaka 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 Cmax LOAEL/Human Notes Cmax Cmax Cmax Cmax AUC 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 Cmax = 0.4 United States, [Voorhees], States, 1977] [FDA, (cleft palate, divided (73.8/205) 1995] GD8-17 United umbilical doses (30 AUC = 0.06 [Binkerd]; Cmax = 410 States, hernia, bilateral mg/kg/dose) (230/4180) [U.S. µg/mLb 1977] talipes, [highest rabbit Depacon AUC = 690 exencephaly, approved Cmax = 2.0 Cmax = 73.8 label] µg∙h/mLb Cmax = 957 hypoplastic dose, U.S. (410/205) a µg/mL µg/mLb ears, Depakote AUC = 0.2 AUC = 230 Cmax = 227 AUC = gastrochisis, and (690/4180) µg∙h/mLa µg/mLa 1610 bilateral Depakene AUC = 707 µg∙h/mL talipes); b labels] LOAEL: µg∙h/mLa visceral rat malformations Cmax = 1.1 (intraventricular Cmax = 205 (227/205) septal defects, µg/mLc AUC = 0.2 misshapen AUC(0-inf) = (707/4180) ventricle, renal 4180 rabbit agenesis); µg∙h/mLd Cmax = 4.7 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): Cmax = 227 µg/mL, AUC = 707 µg·h/mL. PK data are also available on GD8: Cmax = 341µg/mL, AUC = 1019 µg∙h/mL b Extrapolated from reported value after 70 mg/kg valproic acid oral single dose in male New Zealand White rabbits (Bourin): Cmax = 191.3 µg/mL, AUC(0-inf) = 322 µg·h/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): Cmax = 114 µg/mL. d Extrapolated from reported value after 1000 mg valproic acid oral single dose (Nitsche): AUC(0-inf) = 1161 µg·h/mL. 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): Cmax = 86 µg/mL] 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: Cmax = 40.7 µg/mL, AUC(0-inf) = 3458 µg∙min/mL (57.6 µg∙h/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: C0 = 157 µg/mL, AUC(0-inf) = 308 µg∙h/mL] 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: Cmax = 64.2 µg/mL, AUC(0-inf) = 227 µg·h/mL]. 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: Cmax = 238 µg/mL, AUC(0-6h) = 17.5 mg∙min/L (292 µg∙h/mL)] 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 Cmax Dose Dose Cmax LOAEL/Human Cmax AUC Cmax Cmax AUC 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 Cmax = 13.0 NOAEL not fused digits µg/mLb identified Cmax = 7.22 µg/mLa on the hind AUC(0-24h) = 306 rabbit: AUC(0-24h) = 50.5 limb, open µg∙h/mLb no data found µg∙h/mLa perineum, multiple LOAEL: craniofacial rat anomalies Cmax = 0.6 (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): Cmax = 7.22 µg/mL, AUC(0-24h) = 50.5 µg·h/mL. b Reported value after 14 daily oral doses of 150 mg vismodegib (FDA, United States, 2012): Cmax = 30.9 µM (13.0 µg/mL), AUC(0-24h) = 727 µmol∙h/L (306 µg∙h/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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC 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: Cmax: 136 (FDA, United loss in GD6-18 Cmax = 0.33 (45/0.33) States 1989) Cmax = 128 presence of (FDA, µg/mLd AUC: 111 µg/mLa maternal United AUC(0-24h): 3.0 (334/3.02) Cmax = 45 AUC = 1010 toxicity States 1989) µg•hr/mLd Rabbit (135 a b µg/mL µg•h/mL (death, mg/kg/day) AUC = 301 clinical Cmax = 137 Cmax: 415 b µg•h/mL signs) µg/mLc (137/0.33) AUC = 642 AUC: 213 Exposure data µg•h/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 Cmax: 388 oral (128/0.33) Cmax = 4.6 GD6-18 AUC: 334 µg/mLa (FDA, (1010/3.02) AUC = 32 United rabbit µg•h/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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC AUC AUC AUC AUC 25 mg/kg oral Cmax = 15 GD6-15 µg/mLc (FDA, United AUC = 71 States 1989) µg•h/mLc Cmax = 12 45 mg/kg µg/mLa oral AUC = 41 GD6-18 µg•h/mLb (FDA, United States 1989) Cmax = 51 µg/mLc AUC = 116 µg•h/mLc a From reported Cmax 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. Cmax for 8 mg/kg/day 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 Cmax and AUC values in pregnant rabbits exposed from GD6-18 at steady state (GD18) at doses of 25, 45 and 90 mg/kg/day. Cmax and AUC for 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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC 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, Cmax: 10,375 of United Saxagliptin (249/0.024) saxagliptin. Saxagliptin States 2009) Cmax = 0.024 AUC: 8,294 (U.S. Label Cmax = 249 µg/mLd (647/0.078) and µg/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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC AUC AUC AUC AUC AUC0-24 = 647 Cmax = 43 AUC(0-24h): BMS-510849 public µg•h/mLa µg/mLc 0.078 Cmax: 449 assessment AUC0-24 = µg•hr/mLd (21.1/0.047) reports (EU BMS-510849 111 AUC: 673 EPAR) Cmax = 21.1 µg•h/mLa BMS-510849 (144/0.214) Onglyza) µg/mLb Cmax = 0.047 AUC0-24 = 144 BMS-510849 µg/mLd Rabbit (200 µg•h/mLa Cmax = 125 AUC(0-24h): mg/kg/day) µg/mLc 0.214 AUC0-24 = µg•hr/mLd Saxagliptin Exposure data 434 Cmax: 1,792 at lower doses µg•h/mLa (43/0.024) 64 mg/kg oral AUC: 1,423 GD6-15 (111/0.078) Exposure Saxagliptin data at lower BMS-510849 Cmax = 17.7 doses Cmax: 2,659 µg/mLa 8 mg/kg oral (125/0.047) AUC0-24 = 23.6 GD7-19 AUC: 2,028 µg•h/mLa (434/0.214) Saxagliptin BMS-510849 Cmax = 2 LOAEL: Cmax = 1.5 µg/mLc rat µg/mLb AUC0-24 = Not applicable AUC0-24 = 6.3 2.5 rabbit µg•h/mLa µg•h/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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC AUC AUC AUC AUC GD6-15 Cmax = 5 µg/mLc Saxagliptin AUC0-24 = Cmax = 66.3 7.4 µg/mLa µg•h/mLa AUC0-24 = 121 µg•h/mLa 40 mg/kg oral BMS-510849 GD7-19 Cmax = 5.6 µg/mLb Saxagliptin AUC0-24 = 28.9 Cmax = 9 µg•h/mLa µg/mLc AUC0-24 = 12.3 µg•h/mLa BMS-510849 Cmax = 25 µg/mLc AUC0-24 = 47.9 µg•h/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 Cmax 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, corresponding to 50, 78, and 139 µg/mL for saxagliptin and 4.6, 7.9, and 11 µg/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 Cmax values in a rabbit EFD study at steady state (GD19) at 40 mg/kg/day saxagliptin (Cmax 8.5 µg/mL). Saxagliptin Cmax values were linearly 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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC 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(0-24h): 2.06 Rabbit (150 µg•hr/mLb mg/kg/day) AUC0-24 = AUC0-24 = 241 µg•h/mLa 80 µg•h/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 AUC0-24 = 23 GD7-20 µg•h/mLa AUC0-24 = 6 225 mg/kg µg•h/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 Cmax LOAEL/Human Notes Cmax Cmax Findings Findings Cmax Cmax AUC AUC AUC AUC AUC AUC0-24 = 68 µg•h/mLa AUC0-24 = 19 µg•h/mLa a Calculated from exposure ratios compared to human exposure at MRHD (2.06 µg•hr/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