FEED:	Fertility and Early Embryonic Developmental
GD:	Gestation Day
GI:	Gastrointestinal
GLP:	Good Laboratory Practices
ICH:	International Council for Harmonisation of Technical Requirements Pharmaceuticals for Human Use	for
ICH:
IV:	Intravenous
LOAEL:	Lowest Observed Adverse Effect Level
LLO:	Late Life Onset
MOA:	Mechanism of Action
MEFL:	Malformation or Embryo-Fetal Lethality
MFD:	Maximum Feasible Dose
MRHD:	Maximum Recommended Human Dose
NHP:	Non-Human Primate
NOAEL:	No Observed Adverse Effect Level
PD:	Pharmacodynamic
pEFD:	Preliminary Embryo-Fetal Development
PK:	Pharmacokinetic
PND:	Postnatal Day
PPND:	Pre- and Postnatal Developmental
SDLT:	Severely Debilitating or Life-Threatening
TK:	Toxicokinetic

WOCBP: Women of Child Bearing Potential

1. INTRODUCTION & GENERAL PRINCIPLES

The purpose of this document is to recommend international standards for, and promote

harmonization of, the assessment of nonclinical developmental and reproductive toxicity (DART) testing required to support human clinical trials and marketing authorization for pharmaceuticals. The guideline 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 is a revision of the ICH guideline “S5 Detection of Toxicity to Reproduction for Medicinal Products” that was originally published in 1993. This revision brings the guideline into alignment with other ICH guidelines, 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, 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 pharmaceutical and, when appropriate, its metabolites (ICH M3 (1), ICH S6 (2)) on all stages of reproduction and development. No guideline can provide sufficient information

to cover all possible cases, and flexibility in testing strategy is warranted.

1.1. Aim of Studies

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 (3)).

2. SCOPE OF THE GUIDELINE

This guideline 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 guideline, the term “pharmaceutical” is used to encompass all of these treatment modalities. This guideline 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 guideline apply to all compounds for which the conduct of reproductive and/or developmental toxicity studies is appropriate. This guideline should be read in conjunction with ICH M3, ICH S6, and ICH S9 regarding whether and when nonclinical DART studies are warranted.

3. 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 non-mammalian 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 guideline.

To the extent that it does not diminish the overall risk assessment, the experimental strategy should minimize the use of animals. Approaches towards this goal can include the conduct of studies that combine typical study types (see Section 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 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 non-compliance should be identified within the study report and their impact on study results/data interpretation should be considered relative to the overall

safety assessment.

3.1. Target Patient Population/ Therapeutic Indication Considerations

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 post-menopausal female patient population, for use in the pediatric or juvenile pre-pubescent population, or for patient populations in

hospitalized settings where pregnancy can be excluded.

3.2. Pharmacology Considerations

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 pre-term labor. If the intended pharmacologic effects are incompatible with the study endpoints, testing for a particular

reproductive endpoint is not warranted, with justification.

3.3. Toxicity Considerations

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 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.

3.4. Timing Considerations

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 Section

4.2.2 and 4.2.3.

3.5. Toxicokinetics (TK)

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 TK parameters induced by pregnancy, it is recommended to determine if pregnancy alters exposure. If dose selection is based on exposure ratio (see section 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 pre-weaning period.

General concepts regarding TK data collection are discussed in ICH S3A (4).

4. DESIGN AND EVALUATION OF IN VIVO MAMMALIAN STUDIES

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 non-human primate (NHP)) it is not possible to evaluate all stages. For most pharmaceuticals, the 3-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.

4.1. Strategy to Address Fertility and Early Embryonic Development (FEED)

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 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., sperm collection for counts and morphology/motility assessments, measuring hormone levels, or monitoring of the estrous/menstrual cycle) to further characterize potential effects on fertility.

4.1.1. Considerations for Biopharmaceuticals

If the biopharmaceutical ispharmacologically active in rodents or rabbits, a FEED study in one of these species is recommended. Mating evaluations are not generally feasible in non- rodents 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.

4.2. Strategies to Address Embryo-Fetal Development (EFD)

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 non-rodent (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 (Section 5.1) then non-routine species (Section 5.2), genetically modified animals, or use of a species-specific surrogate molecule (Section 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.

4.2.1. Considerations for Biopharmaceuticals

The effect of biopharmaceuticals on EFD should typically be assessed in two species (one rodent and one non-rodent) if both are pharmacologically relevant. However, the rodent is often not pharmacologically relevant, in which case EFD assessment in a single pharmacologically relevant non-rodent species can be conducted. In cases where the NHP is the only relevant species, an enhanced pre-and postnatal development (ePPND) study can be conducted instead of an EFD study. Biopharmaceuticals intended for the treatment of advanced cancer typically need only 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.

4.2.2. Alternative Approaches for Addressing EFD Risk

4.2.2.1. Use of Alternative Assays

A number of alternative in vitro, ex vivo, and non-mammalian 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.

4.2.3. Potential Approaches to Defer Definitive In Vivo Testing as Part of an Integrated Testing

Strategy

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 guideline expands on ICH M3 by allowing two additional options to support inclusion of WOCBP prior to Phase 3 clinical trials:

1) Qualified alternative assays which predict the outcome in one species (see Annex 2), can be combined with a pEFD 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 non-rodent 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 in a 2nd species allows all regions to include

an unlimited number of WOCBP in clinical trials through Phase 2.

4.3. Strategy to Address Effects on Pre- and Postnatal Development (PPND)

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 (5).

4.3.1. Considerations for Biopharmaceuticals

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).

5. TEST SYSTEM SELECTION

5.1. Routine Test Species

Mammalian species should be used to detect DART. The use of the same species and strain

as 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 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, etc.).

5.1.1. Selection of Species for DART Testing

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 non-rodent species is typically evaluated, although there are exceptions (e.g., vaccines and biopharmaceuticals, see Sections 5.1.2 and 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 non- rodent species based on the extensive historical background data, availability of animals,

and practicality.

5.1.2. Species Selection for Preventative and Therapeutic Vaccines

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.

5.2. Non-routine Test Species

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.

NHPs should be considered a non-routine 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).

5.3. Use of Disease Models, Genetically Modified Models, and Surrogate Molecules

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.

6. DOSE LEVEL SELECTION, ROUTE OF ADMINISTRATION AND

SCHEDULE

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.

6.1. Dose Selection

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 concepts set forth in sections 6.1.1 to 6.1.5 below. The selected doses should take into account observations made in previous studies (e.g., repeated-dose, TK, DRF, etc.). There can be instances where fewer than three dose levels are sufficient to provide the necessary 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.

6.1.1. Toxicity–based Endpoint

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.

6.1.2. Saturation of Systemic Exposure Endpoint

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.

6.1.3. Exposure Margin Based Endpoint

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 AUC or 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). 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.

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 the use of total vs. fraction unbound pharmaceutical exposures should be justified and consistent with the entire nonclinical development program as outlined in ICH S3A.

6.1.3.1. Exposure–based Approach for Biopharmaceuticals

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.

6.1.4. Maximum Feasible Dose (MFD) Endpoint

The MFD can be used for high dose selection when the physico-chemical 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 theMFD should maximize exposure in the test species, rather than maximize the administered dose, as per ICH M3 Q&A (1). Note that changes to the frequency of dose administration can be considered to increase the total feasible daily exposure (see Section 6.3).

6.1.5. Limit Dose Endpoint

A limit dose of 1 g/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).

6.1.6. Selection of Lower Dose Levels

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 sub-therapeutic in humans should

be justified.

6.2. Route

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 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.

6.3. Schedule

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.

6.4. Dose Selection and Study Designs for Vaccines

This guideline covers vaccines (adjuvanted or not) used in both preventative and therapeutic indications against infectious diseases. While not within the scope of this guideline, 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, pre-pubertal 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 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 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 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.

7. POSSIBLE COMBINATION STUDY DESIGNS IN RODENTS

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. Further, if treated, dosing of females can be extended until the end of organogenesis, thereby allowing evaluation of EFD endpoints (Annex 1).

8. DATA REPORTING AND STATISTICS

8.1. Data Reporting

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 non-pregnant 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.

8.2. Statistics

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.

9. PRINCIPLES OF RISK ASSESSMENT

As described in the preceding sections of this guideline, 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 can include exposure margins, biological plausibility, evidence of a dose-response relationship, potential for reversibility, the potential for confounding parental toxicity, and 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 , 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 this 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.

Also, 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).

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.

10. ENDNOTES

Note 1: In particular, the testes and epididymides should be sampled and processed using methods which 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. (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. (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.

11. GLOSSARY

Disclaimer: The definitions in this glossary are specific for their use within this guideline.

Alternative assay(s): In vitro, ex vivo or non-mammalian 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.

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.

Developmental toxicity: Any adverse effect induced prior to attainment of adult life. It includes effects induced or manifested from conception to postnatal life.

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).

Malformation: Permanent structural deviation that generally is incompatible with or severely detrimental to normal development or survival.

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).

Surrogate molecule: A molecule showing similar pharmacologic activity in the test species as that shown by the human pharmaceutical in the human.

Vaccine: For the purpose of this guideline, 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.

12. REFERENCES

1. International Council for Harmonisation M3: Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals together with ICH M3 Questions & Answers.

2. International Council for Harmonisation S6: Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals.

3. International Council for Harmonisation S9: Nonclinical Evaluation for Anticancer Pharmaceuticals.

4. International Council for Harmonisation S3A: Note for Guidance on Toxicokinetics: The Assessment of Systemic Toxicity in Toxicity Studies together with ICH S3A Questions and Answers.

5. International Council for Harmonisation S11: Nonclinical Safety Testing in Support of Development of Pediatric Medicines.

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;105:62-8.

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.

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• Widely used for pharmacodynamics and drug discovery• Robust reproductive capacity with short gestation• Large group sizes and litter size• Data available from repeated- dose toxicity study• Suitable for all stages of testing• Widespread laboratory experience and availability• Extensive historical data	• Different placentation to human (e.g., timing, inverted yolk sac)• Dependence on prolactin as the primary hormone for establishment and maintenance of early pregnancy, which makes them sensitive to some pharmaceuticals (e.g., dopamine agonists)• Highly sensitive to pharmaceuticals that disrupt parturition (e.g., nonsteroidal anti-inflammatory drugs in late pregnancy)• Less sensitive than humans to fertility perturbations• Limited application for foreign proteinso Limited or nopharmacologic activityo Potential impact of immunogenicity

Rabbit	• Similar advantages to rats• Non-rodent model• Suitable for serial semen sampling and mating studies• Placental transfer of antibodies more closely approximates primates than rodents, an advantage for DART testing of vaccines	• Limitations similar to rat for foreign proteins• Limited historical data for fertility and pre-/postnatal studies• Sensitive to gastrointestinal disturbances; (e.g., some antibiotics)• Prone to spontaneous abortion• General physical condition difficult to monitor using clinical signs• Should generate PD, toxicity, and TK data as not generally used for toxicology programs (except for vaccines)
Mouse	• Similar advantages to rats• Genetically modified models available or can be generated• Surrogate molecules are often available• Uses small amounts of test material	• Similar limitations to rats• Small fetus size and tissue volumes• Stress sensitivity• Malformation clusters are known to occur

Non-routine Species
Species	Advantages	Disadvantages
Cynomolgus Monkey(NHP)	• Generally morephylogenetically andphysiologically similar to humans than other species• More likely than rodents to show similar pharmacology to humans• Placentation similar to human• Data available from repeated- dose toxicity study• Transfer of antibodies across the placenta similar to humans	• Small group size, hence low statistical power and wide variability across groups• Low fecundityo Single offspring• High background pregnancy lossLimited availability of breeding animals• Long menstrual cycle (30 days) and gestation (165 days)• Impractical for fertility (mating) studies• F1 reproduction function not practical to evaluate due to 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 non-rodent for general toxicity testing• Short period of organogenesis (GD 11-35)• Defined genetic background and specific-pathogen-free animals• 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 number of experienced laboratories• Long gestation (114 days)• Uses a large amount of test material• Minimal to no prenatal transfer of antibodies

Limited Use Species (primarily used for investigative purposes)
Species	Advantages	Disadvantages
Hamster	• Alternate rodent model that can be pharmacologically relevant	• High postnatal loss due to cannibalization• Limited historical control data and laboratory experience
		• Limited postnatal	availability behavioral	of and
		functional tests• IV route difficult• Aggressive• Sensitive to GI disturbances• Should generate PD, toxicity, and TK data as not generally used for toxicology programs• Blood sampling is difficult

Dog

• Usually have repeated-dose toxicity data• Readily amenable to semen collection

• Long gestation (63 days)• Limited historical control data and laboratory experience• 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 fulfil 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. Below 16 litters inter-study results become inconsistent, and above 20 to 24 litters per group, consistency and precision is 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.

Table 2: FEED Study Design: Rodents, combined male and female study

Parameter

Group size

Number of dose groups Administration perioda

at least 16 of each sex 4 (including 1 control)

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.

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 13-15 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 Developmental (EFD) Toxicity Study

The EFD toxicity study is designed to assess maternal toxicity relative to that in non- pregnant 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. 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 Developmental (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).

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.

Table 3: EFD Toxicity Study Designs for Rodent, Rabbit and

ParameterGLP StatusMinimum number of pregnant femalesNumber of dose groupsAdministration perioddAntemortem endpointsClinicalobservations/mortality Body weightFood consumptionToxicokineticsPostmortem endpointsCesarean section fMacroscopic examination Gravid uterine weightCorpora luteaImplant sitesLive and dead conceptusesEarly and late resorptions Gross evaluation of placenta Weight of placentaFetal body weight Fetal sexFetal external evaluationsg

pEFDRodent/RabbitOptionalc 64 (including 1 control) Species appropriate At least once dailyAt least twice weekly At least once weekly Optionalc Species appropriate YesOptional YesYesYesYesYesOptional YesYes Yes

NHP

EFD

Rat (Mouse)Yes 164 (including 1 control) GD6/7-17 (6/7-15) At least once dailyAt least twice weeklye At least once weekly Yes GD20/21 (17/18)YesOptional YesYesYesYesYesOptional YesYes Yes

RabbitYes 164 (including 1 control) GD6/7-19 At least once dailyAt least twice weeklye At least once weekly Yes GD28/29 YesOptional YesYesYesYesYesOptional YesYesYes

NHPa

Yes 16b

At least 2 (including 1 control)

Approximately GD 20 – to at least GD 50

At least once daily

At least once weekly Optional

Yes

GD100 Optional NA

Yes

Optional Yes

Yes Yes

Yes	Yesg	Yes	Yes
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). For NHP, females are dosed from confirmation of pregnancy (approximately GD 20) 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 GD 100.

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.

1.1.3 Pre– and Postnatal Developmental (PPND) Toxicity Study

The PPND toxicity study is designed to assess enhanced toxicity relative to that in non- pregnant 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 (GD 6/7) through weaning

(postnatal day (PND) 20)

F0 Females

Clinical At least once daily

observations/mortality

Body weight At least twice weekly

Food consumption At least once weekly until mid-lactation

Parturition observations GD 21 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 Pre-weaning

Clinical Daily from PND 0

observations/mortality

Pre-and postweaning survival Daily from PND 0

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 Post-weaning
Selection for post-weaning evaluation and group sizeb	PND 21, at least 1 male and 1 female/litter where possible to achieve 16 animals per group/sex
Clinical observations/mortality	Daily
Body weight	Weekly
Optional Food consumption	Weekly
Sexual maturationc	Females: vaginal openingMales: 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 bodyweight, however, measurement of bodyweight 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 Non–Human

Primate (NHP)

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 for information on timing and additional parameters to be evaluated.

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

GD 20) to parturition

F0 Females

Clinical At least once daily

observations/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 TK profiles and/or systemic drug levels should be

Exposure Assessment measured, as appropriate

Clinical Daily from PND 0

observations/mortality

Body weights Weekly

Morphometry/Physical and/or At regular intervals, as appropriate

functionalassessment

Neurobehavioural test battery At least 1 interval during the first 2 weeks

. postpartum

Grip strength

Mother-infant interaction

Exposure assessment

External evaluation Skeletal evaluation Visceral evaluation Necropsy

PND 28

Minimally in early postnatal period to confirm nursing; as appropriate thereafter

Systemic drug levels should be measured, as appropriate

At regular intervals

Approximately PND 28 or later At 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.

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). This study design is most often used with rodents, although it could be used with non-rodents.

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 will need to be evaluated in a separate study.

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