S5A: Detection of Toxicity to Reproduction for Medicinal Products

December 23, 2002

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Notice - Withdrawal of Toxicological Evaluation Guidelines and Re-issuance of ICH¹ Safety Guidances

The Health Canada Toxicological Evaluation guidances (revised 1996) are being withdrawn following an internal review by a Safety Expert Working Group which concluded that they no longer reflected current toxicological methodologies. Furthermore, the review revealed substantial areas of overlap and inconsistency between these guidances and their more recently adopted ICH counterparts.

The following Health Canada-adopted ICH Safety (Nonclinical) guidances, previously available as part of the Toxicological Evaluation guidances, are being re-issued as stand alone documents:

1. S1A Need for Carcinogenicity Studies of Pharmaceuticals
2. S2A Guidance on Specific Aspects of Regulatory Genotoxicity Tests For Pharmaceuticals
3. S3A Note for Guidance on Toxicokinetics: The Assessment of Systemic Exposure in Toxicity Studies
4. S3B Pharmacokinetics: Guidance for Repeated Dose Tissue Distribution Studies
5. S5A Detection of Toxicity to Reproduction for Medicinal Products

These ICH guidances have been developed by the appropriate ICH Expert Working Group and have been subject to consultation by the regulatory parties, in accordance with the ICH Process. The ICH Steering Committee has endorsed the final draft and recommended its adoption by the regulatory bodies of the European Union, Japan and USA.

In adopting these ICH guidances, Health Canada as observer to ICH, endorses the principles and practices described therein. These documents should be read in conjunction with this covering notice and with the relevant sections of other applicable Health Canada guidances.

These and other guidance documents are currently available on the Therapeutic Products Directorate / Biologics and Genetic Therapies Directorate Website (s) (http://www.hc-sc.gc.ca/hpfb-dgpsa/tpd-dpt/). The availability of printed copies of guidance documents may be confirmed by consulting the Guidelines and Publications Order Forms (available on the TPD/BGTD Website) or by contacting the Publications Coordinator².

Should you have any questions regarding the content of the guidance, please contact

Colette F. Strnad, B. Sc., Ph.D.
Title: Senior Scientific Advisor
Office of Science
Therapeutic Products Directorate
Holland Cross, Tower B, 2nd Floor,
A.L. 3102C3 1600 Scott Street
Ottawa, Ontario K1A 1B6
telephone: (613) 941-3693
fax: (613) 941-5035
email: colette_strnad@hc-sc.gc.ca

Guidance for Industry

Published by authority of the Minister of Health

1996

Health Products and Food Branch Guidance Document

© Minister of Public Works and Government Services Canada 1996

Available in Canada through
Health Canada - Publications
Brooke Claxton Building,
A.L. #0913A Tunney's Pasture
Ottawa, Ontario K1A 0K9
Tel: (613) 954-5995
Fax: (613) 941-5366

Foreword

This guidance has been developed by the appropriate ICH Expert Working Group and has been subject to consultation by the regulatory parties, in accordance with the ICH Process. The ICH Steering Committee has endorsed the final draft and recommended its adoption by the regulatory bodies of the European Union, Japan and USA.

In adopting this ICH guidance, Health Canada endorses the principles and practices described therein. This document should be read in conjunction with the accompanying notice and the relevant sections of other applicable guidances.

Guidance documents are meant to provide assistance to industry and health care professionals on how to comply with the policies and governing statutes and regulations. They also serve to provide review and compliance guidance to staff, thereby ensuring that mandates are implemented in a fair, consistent and effective manner.

Guidance documents are administrative instruments not having force of law and, as such, allow for flexibility in approach. Alternate approaches to the principles and practices described in this document may be acceptable provided they are supported by adequate scientific justification. Alternate approaches should be discussed in advance with the relevant program area to avoid the possible finding that applicable statutory or regulatory requirements have not been met.

As a corollary to the above, it is equally important to note that Health Canada reserves the right to request information or material, or define conditions not specifically described in this guidance, in order to allow the Department to adequately assess the safety, efficacy or quality of a therapeutic product. Health Canada is committed to ensuring that such requests are justifiable and that decisions are clearly documented.

Table of Contents

1. Introduction
   
   • 1.1 Purpose of This Guidance Document
   • 1.2 Aim of Studies
   • 1.3 Choice of Studies
2. Animal Criteria
   
   • 2.1 Selection and Number of Species
   • 2.2 Other Test Systems
3. General recommendations concerning treatment
   
   • 3.1 Dosages
   • 3.2 Route and Frequency of Administration
   • 3.3 Kinetics
   • 3.4 Control Groups
4. Proposed Study Designs - Combination of Studies
   
   • 4.1 The Most Probable Option
     • 4.1.1 Study of Fertility and Early Embryonic Development to Implantation
     • 4.1.2 Study for Effects on Pre-and Post-natal Development, Including Maternal Function
     • 4.1.3 Study for Effects on Embryo-Fetal Development
   • 4.2 Single Study Design (Rodents)
   • 4.3 Two Study Design (Rodents)
5. Statistics
6. Data Presentation
7. Terminology

1. Introduction

1.1 Purpose of This Guidance Document

There is a considerable overlap in the methodology that could be used to test chemicals and medicinal products for potential reproductive toxicity. As a first step to using this wider methodology for efficient testing, this guidance document attempts to consolidate a strategy based on study designs currently in use for testing of medicinal products; it should encourage the full assessment on the safety of chemicals on the development of the offspring. It is perceived that tests in which animals are treated during defined stages of reproduction better reflect human exposure to medicinal products and allow more specific identification of stages at risk. While this approach may be useful for most medicines, long-term exposure to low doses does occur and may be represented better by a one or two generation study approach.

The actual testing strategy should be determined by:

• anticipated drug use especially in relation to reproduction,
• the form of the substance and route(s) of administration intended for humans, and
• making use of any existing data on toxicity, pharmaco-dynamics, kinetics, and similarity to other compounds in structure/activity.

To employ this concept successfully, flexibility is needed (Note 1). No guidance can provide sufficient information to cover all possible cases. All persons involved should be willing to discuss and consider variations in test strategy according to the state of the art and ethical standards in human and animal experimentation.

Note 1 (1.1) Scientific Flexibility

These guidances are not mandatory rules; they are a starting point rather than an end point. They provide a basis from which an investigator can devise a strategy for testing according to available knowledge of the test material and the "state of the art." For encouragement, some alternative test designs have been mentioned in this document but there are others that can be sought out or devised. In devising a strategy, the primary objective should be to detect and bring to light any indication of toxicity to reproduction.

Fine details of study design and technical procedures have been omitted from the text. Such decisions rightly belong in the field of the investigator, since a technique that may be suitable for one laboratory may not be suitable for another. The investigator needs to utilize staff and resources to do the best he or she can achieve, and should know how to do this better than any outsider; human attributes of attitude, ability, and consistency are more important than material facilities. For necessary compliance to GLP, reference is made to such regulations.

1.2 Aim of Studies

The aim of reproduction toxicity studies is to reveal any effect of one or more active substance(s) on mammalian reproduction. For this purpose both the investigations and the interpretation of the results should be related to all other pharmacological and toxicological data available to determine whether potential reproductive risks to humans are greater, lesser, or equal to those posed by other toxicological manifestations. Further, repeated dose toxicity studies can provide important information regarding potential effects on reproduction, particularly male fertility. To extrapolate the results to humans (assess the relevance), data on likely human exposures, comparative kinetics, and mechanisms of reproductive toxicity may be helpful.

The combination of studies selected should allow exposure of mature adults and all stages of development from conception to sexual maturity. To allow detection of immediate and latent effects of exposure, observations should be continued through one complete life cycle, (i.e., from conception in one generation through conception in the following generation). For convenience of testing, this integrated sequence can be subdivided into the following stages.

1. Premating to conception (adult male and female reproductive functions, development and maturation of gametes, mating behaviour, fertilisation).
2. Conception to implantation (adult female reproductive functions, preimplantation development, implantation).
3. Implantation to closure of the hard palate (adult female reproductive functions, embryonic development, major organ formation).
4. Closure of the hard palate to the end of pregnancy (adult female reproductive functions, fetal development and growth, organ development and growth).
5. Birth to weaning (adult female reproductive functions, neonate adaptation to extrauterine life, preweaning development and growth).
6. Weaning to sexual maturity (postweaning development and growth, adaptation to independent life, attainment of full sexual function).

For timing conventions, see Note 2.

Note 2 (1.2) Timing Conventions

In this guidance document, the convention for timing of pregnancy is to refer to the day that a sperm-positive vaginal smear and/or plug is observed as day 0 of pregnancy even if mating occurs overnight. Unless shown otherwise, it is assumed that, for rats, mice, and rabbits, implantation occurs on day 6 to 7 of pregnancy, and closure of the hard palate on day 15 to 18 of pregnancy.

Other conventions are equally acceptable but MUST be defined in reports. Also, the investigator must be consistent in different studies to assure that no gaps in treatment occur. It is an advisable precaution to provide an overlap of at least one day in the exposure period of related studies.

The accuracy of the time of mating should be specified since this will affect the variability of fetal and neonatal parameters.

Similarly, for reared litters, the day offspring are born will be considered as post-natal or lactation day 0, unless otherwise specified. However, particularly with regard to delays in, or prolongation of, parturition, reference to a postcoital time frame may be useful.

1.3 Choice of Studies

The guidance addresses the design of studies primarily for detection of effects on reproduction. When an effect is detected, further studies to characterise fully the nature of the response have to be designed on a case-by-case basis (Note 3). The rationale for the set of studies chosen should be given and should include an explanation for the choice of dosages.

Studies should be planned according to the "state of the art," and take into account pre-existing knowledge of class-related effects on reproduction. They should avoid suffering and should use the minimum number of animals necessary to achieve the overall objectives. If a preliminary study is performed, the results should be considered and discussed in the overall evaluation (Note 4).

Note 3 (1.3) First Pass and Secondary Testing

To a greater or lesser degree all first pass (guideline) tests are apical in nature (i.e., an effect on one endpoint may have several different origins). A reduced litter size at birth may be due to a reduced ovulation rate (corpora lutea count), higher rate of pre-implantation deaths, higher rate of post-implantation deaths or immediate post-natal deaths. In turn, these deaths may be the consequence of an earlier physical malformation that can no longer be observed due to subsequent secondary changes, and so on. Particularly for effects with a natural low frequency among controls, discrimination between treatment induced and coincidental occurrence is dependent upon association with other types of effects.

A toxicant usually induces more than one type of effect in a dose-dependent manner. For example, induction of malformation is almost invariably associated with increased embryonic death and an increased incidence of less severe structural changes. Given an effect on one endpoint, secondary investigations for possible associations should be considered, (i.e., the nature, scope, and origins of the substance's toxicity should be characterized). Characterization should also include identification of dose-response relationships to facilitate risk assessment; this is different from the situation in first pass tests where the presence or absence of a doseresponse assists discrimination between treatment-related and coincidental differences.

Note 4 (1.3) Preliminary Studies

At the time most reproduction studies are planned or initiated, there is usually information available from acute and repeated dose toxicity studies of at least one month's duration. This information can be expected to be sufficient in identifying doses for reproductive studies. If adequate preliminary studies are performed, they are part of the justification of the choice of dose for the main study. Such studies should be submitted regardless of their GLP status in principle. This may avoid unnecessary use of animals.

2. Animal Criteria

The animals used must be well defined with respect to their health, fertility, fecundity, prevalence of abnormalities, embryofetal deaths and the consistency they display from study to study. Within and between studies, animals should be of comparable age, weight, and parity at the start; the easiest way to fulfil these criteria is to use animals that are young, mature adults at the time of mating, with the females being virgin.

2.1 Selection and Number of Species

Studies should be conducted in mammalian species. It is generally desirable to use the same species and strain as in other toxicological studies. Reasons for using rats as the predominant rodent species are practicality, comparability with other results obtained in this species, and the large amount of background knowledge accumulated.

In embryotoxicity studies only, a second mammalian species traditionally has been required, the rabbit being the preferred choice as a "non-rodent." Reasons for using rabbits in embryotoxicity studies include the extensive background knowledge that has accumulated, as well as availability and practicality. Where the rabbit is unsuitable, an alternative non-rodent or a second rodent species may be acceptable and should be considered on a case by case basis (Note 5).

Note 5 (2.1) Selection of Species and Strains

In choosing an animal species and strain for reproductive toxicity testing, care should be given to select a relevant model. Selection of the species and strain used in other toxicology studies may avoid the need for additional preliminary studies. If it can be shown - by means of kinetic, pharmacological, and toxicological data - that the species selected is a relevant model for the human, a single species can be sufficient. There is little value in using a second species if it does not show the same similarities to humans. Advantages and disadvantages of species (strains) should be considered in relation to the substance to be tested, the selected study design, and in the subsequent interpretation of the results.

All species have their advantages. Rats, and to a lesser extent mice, are good, general purpose models; the rabbit has been somewhat neglected as a "non-rodent" species for repeated dose toxicity and other reproduction studies than embryotoxicity testing. It has attributes that would make it a useful model for fertility studies, especially male fertility. For both rabbits and dogs (which are often used as a second species for chronic toxicity studies) it is feasible to obtain semen samples without resorting to painful techniques (electro ejaculation) for longitudinal semen analysis. Most of the other species are not good, general purpose models and probably are best used for very specific investigations only.

All species have their disadvantages, for example:

Rats:

sensitivity to sexual hormones; unsuitable for dopamine agonists due to dependence on prolactin as the primary hormone for establishment and maintenance of early pregnancy; highly susceptible to non-steroidal, anti-inflammatory drugs in late pregnancy.

Mice:

fast metabolic rate; stress sensitivity; malformation clusters (which occur in all species) particularly evident; small fetus.

Rabbits:

often lack of kinetic and toxicity data; susceptibility to some antibiotics and to disturbance of the alimentary tract; clinical signs can be difficult to interpret.

Guinea pigs:

often lack of kinetic and toxicity data; susceptibility to some antibiotics and to disturbance of the alimentary tract; long fetal period; insufficient historical background data.

Domestic and/or mini pigs:

malformation clusters with variable background rate; large amounts of compound required; large housing necessary; insufficient historical background data.

Ferrets:

seasonal breeder unless special management systems used (success highly dependent on human/animal interaction); insufficient historical background data.

Hamsters:

Intravenous route difficult if not impossible; can hide doses in the cheek pouches and can be very aggressive; sensitive to intestinal disturbance; overly sensitive teratogenic response to many chemicals; small fetus.

Dogs:

seasonal breeders; inbreeding factors; insufficient historical background data.

Non-human primates:

kinetically they can differ from humans as much as other species; insufficient historical background data; often numbers too low for detection of risk. They are best used when the objective of the study is to characterize a relatively certain reproductive toxicant, rather than detect a hazard.

2.2 Other Test Systems

Other test systems are considered to be any developing mammalian and non-mammalian cell systems, tissues, organs, or organism cultures developing independently in vitro or in vivo. Integrated with whole animal studies either for priority selection within homologous series or as secondary investigations to elucidate mechanisms of action, these systems can provide invaluable information and, indirectly, reduce the numbers of animals used in experimentation. However, they lack the complexity of the developmental processes and the dynamic interchange between the maternal and the developing organisms. These systems cannot provide assurance of the absence of effect, nor provide perspective with respect to risk or exposure. In short, there are no alternative test systems to whole animals currently available for reproduction toxicity testing with the aims set out in the introduction (Note 6).

Note 6 (2.2) Uses of Other Test Systems than Whole Animals

Other test systems have been developed and used in preliminary investigations ("pre-screening" or priority selection) and secondary testing.

For preliminary investigation of a range of analogue series of substances it is essential that the potential outcome in whole animals is known for at least one member of the series to be studied (by inference, effects are expected). With this strategy substances can be selected for higherlevel testing.

For secondary testing or further substance characterization other test systems offer the possibility to study some of the observable developmental processes in detail, (e.g., to reveal specific mechanisms of toxicity, to establish concentration-response relationships, to select "sensitive periods", or to detect effects of defined metabolites).

3. General Recommendations Concerning Treatment

3.1 Dosages

Selection of dosages is one of the most critical issues in design of the reproductive toxicity study. The choice of the high dose should be based on data from all available studies (pharmacology, acute and chronic toxicity, and kinetic studies) (Note 7). A repeated dose toxicity study of about 2 to 4 weeks duration provides a close approximation to the duration of treatment in segmental designs of reproductive studies. When sufficient information is not available, preliminary studies are advisable (see Note 4).

Having determined the high dosage, lower dosages should be selected in a descending sequence, the intervals depending on kinetic and other toxicity factors. While it is desirable to be able to determine a "no observed adverse effect level," priority should be given to setting dosage intervals close enough to reveal any dosage related trends that may be present (Note 8).

Note 7 (3.1) Selection of Dosages

Using similar doses in the reproductive toxicity studies as in the repeated dose toxicity studies will allow interpretation of any potential effects on fertility in context with general systemic toxicity.

Some minimal toxicity is expected to be induced in the high-dose dams.

According to the specific compound, factors limiting the high dosage determined from repeat dose toxicity studies or from preliminary reproduction studies could include:

• reduction in bodyweight gain;
• increased bodyweight gain, particularly when related to perturbation of homeostatic mechanisms;
• specific target organ toxicity;
• hematology, clinical chemistry;
• exaggerated pharmacological response, which may or may not be reflected as marked clinical reactions (e.g., sedation, convulsions);
• the physico-chemical properties of the test substance or dosage formulation which, allied to the route of adminstration, may impose practical limitations in the amount that can be administered. Under most circumstances 1 g/kg/day should be an adequate limit dose;
• kinetics, they can be useful in determining high dose exposure for low toxicity compounds. There is, however, little point in increasing administered dosage if it does not result in increased plasma or tissue concentration;
• marked increase in embryo-fetal lethality in preliminary studies.

Note 8 (3.1) Determination of Dose-Response Relationships

For many of the variables in reproduction studies, the power to discriminate between random variation and treatment effect is poor and the presence or absence of a dosage-related trend can be a critical means of determining the probability of a treatment effect. It has to be kept in mind that in these studies, dose responses may be steep, and wide intervals between doses would be inadvisable. If an analysis of dose-response relationships for the effects observed is attempted in a single study, it is recommended to use at least three dose levels and appropriate control groups. If in doubt, a fourth dose group should be added to avoid excessive dosage intervals. Such a strategy should provide a "no observed adverse effect level" for reproductive aspects. If not, the implication is that the test substance merits a greater depth of investigation and further studies.

3.2 Route and Frequency of Administration

In general the route or routes of administration should be similar to those intended for human usage. One route of substance administration may be acceptable if it can be shown that a similar distribution (kinetic profile) results from different routes (Note 9).

The usual frequency of administration is once daily but consideration should be given to use either more frequent or less frequent administration taking kinetic variables into account (Note 10).

Note 9 (3.2) Exposure by Different Routes of Administration

If it can be shown that one route provides a greater body burden, (e.g., area under the curve (AUC)), there seems little reason to investigate routes that would provide a lesser body burden or which present severe practical difficulties (e.g., inhalation). Before designing new studies for a new route of adminstration, existing data on kinetics should be used to determine the necessity of another study.

3.3 Kinetics

It is preferable to have some information on kinetics before initiating reproduction studies, since this may suggest the need to adjust choice of species, study design, and dosing schedules. At this time the information need not be sophisticated nor derived from pregnant or lactating animals.

At the time of study evaluation, further information on kinetics in pregnant or lactating animals may be required according to the results obtained (Note 10).

Note 10 (3.3) Kinetics in Pregnant Animals

Kinetic investigations in pregnant and lactating animals may pose some problems due to the rapid changes in physiology. It is best to consider this as a two-or three-phase approach. In planning studies, kinetic data (often from non-pregnant animals) provide information on the general suitability of the species, and can assist in deciding study designs and choice of dosage. During a study, kinetic investigations can provide assurance of accurate dosing or indicate marked deviations from expected patterns.

3.4 Control Groups

It is recommended that control animals be dosed with the vehicle at the same rate as test-group animals. When the vehicle may cause effects or affect the action of the test substance, a second (sham or untreated) control group should be considered.

4. Proposed Study Designs - Combination of Studies

All available pharmacological, kinetic, and toxicological data for the test compound and similar substances should be considered in deciding the most appropriate strategy and choice of study design. It is anticipated that, initially, preference will be given to designs that do not differ too radically from those of established guidances for medicinal products (the most probable option). For most medicinal products, the three-study design will usually be adequate. Other strategies, combinations of studies, and study designs could be as valid or more valid as the "most probable option" according to circumstances. The key factor is that, in total, they leave no gaps between stages and allow direct or indirect evaluation of all stages of the reproductive process (Note 11).

Designs should be justified.

Note 11 (4) Examples of Choosing Other Options

For compounds causing no lethality at 2 g/kg and no evidence of repeated dose toxicity at 1 g/kg, conduct of a single two-generation study with one control and two test groups (0.5 and 1.0 g/kg) would seem sufficient. However, it might pose the question as to whether the correct species had been chosen or whether the compound was an effective medicine.

For compounds that may be given as a single dose once-in-a-lifetime (e.g., diagnostics, medicines used in operations) it may be impossible to administer repeated dosages more than twice the human therapeutic dosage for any length of time. A reduced period of treatment allowing a higher dose would seem more appropriate. For females, considerations of human exposure suggest little or no need for exposures beyond the embryonic period.

For dopamine agonists or compounds reducing circulating prolactin levels, female rats are poor models; the rabbit would probably make a better choice for all the reproductive toxicity studies, but it does not appear to have been attempted. This also applies to other types of compound when the rabbit shows a pattern of metabolism considerably closer to humans than the rat.

For drugs where alterations in plasma kinetics are seen following repeated administration, the potential for adverse effects on embryo-fetal development may not be fully evaluated in studies according to 4.1.3. In such cases, it may be desirable to extend the period of drug adminstration to females in a 4.1.1 study to day 17. With sacrifice at term, both fertility and embryo-fetal development can be assessed.

4.1 The Most Probable Option

The "most probable option" can be equated to a combination of studies for effects on:

• Fertility and early embryonic development;
• Pre- and post-natal development, including maternal function; and
• Embryo-fetal development.

4.1.1 Study of Fertility and Early Embryonic Development to Implantation

Aim

To test for toxic effects/disturbances resulting from treatment from before mating (males/females) through mating, and implantation. This comprises evaluation of stages A and B of the reproductive process (see Section 1.2). For females, this should detect effects on the oestrous cycle, tubal transport, implantation, and development of preimplantation stages of the embryo. For males, it will permit detection of functional effects (e.g., on libido, epididymal sperm maturation) that may not be detected by histological examinations of the male reproductive organs (Note 12).

Assessment

• maturation of gametes,
• mating behaviour,
• fertility,
• pre-implantation stages of the embryo,
• implantation.

Animals

At least one species, preferably rats.

Number of Animals

The number of animals per sex per group should be sufficient to allow meaningful interpretation of the data (Note 13).

Administration Period

It is assumed that, especially for effects on spermatogenesis, use will be made of available data from toxicity studies (e.g. histopathology, weight of reproductive organs, in some cases hormone assays and genotoxicity data). Provided no effects have been found in repeated dose toxicity studies of at least 4 weeks duration that preclude this, a premating treatment interval of 2 weeks for females and 4 weeks for males can be used (Note 12). Selection of the length of the premating administration period should be stated and justified. Treatment should continue throughout mating to termination of males and at least through implantation for females. This will permit evaluation of functional effects on male fertility that cannot be detected by histopathologic examination in repeated dose toxicity studies and effects on mating behaviour in both sexes. If data from other studies show there are effects on weight or histology of reproductive organs in males or females, or if the quality of examinations is dubious or if there are no data from other studies, then the need for a more comprehensive study should be considered (Note 12).

Mating

A mating ratio of 1:1 is advisable and procedures should allow identification of both parents of a litter (Note 14).

Terminal Sacrifice

Females may be sacrificed at any point after mid-pregnancy.

Males may be sacrificed at any time after mating but it is advisable to ensure successful induction of pregnancy before taking such an irrevocable step (Note 15).

Observations

During study:

• signs and mortalities at least once daily;
• body weight and body weight change at least twice weekly (Note 16);
• food intake at least once weekly (except during mating);
• record vaginal smears daily, at least during the mating period, to determine whether there are effects on mating or precoital time;
• observations that have proved of value in other toxicity studies.

At terminal examination:

• perform necropsy (macroscopic examination) of all adults,
• preserve organs with macroscopic findings for possible histopathological evaluation; keep corresponding organs of sufficient controls for comparison,
• preserve testes, epididymides, ovaries and uteri from all animals for possible histopathological examination and evaluation on a case by case basis,
• count corpora lutea, implantation sites (Note 16),
• count live and dead conceptuses.

Sperm analysis can be used as an optional procedure for confirmation or better characterisation of the effects observed (Note 12).

Note 12 (4.1.1) Premating Treatment

The design of the fertility study, especially the reduction in the premating period for males, is based on evidence accumulated and on re-appraisal of the basic research on the process of spermatogenesis. Compounds inducing selective effects on male reproduction are rare; compounds affecting spermatogenesis almost invariably affect post meiotic stages and weight of testis; mating with females is an insensitive means of detecting effects on spermatogenesis. Histopathology of the testis has been shown to be the most sensitive method for the detection of effects on spermatogenesis. Good pathological and histopathological examination (e.g. by employing Bouin's fixation, paraffin embedding, transverse section of 2-4 microns for testes, longitudinal section for epididymides, PAS and haematoxylin staining) of the male reproductive organs provides a direct means of detection. Sperm analysis (sperm counts, sperm motility, sperm morphology) can be used as an optional method to confirm findings by other methods and to characterise effects further. Sperm analysis data are considered more relevant for fertility assessment when samples from vas deferens or from cauda epididymis are used. Information on potential effects on spermatogenesis (and female reproductive organs) can be derived from repeated dose toxicity studies or reproductive toxicity studies.

For detection of effects not detectable by histopathology of male reproductive organs and sperm analysis, mating with females after a premating treatment of 4 weeks has been shown to be least as efficient as mating after a longer duration of treatment. Two weeks may be acceptable in some cases. However, when a 2 weeks treatment period is selected, more convincing justification should be provided. When the available evidence suggests that the scope of investigations in the fertility study should be increased, appropriate studies should be designed to characterise the effects further.

Note 13 (4.1.1, 4.1.2, 4.1.3) Number of Animals

There is very little scientific basis underlying specified group sizes in past and existing guidances nor in this one. The numbers specified are educated guesses governed by the maximum study size that can be managed without undue loss of overall study control. This is indicated by the fact that the more expensive the animal is to obtain or keep, the smaller the group size proposed. Ideally, at least the same group size should be required for all species, and there is a case for using larger group sizes for less frequently-used species such as primates.

It should also be made clear that the numbers required depend on whether or not the group is expected to demonstrate an effect. For a high frequency effect few animals are required. To presume the absence of an effect, the number required varies according to the variable (endpoint) being considered, its prevalence in control populations (rare or categorical events) or dispersion around the central tendency (continuous or semicontinuous variables). (See also Note 23.)

For all but the rarest events (such as malformations, abortions, total litter loss), evaluation of between 16 to 20 litters for rodents and rabbits tends to provide a degree of consistency between studies. Below 16 litters per evaluation, between-study results become inconsistent. Above 20 to 24 litters per group, consistency and precision are not greatly enhanced. These numbers relate to evaluation. If groups are subdivided for different evaluations, the number of animals starting the study should be doubled. Similarly, in studies with two breeding generations, 16 to 20 litters would be required for the final evaluation of the litters of the F1 generation. To allow for natural wastage, the starting group size of the F0 generation must be larger.

Note 14 (4.1.1) Mating

Mating Ratios:

When both sexes are being dosed or are of equal consideration in separate male and female studies, the preferred mating ratio is 1:1 since this is the safest option in respect of obtaining good pregnancy rates and avoiding incorrect analysis and interpretation of results.

Mating Period and Practices:

Most laboratories would use a mating period of between 2 to 3 weeks; some remove females as soon as a positive vaginal smear or plug is observed while others leave the pairs together. Most rats will mate within the first five days of cohabitation (i.e., at the first available estrus), but in some cases females may become pseudopregnant. Leaving the female with the male for about 20 days allows these females to restart estrus cycles and become pregnant.

Note 15 (4.1.1) Terminal Sacrifice

Females:

When exposure of the females ceases at implantation, termination of females between days 13 to 15 of pregnancy in general is adequate to assess effects on fertility or reproductive function, (e.g., to differentiate between implantation and resorption sites).

In general, for detection of adverse effects, it is not thought necessary, in a fertility study, to sacrifice females at day 20 or 21 of pregnancy in order to gain information on late embryo loss, fetal death, and structural abnormalities.

Males:

It would be advisable to delay sacrifice of the males until the outcome of mating is known. In the event of an equivocal result, males could be mated with untreated females to ascertain their fertility or infertility. The males treated as part of study 4.1.1 may also be used for evaluation of toxicity to the male reproductive system if dosing is continued beyond mating and sacrifice is delayed.

Note 16 (4.1.1, 4.1.2, 4.1.3) Observations

Daily weighing of pregnant females during treatment can provide useful information. Weighing an animal more frequently than twice weekly during periods other than pregnancy (premating, mating, lactation) may also be advisable for some compounds.

For apparently non-pregnant rats or mice (but not rabbits), ammonium sulphide staining of the uterus might be useful to identify peri-implantation death of embryos.

4.1.2 Study for Effects on Pre-and Post-natal Development, Including Maternal Function

Aim

To detect adverse effects on the pregnant/lactating female and on development of the conceptus and the offspring following exposure of the female from implantation through weaning. Since manifestations of effect induced during this period may be delayed, observations should be continued through sexual maturity (i.e., stages C to F listed in Section 1.2) (Notes 17, 18).

Adverse Effects to be Assessed

• enhanced toxicity relative to that in non-pregnant females;
• pre- and post-natal death of offspring;
• altered growth and development;
• functional deficits in offspring, including behaviour, maturation (puberty) and reproduction (F1).

Animals

At least one species, preferably rats.

Number of Animals

The number of animals per sex per group should be sufficient to allow meaningful interpretation of the data (Note 13).

Administration Period

Females are exposed to the test substance from implantation to the end of lactation (i.e., stages C to E listed in Section 1.2).

Experimental Procedure

The females are allowed to deliver and rear their offspring to weaning, at which time one male and one female offspring per litter should be selected (document method used) for rearing to adulthood and mating to assess reproductive competence (Note 19).

Observations

During study (for maternal animals):

• signs and mortalities at least once daily;
• body weight and body weight change at least twice weekly (Note 16);
• food intake at least once weekly at least until delivery;
• observations that have proved of value in other toxicity studies;
• duration of pregnancy;
• parturition.

At terminal examination (for maternal animals and where applicable for offspring):

• necropsy (macroscopic examination) of all adults;
• preservation and possibly histological evaluation of organs with macroscopic findings; keep corresponding organs of sufficient controls for comparison;
• implantations (Note 16);
• abnormalities;
• live offspring at birth;
• dead offspring at birth;
• body weight at birth;
• pre- and post-weaning survival and growth/body weight (Note 20), maturation, and fertility;
• physical development (Note 21);
• sensory functions and reflexes (Note 21); and
• behaviour (Note 21).

Note 17 (4.1.2) Treatment of Offspring

Consequent to derivation from existing guidances for medicines, this guidance document does not fully cover exposures from weaning through puberty, nor does it deal with the possibility of reduced reproductive life span.

To detect adverse effects for medicinal products that may be used in infants and juveniles, special studies (case by case designs) involving direct treatment of offspring, at ages to be specified, should be considered.

Note 18 (4.1.2) Separate Embryotoxicity and Peri- Post-natal Studies

If a pre- and post-natal study is separated into two studies, one covering the embryonic period the other the fetal period, parturition, and lactation, post-natal evaluation of offspring is required in both studies.

Note 19 (4.1.2) F1-Animals

The guidance suggests selection of one male and one female per litter, on the evidence that it is feasible to conduct behavioural and other functional tests on the same F1 individuals that will be used for assessment of reproductive function. This has the advantage of allowing cross-referencing of performance in different tests at the individual level. It is recognized, however, that some laboratories prefer to select separate sets of animals for behaviour testing and for assessment of reproductive function. Whichever is the most suitable for an individual laboratory will depend upon the combination of tests used and the resources available.

Note 20 (4.1.2) Reduction of Litter Size

The value of culling or not culling for detection of effects on reproduction is still under discussion. Whether or not culling is performed, it should be explained by the investigator.

Note 21 (4.1.2) Physical Development, Sensory Functions, Reflexes, and Behaviour

The best indicator of physical development is bodyweight. Achievement of preweaning landmarks of development such as pinna unfolding, coat growth, incisor eruption, etc., is highly correlated with pup bodyweight. This weight is better related to postcoital time than post-natal time, at least when significant differences in gestation length occur. Reflexes, surface righting, auditory startle, air righting, and response to light are also dependent on physical development.

Two post-weaning landmarks of development that are advised are vaginal opening of females and cleavage of the balanopreputial gland of males. The latter is associated with increasing testosterone levels, whereas testis descent is not. These landmarks indicate the onset of sexual maturity and it is advised that bodyweight be recorded at the time of attainment to determine whether any differences from control are specific or related to general growth.

Functional tests:

To date, functional tests have been directed almost exclusively to behaviour. Even though a great deal of effort has been expended in this direction, it is not possible to recommend specific test methods. Investigators are encouraged to find methods that will assess sensory functions, motor activity, learning, and memory.

4.1.3 Study for Effects on Embryo-Fetal Development

Aim

To detect adverse effects on the pregnant female and development of the embryo and fetus consequent to exposure of the female from implantation to closure of the hard palate (i.e., stages C to D listed in Section 1.2).

Adverse Effects to be Assessed

• enhanced toxicity relative to that in non-pregnant females;
• embryo-fetal death;
• altered growth;
• structural changes.

Animals

Usually, two species: one rodent, preferably rats; one non-rodent, preferably rabbits (Note 5). Justification should be provided when using one species.

Number of Animals

The number of animals should be sufficient to allow meaningful interpretation of the data (Note 13).

Administration Period

The treatment period extends from implantation to the closure of the hard palate (i.e., end of C). (See Section 1.2.)

Experimental Procedure

Females should be killed and examined about one day prior to parturition. All fetuses should be examined for viability and abnormalities. To allow subsequent assessment of the relationship between observations made by different techniques fetuses should be individually identified (Note 22).

When using techniques requiring allocation to separate examination for soft tissue or skeletal changes, it is preferable that 50% of fetuses from each litter be allocated for skeletal examination. A minimum of 50% rat fetuses should be examined for visceral alterations, regardless of the technique used. When using fresh microdissection techniques for soft tissue alterations - which is the strongly preferred method for rabbits - 100% of rabbit fetuses should be examined for soft tissue and skeletal abnormalities.

Observations

During study (for maternal animals):

• signs and mortalities at least once daily;
• body weight and body weight change at least twice weekly (Note 16);
• food intake at least once weekly;
• observations that have proved of value in other toxicity studies.

At terminal examination:

• necropsy (macroscopic examination) of all adults;
• preserve organs with macroscopic findings for possible histological evaluation; keep corresponding organs of sufficient controls for comparison;
• count corpora lutea, numbers of live and dead implantations (Note 16);
• individual fetal body weight;
• fetal abnormalities (Note 22);
• gross evaluation of placenta.

Note 22 (4.1.3) Individual Identification and Evaluation of Fetuses

It must be possible to relate all findings by different techniques (i.e., body weight, external inspection, visceral and/or skeletal examinations) to a single specimen in order to detect patterns of abnormalities. The examination of mid- and low-dose fetuses for visceral and/or skeletal abnormalities may not be necessary where the evaluation of the high dose and the control groups did not reveal any relevant differences. It is advisable, however, to store the fixed specimen for possible later examination. If fresh dissection techniques are normally used, difficulties with later comparisons involving fixed fetuses should be anticipated.

4.2 Single Study Design (Rodents)

If the dosing period of the fertility study and pre- and post-natal study are combined into a single investigation, this comprises evaluation of stages A to F of the reproductive process (see Section 1.2). If such a study, if it includes fetal examinations, provided clearly negative results at sufficiently high exposure no further reproduction studies in rodents should be required. Fetal examinations for structural abnormalities can also be supplemented with an embryo-fetal development study (or studies) to make a two-study approach (Notes 3, 11).

Results from a study for effects on embryo-fetal development in a second species are expected (see also Section 4.1.3).

4.3 Two Study Design (Rodents)

The simplest two segment design would consist of the fertility study and the pre- and post-natal development study, if it includes fetal examinations. It can be assumed, however, that if the preand post- natal development study provided no indication of prenatal effects at adequate margins above human exposure, the additional fetal examinations (as made in Section 4.1.3) are most unlikely to provide a major change in the assessment of risk.

Alternatively, female treatment in the fertility study (Section 4.1.1) could be continued until closure of the hard palate, and fetuses examined according to the procedures of the embryofetal development study (in Section 4.1.3). This, combined with the pre- and post-natal study (Section 4.1.2), would provide all the examinations required in "the most probable option" but would use considerably less animals (Notes 3, 11).

Results from a study for effects on embryo-fetal development in a second species are expected (see also Section 4.1.3).

5. Statistics

Analysis of the statistics of a study is the means by which results are interpreted. The most important part of this analysis is to establish the relationship between the different variables and their distribution (descriptive statistics), since these determine how groups should be compared. The distributions of the endpoints observed in reproductive tests are usually non-normal, and extend from almost continuous to the extreme categorical.

When employing inferential statistics (determination of statistical significance) the mating pair or litter, not the fetus or neonate, should be used as the basic unit of comparison. The tests used should be justified (Note 23).

Note 23 (5) Inferential Statistics

"Significance" tests (inferential statistics) can be used only as a support for the interpretation of results. The interpretation itself must be based on biological plausibility. It is unwise to assume that a difference from control values is not biologically relevant simply because it is not "statistically significant." To a lesser extent, it can be unwise to assume that a "statistically significant" difference must be biologically relevant. Particularly for low-frequency events (e.g., embryonic death, malformations) with one-sided distributions, the statistical power of studies is low. Confidence intervals for relevant quantities can indicate the likely size of the effect. When using statistical procedures, experimental units of comparison should be considered: the litter, not the individual conceptus, the mating pair, when both sexes are treated, the mating pair of the parent generation in a two generation study.

6. Data Presentation

The key to good reporting is the tabulation of individual values in a clear, concise manner to account for every animal that was entered into the study. A reader should be able to follow the history of any individual animal from initiation to termination and should be able to deduce with ease the contribution that the individual has made to any group summary values. Group summary values should be presented in a form that is biologically plausible (i.e., avoid false precision) and that reflects the distribution of the variable. Appendices or tabulations of individual values such as bodyweight, food consumption, litter values should be concise and, as far as possible, consist of absolute rather than calculated values; unnecessary duplication should be avoided.

For tabulation of low-frequency observations such as clinical signs, autopsy findings, abnormalities, etc., it is advisable to group together the (few) individuals with a positive recording. Especially in the presentation of data on structural changes (fetal abnormalities), the primary listing (tabulation) should clearly identify the litters containing abnormal fetuses, identify the affected fetus in the litter, and report all the changes observed in the affected fetuses. Secondary listings by type of change can be derived from this, if necessary.

7. Terminology

Besides effects on the reproductive competence of adult animals, toxicity to reproduction includes:

Developmental Toxicity

Any adverse effect induced prior to attainment of adult life. It includes effects induced or manifested in the embryonic or fetal period and those induced or manifested post-natally.

Embryotoxicity, Fetotoxicity, Embryo-Fetal Toxicity

Any adverse effect on the conceptus resulting from prenatal exposure, including structural or functional abnormalities or post-natal manifestations of such effects.

Terms such as "embryotoxicity" and "fetotoxicity" relate to the timepoint/period of induction of adverse effects, irrespective of the time of detection.

One, Two, Or Three Generation Studies

Are defined according to the number of adult breeding generations directly exposed to the test material. For example, in a one generation study there is direct exposure of the F0 generation and indirect exposure (via the mother) of the F1 generation; the study is usually terminated at weaning of the F1 generation. In a two generation study as used for agro-chemicals and industrial chemicals, there is direct exposure of the F0 generation, indirect and direct exposure of the F1 generation and indirect exposure of the F2 generation. A three generation study is defined accordingly.

Body Burden

The total internal dosage of an individual arising from the administration of a substance, comprising parent compound and metabolites, taking distribution and accumulation into account.

Kinetics

The term "kinetics" is used consistently throughout this guidance document, irrespective of intending to mean pharmaco-and/or toxicokinetics. No better single term was available.