Guideline for Industry

Detection of Toxicity to

Reproduction for Medicinal

Products 

ICH-S5A

September 1994
i

TABLE OF CONTENTS

I. INTRODUCTION ........................................ 1

A. Purpose of the Guideline ............................. 1

B. Aim of Studies .................................... 2

C. Choice of Studies .................................. 3

II. ANIMAL CRITERIA ...................................... 4

A. Selection and Number of Species ....................... 4

B. Other Test Systems ................................. 4

III. GENERAL RECOMMENDATIONS CONCERNING TREATMENT ......... 5

A. Dosages ........................................ 5

B. Route and Frequency of Administration ................... 5

C. Kinetics ......................................... 5

D. Control Groups .................................... 6

IV. PROPOSED STUDY DESIGNS-COMBINATION OF STUDIES .......... 6

A. The Most Probable Option ............................ 6

1. Study of Fertility and Early Embryonic Development to

Implantation ................................. 6

a. Aim ................................... 6

b. Assessment of: ........................... 7

c. Animals ................................ 7

d. Number of Animals ........................ 7
ii

e. Administration Period ....................... 7

f. Mating ................................. 8

g. Terminal Sacrifice ......................... 8

h. Observations ............................ 8

2. Study for Effects on Prenatal and Postnatal Development,

Including Maternal Function ....................... 9

a. Aim ................................... 9

b. Adverse Effects To Be Assessed ............... 9

c. Animals ............................... 10

d. Number of Animals ....................... 10

e. Administration Period ...................... 10

f. Experimental Procedure .................... 10

g. Observations ........................... 10

3. Study for Effects on Embryo-Fetal Development ........ 11

a. Aim .................................. 11

b. Adverse Effects To Be Assessed .............. 12

c. Animals ............................... 12

d. Number of Animals ....................... 12

e. Administration Period ...................... 12

f. Experimental Procedure .................... 12

g. Observations ........................... 13
iii

B. Single Study Design (rodents) ......................... 13

C. Two Study Design (rodents) .......................... 14

V. STATISTICS ......................................... 14

VI. DATA PRESENTATION .................................. 15

VII. TERMINOLOGY ....................................... 15

VIII. REFERENCE ......................................... 16

NOTES FOR CLARIFICATION OF GUIDELINE. . . . . . . . . . . . . . . . . . . Appendix A

ADDITIONAL CLARIFICATION FOR USE OF GUIDELINE . . . . . . . . . . . Appendix B
This guideline was developed within the Expert Working Group (Safety) of †

the International Conference on Harmonisation of Technical Requirements for

Registration of Pharmaceuticals for Human Use (ICH) and has been subject to

consultation by the regulatory parties, in accordance with the ICH process. This

document has been endorsed by the ICH Steering Committee at Step 4 of the ICH

process, June 24, 1993. At Step 4 of the process, the final draft is recommended

for adoption to the regulatory bodies of the European Union, Japan and the USA. 

This guideline was published in the Federal Register on September 22, 1994 (59

FR 48746) and is applicable to drug and biological products. In the past,

guidelines have generally been issued under § 10.90(b) [21 CFR 10.90(b)], which

provides for the use of guidelines to state procedures or standards of general

applicability that are not legal requirements but that are acceptable to FDA. The

agency is now in the process of revising §10.90(b). Therefore, this guideline is

not being issued under the authority of §10.90(b), and it does not create or confer

any rights, privileges or benefits for or on any person, nor does it operate to bind

FDA in any way. For additional copies of this guideline contact the Executive

Secretariat Staff, HFD-8, Center for Drug Evaluation and Research, 7500 Standish

Place, Rockville, MD, 20855, 301-594-1012. An electronic version of this

guideline is also available via Internet by connecting to the CDER FTP server

(CDVS2.CDER.FDA.GOV) using the FTP protocol.

To help facilitate understanding of the guideline, the agency is providing ††

further clarification of important questions that have been raised since initial

general distribution of the document at ICH 2 by both industry and regulatory

scientists. See Appendix B for these clarifications.

GUIDELINE FOR INDUSTRY†

DETECTION OF TOXICITY TO REPRODUCTION FOR

MEDICINAL PRODUCTS††

I. INTRODUCTION (1)

A. Purpose of the Guideline (1.1)

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 guideline attempts to consolidate a strategy based on

study designs currently in use for testing of medicinal products; it
2

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,

pharmacodynamics, kinetics, and similarity to other compounds

in structure/activity.

To employ this concept successfully, flexibility is needed. No 1

guideline 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. Areas where more

basic research would be useful for optimization of test designs are

male fertility assessment, and kinetic and metabolism in

pregnant/lactating animals. 

B. Aim of Studies (1.2)

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
3

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(A). Premating to conception (adult male and female reproductive

functions, development and maturation of gametes, mating

behavior, fertilization).

2(B). Conception to implantation (adult female reproductive

functions, preimplantation development, implantation).

3(C). Implantation to closure of the hard palate (adult female

reproductive functions, embryonic development, major organ

formation).

4(D). Closure of the hard palate to the end of pregnancy (adult

female reproductive functions, fetal development and growth,

organ development and growth).

5(E). Birth to weaning (adult female reproductive functions, neonate

adaption to extrauterine life, preweaning development and

growth).

6(F). Weaning to sexual maturity (postweaning development and

growth, adaption to independent life, attainment of full sexual

function).

For timing conventions see Note 2.2

C. Choice of Studies (1.3)

The guideline addresses the design of studies primarily for detection

of effects on reproduction. When an effect is detected, further

studies to characterize fully the nature of the response have to be

designed on a case-by-case basis. The rationale for the set of 3

studies chosen should be given and should include an explanation for

the choice of dosages. 
4

Studies should be planned according to the "state-of-the art," and

take into account preexisting 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.4

II. ANIMAL CRITERIA (2)

The animals used should 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 fulfill these criteria is to use animals that are young, mature

adults at the time of mating with the females being virgin.

A. Selection and Number of Species (2.1)

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 "nonrodent." 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 nonrodent or a second rodent species may be

acceptable and should be considered on a case-by-case basis.5

B. Other Test Systems (2.2)

Other test systems are considered to be any developing mammalian

and nonmammalian 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,
5

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 in respect of

risk/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.6

III. GENERAL RECOMMENDATIONS CONCERNING TREATMENT (3)

A. Dosages (3.1)

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 ). A repeated dose toxicity study 7

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.4

Having determined the high dosage, lower dosages should be selected

in a descending sequence, the intervals depending on kinetic and

other toxicity factors. Whilst 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.8

B. Route and Frequency of Administration (3.2)

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.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.10
6

C. Kinetics (3.3)

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.10

D. Control Groups (3.4)

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. 

IV. PROPOSED STUDY DESIGNS-COMBINATION OF STUDIES (4)

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 guidelines 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. 

11

Designs should be justified. 

A. The Most Probable Option (4.1)

The most probable option can be equated to a combination of studies

for effects on:
7

Fertility and early embryonic development,

Prenatal and postnatal development, including maternal

function, and

Embryo-fetal development.

1. Study of Fertility and Early Embryonic Development to

Implantation (4.1.1) 

a. 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 I.B.

(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.12

b. Assessment of:

Maturation of gametes,

Mating behavior, 

Fertility,

Preimplantation stages of the embryo, and

Implantation.

c. Animals

At least one species, preferably rats. 

d. Number of Animals
8

The number of animals per sex per group should be

sufficient to allow meaningful interpretation of the data.13

e. Administration Period

The design assumes that, especially for effects on

spermatogenesis, use will be made of data from repeated

dose toxicity studies of at least 1-month duration. 

Provided no effects have been found that preclude this, a

premating treatment interval of 2 weeks for females and

4 weeks for males can be used. Selection of the length 12

of the premating administration period should be stated

and justified [see also I.A. (1.1), pointing out the need

for research]. 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 histologic examination in repeated dose

toxicity studies and effects on mating behavior in both

sexes. If data from other studies show there are effects

on weight or histologic appearance 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 a more comprehensive study should

be designed.12

f. Mating

A mating ratio of 1:1 is advisable and procedures should

allow identification of both parents of a litter. 

14

g. Terminal Sacrifice

Females may be sacrificed at any point after

midpregnancy. 

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.15

h. Observations 
9

During study:

Signs and mortalities at least once daily;

Body weight and body weight changes at

least twice weekly;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;

and

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;

Preserve testes, epididymides, ovaries and

uteri from all animals for possible

histological examination and evaluation on a

case-by-case basis; tissues can be discarded

after completion and reporting of the study;

Sperm count in epididymides or testes, as

well as sperm viability;

Count corpora lutea, implantation sites;16

and

Live and dead conceptuses. 
10

2. Study for Effects on Prenatal and Postnatal Development,

Including Maternal Function (4.1.2)

a. 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 3 to 6 (C to F) listed in I.B.

(1.2)].17,18

b. Adverse Effects To Be Assessed

Enhanced toxicity relative to that in nonpregnant

females;

Prenatal and postnatal death of offspring;

Altered growth and development; and

Functional deficits in offspring, including behavior,

maturation (puberty), and reproduction (F1). 

c. Animals

At least one species, preferably rats.

d. Number of Animals

The number of animals per sex per group should be

sufficient to allow meaningful interpretation of the data.13

e. Administration Period

Females are exposed to the test substance from

implantation to the end of lactation [i.e., stages 3 to 5 (C

to E) listed in I.B. (1.2)].
11

f. 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.19

g. Observations

During study (for maternal animals):

Signs and mortalities at least once daily,

Body weight and body weight change at

least twice weekly,16

Food intake at least once weekly at least

until delivery,

Observations that have proved of value in

other toxicity studies,

Duration of pregnancy, and

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;16

Abnormalities;
12

Live offspring at birth; 

Dead offspring at birth;

Body weight at birth;

Preweaning and postweaning survival and

growth/body weight, maturation, and 20

fertility;

Physical development;21

Sensory functions and reflexes; and 21

Behavior.21

3. Study for Effects on Embryo-Fetal Development (4.1.3)

a. Aim 

To detect adverse effects on the pregnant female and

development of the embryo and fetus consequent to

exposure of the female implantation to closure of the

hard palate [i.e., stages 3 to 4 (C to D) listed in I.B.

(1.2)].

b. Adverse Effects To Be Assessed 

Enhanced toxicity relative to that in nonpregnant

females,

Embryofetal death,

Altered growth, and 

Structural changes.

c. Animals

Usually, two species: one rodent, preferably rats; one
13

nonrodent, preferably rabbits. Justification should be 5

provided when using one species.

d. Number of Animals

The number of animals should be sufficient to allow

meaningful interpretation of the data.13

e. Administration Period

The treatment period extends from implantation to the

closure of the hard palate [i.e., end of 3 (C), see I.B.

(1.2)].

f. Experimental Procedure

Females should be sacrificed and examined about 1 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.22

When using techniques requiring allocation to separate

examination for soft tissue or skeletal changes, it is

preferable that 50 percent of fetuses from each litter be

allocated for skeletal examination. A minimum of 50

percent 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 percent of rabbit fetuses should be

examined for soft tissue and skeletal abnormalities.

g. Observations 

During study (for maternal animals):

Signs and mortalities at least once daily,

Body weight and body weight change at
14

least twice weekly,16

Food intake at least once weekly, and 

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;16

Individual fetal body weight;

Fetal abnormalities; and 22

Gross evaluation of placenta. 

B. Single Study Design (rodents) (4.2)

If the dosing period of the fertility study and prenatal and postnatal

study are combined into a single investigation, this comprises

evaluation of stages 1 to 6 (A to F) of the reproductive process [see

I.B. (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.3,11

Results from a study for effects on embryo-fetal development in a

second species are expected [see also IV.A.3. (4.1.3)]. 
15

C. Two Study Design (rodents) (4.3)

The simplest two-segment design would consist of the fertility study

and the prenatal and postnatal development study, if it includes fetal

examinations. It can be assumed, however, that if the prenatal and

postnatal development study provided no indication of prenatal

effects at adequate margins above human exposure, the additional

fetal examinations [see IV.A.3. (4.1.3)] are most unlikely to provide a

major change in the assessment of risk.

Alternatively, female treatment in the fertility study [IV.A.1. (4.1.1)]

could be continued until closure of the hard palate and fetuses

examined according to the procedures of the embryo-fetal

development study [IV.A.3. (4.1.3)]. This, combined with the

prenatal and postnatal study [IV.A.2. (4.1.2)] would provide all the

examinations required in "the most probable option" but use

considerably less animals.3,11

Results from a study for effects on embryo-fetal development in a

second species are expected [see also IV.A.3. (4.1.3)].

V. STATISTICS (5) 

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), because these determine how groups should be compared. The

distributions of the endpoints observed in reproductive tests are usually

nonnormal 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.23

VI. DATA PRESENTATION (6)

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
16

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 fetuses in the litter, and report all the changes observed in the

affected fetus. Secondary listings by type of change can be derived from

this, if necessary.

VII. TERMINOLOGY (7) 

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 postnatally.

Embryotoxicity, fetotoxicity, embryo-fetal toxicity: Any adverse effect on

the conceptus resulting from prenatal exposure, including structural or

functional abnormalities or postnatal manifestations of such effects. Terms

like "embryotoxicity" or "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, and

the study is usually terminated at the 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
17

administration of a substance, comprising parent compound and

metabolites, taking distribution and accumulation into account. 

Kinetics: The term "kinetics" is used consistently throughout this guideline,

irrespective of intending to mean pharmaco- and/or toxicokinetics. No

better single term was available.

VIII. REFERENCE

Federal Register, Vol. 59, No.183, Thursday, September 22, 1994, pages

48746-48752. 
B-1

APPENDIX B

ADDITIONAL CLARIFICATION FOR USE OF GUIDELINE

To help facilitate understanding of the guideline, the agency is providing further

clarification of important questions that have been raised since initial general

distribution of the document at ICH 2 by both industry and regulatory scientists.

General Comments 

First pass tests in the guideline are those tests that will are likely be

performed as general screens (i.e., the three-study design or "most probable

option") to identify potential treatment related effects. Secondary tests are

those designed to characterize, e.g., the nature, scope, and/or origin of the

toxic effect. In general, repeated dose general toxicity studies of 2 to 4

weeks duration may provide a close approximation of the doses to be used

in the reproductive toxicology studies.

Male Fertility 

As stated in the introduction to the guideline, studies are ongoing to

optimize parameters to be used in fertility studies, including the optimal

treatment period for males prior to mating, histological techniques for the

evaluation of sex organs, and techniques to evaluate sperm. It is expected

that, in most cases, viability will be measured indirectly by evaluating sperm

motility. A variety of methods will be acceptable to evaluate sperm,

including vital dye staining, flow cytometric analysis, and nonautomated and

automated methods to measure the percent of motile sperm. Sponsors

should justify the methods used and define the objective criteria established

to assess the data obtained. It is expected that improvements in methods

to assess male reproductive performance will evolve over the next few

years.

The design of the study of fertility [IV.A.1. (4.1.1)] assumes that, especially

for effects on spermatogenesis, use will be made of data from repeated

dose toxicity studies of at least 1-month duration. The agency encourages

the use of good pathological and histopathological examination techniques

in the repeated dose toxicity studies in addition to the staging of

spermatogenesis which is routinely employed. The preservation of testes

and epididymides from all animals from ICH study IV.A.1. (4.1.1) provides
B-2

an opportunity for more detailed histopathological examination on a

case-by-case basis; for example, if unexpected effects on sperm count or

viability are observed. There may be cases due to species-specific effects

or technical considerations (e.g., multiple samplings are required over time)

when sperm evaluation in nonrodents may be more appropriate.

The duration of pretreatment for males in ICH study IV.A.1. (4.1.1) is 4

weeks, unless data from other studies suggest that this should be modified. 

Males should be treated throughout the mating period (generally between 2

and 3 weeks) and at least through implantation of the females. Thus, males

will generally be sacrificed following at least 7 to 9 weeks dosing. 

Evaluations should generally include organ weights and macroscopic

examinations of testis, epididymis, seminal vesicle, and prostate. Sperm

counts and sperm viability (e.g., motility) should be assessed. Tissues

should be saved for potential histological assessment, as such assessments

may be required on a case-by-case basis. If histological data are not

available from previous studies or the quality of the data are dubious, then

histological evaluation should be performed in this study.

Prenatal and Postnatal Development

When studying the effect on postnatal development, the reduction of litter

size by culling is still under discussion. If culling is performed, it should be

randomized. Whether or not it is performed, it should be explained by the

investigator. Observations on offspring in ICH study IV.A.2. (4.1.2) include

sensory functions and reflexes and behavior, consistent with previous

guidelines from Japan and the European Union. Specific functional tests

have not been recommended in the ICH guideline. Investigators are

encouraged to use methods that will assess sensory functions, motor

activity, learning, and memory to help characterize functional deficits in

offspring. Under the terminology section of the guideline, a three-generation

study is defined as direct exposure of the F0 generation, indirect and direct

exposure of the F1 and F2, and indirect exposure of the F3 generation. 
A-1

1. Scientific Flexibility [I.A. (1.1)]

These guidelines are not mandatory rules, they are a starting point rather

than an endpoint. 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 field of the investigator since

a technique that may be suitable for one laboratory may not be suitable in

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 good

laboratory practices (GLP), reference is made to such regulations.

2. Timing Conventions [I.B. (1.2)]

In this guideline 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 6-7 of

pregnancy, and closure of the hard palate on day 15-18 of pregnancy. 

Other conventions are equally acceptable if defined in reports. Also, the

investigator should be consistent in different studies to ensure that no gaps

in treatment occur. It is an advisable precaution to provide an overlap of at

least 1 day in the exposure period of related studies.

The accuracy of the time of mating should be specified because this will

affect the variability of fetal and neonatal parameters. 

Similarly, for reared litters, the day offspring are born will be considered as

APPENDIX A

NOTES FOR CLARIFICATION OF GUIDELINE
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postnatal or lactation day 0 unless otherwise specified. However,

particularly with regard to delays in, or prolongation of, parturition,

reference to a postcoital timeframe may be useful.

3. First Pass and Secondary Testing [I.C. (1.3)]

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 preimplantation deaths, higher rate of

postimplantation deaths, or immediate postnatal 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

dose response assists discrimination between treatment-related and

coincidental differences.

4. Preliminary Studies [I.C. (1.3)]

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 1-month 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. 

5. Selection of Species and Strains [II.A. (2.1)]
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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

"nonrodent" 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 nonsteroidal 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
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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 foetus.

Dogs: Seasonal breeders, inbreeding factors, insufficient historical

background data.

Nonhuman 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.

6. Uses of Other Test Systems Than Whole Animals [II.B. (2.2)]

Other tests systems have been developed and used in preliminary

investigations ("prescreening" 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 higher level 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.

7. Selection of Dosages [III.A. (3.1)]

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.
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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;

Haematology, 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 administration, may impose

practical limitations in the amount that can be administered; under

most circumstances 1 gram per kilogram per day (g/kg/day) should be

an adequate limit dose;

Kinetics 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; and

Marked increase in embryo-fetal lethality in preliminary studies.

8. Determination of Dose-Response Relationships [III.A. (3.1)]

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
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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.

9. Exposure by Different Routes of Administration [III.B. (3.2)]

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 administration, existing data on kinetics should be used to determine the

necessity of another study.

10. Kinetics in Pregnant Animals [III.C. (3.3)]

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 nonpregnant 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.

11. Examples for Choosing Other Options [IV. (4)]

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
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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 IV.A.3.

(4.1.3). In such cases it may be desirable to extend the period of drug

administration to females in a IV.A.1. (4.1.1) study to day 17. With

sacrifice at term, both fertility and embryo-fetal development can be

assessed.

12. Premating Treatment [IV.A.1. (4.1.1)]

The design of the fertility study, especially the reduction in the premating

period for males, is based on evidence accumulated and reappraisal of the

basic research on the process of spermatogenesis that originally prompted

the demand for a prolonged premating treatment period. Compounds

inducing selective effects on male reproduction are rare; mating with

females is an insensitive means of detecting effects on spermatogenesis;

good pathological and histopathological examination (e.g., by employing

Bouin's fixation, paraffine embedding, transverse sections of 2 to 4 microns

for testes, longitudinal sections for epididymides, PAS, and haematoxylin

staining) of the male reproductive organs provides a more sensitive and

quicker means of detecting effects on spermatogenesis; compounds

affecting spermatogenesis almost invariably affect postmeiotic stages; there

is no conclusive example of a male reproductive toxicant the effects of

which could be detected only by dosing males for 9 to 10 weeks and

mating them with females.

Information on potential effects on spermatogenesis can be derived from

repeated dose toxicity studies. This allows the investigations in the fertility

study to be concentrated on other, more immediate, causes of effect. It is

noted that the full sequence of spermatogenesis (including sperm

maturation) in rats lasts 63 days. When the available evidence, or lack of it,

suggests that the scope of investigations in the fertility study should be

increased, or extended from detection to characterization, appropriate

studies should be designed to further characterize the effects.

13. Number of Animals [IV.A.1. (4.1.1), IV.A.2. (4.1.2), IV.A.3. (4.1.3)]

There is very little scientific basis underlying specified group sizes in past
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and existing guidelines 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 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 2 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.

14. Mating [IV.A.I. (4.1.1)]

Mating ratios: When both the sexes are being dosed or are of equal

consideration in separate male and female studies, the preferred mating ratio

is 1:1 because 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 and 3 weeks, some remove females as soon as a positive

vaginal smear or plug is observed whilst others leave the pairs together. 

Most rats will mate within the first 5 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
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restart estrus cycles and become pregnant.

15. Terminal Sacrifice [IV.A.1. (4.1.1)]

Females: When exposure of the females ceases at implantation, termination

of females between days 13 and 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 20/21 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 IV.A.1. may also be used for

evaluation of toxicity to the male reproductive system if dosing is continued

beyond mating and sacrifice delayed.

16. Observations [IV.A.1. (4.1.1), IV.A.2. (4.1.2), IV.A.3. (4.1.3)]

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 nonpregnant rats or mice (but not rabbits), ammonium

sulphide staining of the uterus might be useful to identify peri-implantation

death of embryos.

17. Treatment of Offspring [IV.A.2. (4.1.2)]

Consequent to derivation from existing guidelines for medicines, this

guideline 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.
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18. Separate Embryotoxicity and Peripostnatal Studies [IV.A.2. (4.1.2)]

If a prenatal and postnatal study is separated into two studies, one covering

the embryonic period the other the fetal period, parturition, and lactation,

postnatal evaluation of offspring is required in both studies. 

19. F1-Animals [IV.A.2. (4.1.2)]

The guideline suggests selection of one male and one female per litter on

the evidence that it is feasible to conduct behavioral 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

behavior testing and for assessment of reproductive function. Which is the

most suitable for an individual laboratory will depend upon the combination

of tests used and the resources available.

20. Reduction of Litter Size [IV.A.2. (4.1.2)]

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.

21. Physical Development, Sensory Functions, Reflexes, and Behavior [IV.A.2.

(4.1.2)]

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 postnatal 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 postweaning 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.
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Functional tests: To date, functional tests have been directed almost

exclusively to behavior. 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.

22. Individual Identification and Evaluation of Fetuses [IV.A.3. (4.1.3)]

It must be possible to relate all findings by different techniques (i.e., body

weight, external inspection, visceral, and/or skeletal examinations) to 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.

23. Inferential Statistics [V (5)]

"Significance" tests (inferential statistics) can be used only as a support for

the interpretation of results. The interpretation itself is to 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.