International Conference on Harmonisation; Guideline on Detection of Toxicity to Reproduction for Medicinal Products; Availability; Notice - [Docket No. 93D-0140]

[Docket No. 93D-0140]

International Conference on Harmonisation; Guideline on Detection

of Toxicity to Reproduction for Medicinal Products; Availability

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

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SUMMARY: The Food and Drug Administration (FDA) is publishing a final

guideline entitled ``Guideline on Detection of Toxicity to Reproduction

for Medicinal Products.'' This guideline was prepared under the

auspices of the International Conference on Harmonisation of Technical

Requirements for Registration of Pharmaceuticals for Human Use (ICH).

The guideline is intended to reflect sound scientific principles for

reproductive toxicity testing. The guideline is applicable to sponsors

submitting applications to both the Center for Drug Evaluation and

Research (CDER) and the Center for Biologics Evaluation and Research

(CBER).

DATES: Effective September 22, 1994. Submit written comments at any

time.

ADDRESSES: Submit written comments on the guideline to the Dockets

Management Branch (HFA-305), Food and Drug Administration, rm. 1-23,

12420 Parklawn Dr., Rockville, MD 20857. Copies of the guideline are

available from the CDER Executive Secretariat Staff (HFD-8), Center for

Drug Evaluation and Research, Food and Drug Administration, 7500

Standish Pl., Rockville, MD 20855.

FOR FURTHER INFORMATION CONTACT:

Regarding the guideline: Joy A. Cavagnaro, Center for Biologics

Evaluation and Research (HFM-500), Food and Drug Administration, 1401

Rockville Pike, Rockville, MD 20852, 301-594-2860.

Regarding the ICH: Janet J. Showalter, Office of Health Affairs

(HFY-20), Food and Drug Administration, 5600 Fishers Lane, Rockville,

MD 20857, 301-443-1382.

SUPPLEMENTARY INFORMATION: In recent years, many important initiatives

have been undertaken by regulatory authorities and industry

associations to promote international Harmonisation of regulatory

requirements. FDA has participated in many meetings designed to enhance

Harmonisation and is committed to seeking scientifically based

harmonized technical procedures for pharmaceutical development. One of

the goals of Harmonisation is to identify and then reduce differences

in technical requirements for drug development.

ICH was organized to provide an opportunity for tripartite

Harmonisation initiatives to be developed with input from both

regulatory and industry representatives. FDA also seeks input from

consumer representatives and others. ICH is concerned with

Harmonisation of technical requirements for the registration of

pharmaceutical products among three regions: The European Union, Japan,

and the United States. The six ICH sponsors are the European

Commission, the European Federation of Pharmaceutical Industry

Associations, the Japanese Ministry of Health and Welfare, the Japanese

Pharmaceutical Manufacturers Association, FDA, and the U.S.

Pharmaceutical Research and Manufacturers of America. The ICH

Secretariat, which coordinates the preparation of documentation, is

provided by the International Federation of Pharmaceutical

Manufacturers Association (IFPMA).

The ICH Steering Committee includes representatives from each of

the ICH sponsors and the IFPMA, as well as observers from the World

Health Organization, the Canadian Health Protection Branch, and the

European Free Trade Area.

Harmonisation of reproductive toxicology testing was selected as a

priority topic during the early stages of the ICH initiative. In the

Federal Register of April 16, 1993 (58 FR 21074), FDA published a draft

tripartite guideline entitled, ``Guideline on Detection of Toxicity to

Reproduction for Medicinal Products.'' The notice gave interested

persons an opportunity to submit comments by May 17, 1993.

After consideration of the comments received and revisions to the

guideline, a final draft of the guideline was submitted to the ICH

Steering Committee in June 1993 and endorsed by the three participating

regulatory agencies. The final guideline was subsequently presented at

the second ICH meeting held in October 1993. The guideline provides

information applicable to sponsors submitting applications to both CDER

and CBER. Sponsors submitting future applications may be asked to

explain differences from the approach suggested in the 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 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 (ICH 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 4.1.1 provides 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 overtime) when sperm evaluation in nonrodents

may be more appropriate.

The duration of pretreatment for males in ICH study 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 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.

In the past, guidelines have generally been issued under

Sec. 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 are acceptable to FDA. The agency

is now in the process of revising Sec. 10.90(b). Therefore, this

guideline is not being issued under the authority of Sec. 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.

As with all of FDA's guidelines, the public is encouraged to submit

written comments with new data or other new information pertinent to

this guideline. The comments in the docket will be periodically

reviewed, and, where appropriate, the guideline will be amended. The

public will be notified of any such amendments through a notice in the

Federal Register.

Interested persons may, at any time, submit written comments on the

guideline to the Dockets Management Branch (address above). Two copies

of any comments are to be submitted, except that individuals may submit

one copy. Comments are to be identified with the docket number found in

brackets in the heading of this document. The guideline and received

comments may be seen in the office above between 9 a.m. and 4 p.m.,

Monday through Friday.

The text of the guideline follows:

Guideline on Detection of Toxicity to Reproduction for Medical Products

1. Introduction

1.1 Purpose of the Guideline

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 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 (Note

1). No 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.

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.

A. Premating to conception (adult male and female reproductive

functions, development and maturation of gametes, mating behavior,

fertilization).

B. Conception to implantation (adult female reproductive

functions, preimplantation development, implantation).

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

reproductive functions, embryonic development, major organ

formation).

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

female reproductive functions, fetal development and growth, organ

development and growth).

E. Birth to weaning (adult female reproductive functions,

neonate adaption to extrauterine life, preweaning development and

growth).

F. Weaning to sexual maturity (postweaning development and

growth, adaption to independent life, attainment of full sexual

function).

For timing conventions see Note 2.

1.3 Choice of Studies

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 (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 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 (Note 4).

2. Animal Criteria

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.

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 ``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 (Note 5).

2.2 Other Test Systems

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,

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 (Note 6).

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. 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 (Note 8).

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 (see

also Note 10).

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).

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

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 (Note

11).

Designs should be justified.

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,

Prenatal and postnatal 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 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 of

Maturation of gametes,

Mating behavior,

Fertility,

Preimplantation stages of the embryo, and

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

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 (Note 12).

Selection of the length of the premating administration period

should be stated and justified (see also 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 (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 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 (Note 15).

Observations

During study:

Signs and mortalities at least once daily;

Body weight and body weight changes 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; 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 (Note 16); and

Live and dead conceptuses.

4.1.2 Study for Effects on Prenatal and Postnatal 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 1.2) (Notes 17 and 18).

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).

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 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, 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 (Note 16);

Abnormalities;

Live offspring at birth;

Dead offspring at birth;

Body weight at birth;

Preweaning and postweaning survival and growth/body

weight (Note 20), maturation, and fertility;

Physical development (Note 21);

Sensory functions and reflexes (Note 21); and

Behavior (Note 21).

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 1.2).

Adverse Effects To Be Assessed

Enhanced toxicity relative to that in nonpregnant

females,

Embryofetal death,

Altered growth, and

Structural changes.

Animals

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

nonrodent, 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 1.2).

Experimental Procedure

Females should be sacrificed and examined about 1 day prior to

parturition. Eleven 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 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.

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, 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 (Note 16);

Individual fetal body weight;

Fetal abnormalities (Note 22); and

Gross evaluation of placenta.

4.2 Single Study Design (rodents)

If the dosing period of the fertility study and prenatal and

postnatal study are combined into a single investigation, this

comprises evaluation of stages A to F of the reproductive process

(see 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 and 11).

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

a second species are expected (see also 4.1.3).

4.3 Two Study Design (rodents)

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 4.1.3) are most unlikely to

provide a major change in the assessment of risk.

Alternatively, female treatment in the fertility study (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 (4.1.3). This, combined with the prenatal and postnatal study

(4.1.2) would provide all the examinations required in ``the most

probable option'' but use considerably less animals (Notes 3 and

11).

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

a second species are expected (see also 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), 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 (Note 23).

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 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.

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

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 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.

Notes

Note 1 (1.1) Scientific Flexibility

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 the 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.

Note 2 (1.2) Timing Conventions

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 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.

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

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.

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 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.

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 ``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

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.

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

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 east 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.

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;

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.

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.

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 administration,

existing data on kinetics should be used to determine the necessity

of another study.

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 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.

Note 11 (4) Examples for 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 administration to females in a 4.1.1 study to day 17.

With sacrifice at term, both fertility and embryo-fetal development

can be assessed.

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

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.

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 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

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.

Note 14 (4.1.1) Mating

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 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 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 4.1.1

may also be used for evaluation of toxicity to the male reproductive

system if dosing is continued beyond mating and sacrifice 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 nonpregnant rats or mice (but not rabbits),

ammonium sulphide staining of the uterus might be useful to identify

peri-implantation death of embryos.

Note 17 (4.1.2) Treatment of Offspring

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.

Note 18 (4.1.2) Separate Embryotoxicity and Peripostnatal Studies

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.

Note 19 (4.1.2) F1-Animals

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.

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

Behavior

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.

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.

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 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.

Note 23 (5) Inferential Statistics

``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.

Dated: September 15, 1994.

William K. Hubbard,

Interim Deputy Commissioner for Policy.

[FR Doc. 94-23379 Filed 9-21-94; 8:45 am]

BILLING CODE 4160-01-F