Users of this Test Guideline should consult the Preface, 

in particular paragraphs 3, 4, 7 and 8.

415

Adopted:

OECD GUIDELINE FOR TESTING OF CHEMICALS 26 May 1983

"One-Generation Reproduction Toxicity Study"

1. I N T R O D U C T O R Y I N F O R M A T I O N

• P r e r e q u i s i t e s

– Solid, liquid, gaseous or vapour test substance

– Chemical identification of test substance

– Purity (impurities) of test substance

– Solubility characteristics

– Melting point/boiling point (where appropriate)

– pH (where appropriate)

• S t a n d a r d d o c u m e n t s

There are no relevant international standards.

2. M E T H O D

A. INTRODUCTION, PURPOSE, SCOPE, RELEVANCE,

APPLICATION AND LIMITS OF TEST

This Test Guideline for reproduction testing is designed to provide general information

concerning the effects of a test substance on male and female reproductive performance, such

as gonadal function, oestrous cycle, mating behaviour, conception, parturition, lactation and

weaning. It is not designed to determine specific cause and effects in all cases and will require

modifications to study substances administered by the inhalation route. The study may also

provide preliminary information about developmental toxic effects of the test substance, such

as neonatal morbidity, mortality, behaviour and teratogenesis and to serve as a guide for

subsequent tests.

• P r i n c i p l e o f t h e t e s t m e t h o d

The test substance is administered in graduated doses to several groups of males and

females. Males should be dosed during growth and for at least one complete spermatogenic

cycle (approximately 56 days in the mouse and 70 days in the rat) in order to elicit any adverse

effects on spermeogenesis by the test substance.
415

page 2 "One-Generation Reproduction Toxicity Study"

Females of the P generation should be dosed for at least two complete oestrous cycles

in order to elicit any adverse effects on oestrus by the test substance. The animals are then

mated. The test substance is administered to both sexes during the mating period and thereafter

only to females during pregnancy and for the duration of the nursing period.

B. DESCRIPTION OF THE TEST PROCEDURE

• P r e p a r a t i o n s

Healthy young adult animals are randomised and assigned to the treatment groups. The

animals are kept in cages for at least five days to allow for acclimatisation. It is recommended

that the test substance be administered in the diet or drinking water. Other routes of administration are also acceptable. All animals should be dosed by the same method during the

appropriate experimental period. If a vehicle or other additives are used to facilitate dosing, they

should be known not to produce toxic effects. Dosing should be on a seven-day per week basis.

• E x p e r i m e n t a l a n i m a l s

Selection of species

This Test Guideline is designed for use with the rat or mouse. If other species are used,

appropriate modifications will be necessary. Strains with low fecundity should not be used.

Healthy animals, not subjected to previous experimental procedures, should be used. The test

animals should be characterised as to species, strain, sex, weight and/or age.

Number and sex

Each test and control group should contain a sufficient number of animals to yield about

20 pregnant females at or near term. For substances that cause sterility this may not be possible.

The objective is to produce enough pregnancies and offspring to assure a meaningful evaluation

of the potential of the substance to affect fertility, pregnancy and maternal behaviour in P

generation animals and suckling, growth and development of the F1 offspring from conception

to weaning.
415

page 3 "One-Generation Reproduction Toxicity Study"

Housing and feeding conditions

The temperature in the experimental animal room should be 22°C (± 3°) and the relative

humidity 30 to 70 per cent. When the lighting is artificial the sequence should be 12 hours light,

12 hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply

of drinking water. Pregnant females should be caged individually and may be provided with

nesting materials.

• T e s t c o n d i t i o n s

Dose levels

At least three treatment groups and a control group should be used. If a vehicle is used

in administering the test substance, the control group should receive the vehicle in the highest

volume used. If a test substance causes reduced dietary intake or utilisation, then the use of a

paired-fed control group may be considered necessary. Ideally, unless limited by the physicalchemical nature or biological effects of the test substance, the highest dose level should induce

toxicity but not mortality in the parental (P) animals.

Ideally, the intermediate dose(s) should induce minimal toxic effects attributable to the

test substance, and the low dose should not induce any observable adverse effects on the parents

or offspring.

When administered by gavage or capsule the dose given each animal should be based

on the individual animal's body weight and adjusted weekly for changes in body weight. For

females during pregnancy, dosage may be based on daily body weight or on body weight at day

0 or 5 of pregnancy, if desired.

Limit test

In the case of substances of low toxicity as demonstrated in repeated-dose studies, if a

dose of at least 1000 mg/kg produces no evidence of interference with reproductive

performance, studies at other dose levels may not be considered necessary. If a preliminary

study at the high dose level, with definite evidence of maternal toxicity, shows no adverse

effects on fertility, studies at other dose levels may not be considered necessary.
415

page 4 "One-Generation Reproduction Toxicity Study"

• P e r f o r m a n c e o f t h e t e s t

Experimental schedules

Daily dosing of the parental (P) males should begin when they are about five to nine

weeks old, after they have been weaned and acclimatised for at least five days. In rats dosing

is continued for ten weeks prior to the mating period (for mice, eight weeks). Males should be

killed and examined either at the end of the mating period or, alternatively, males may be

retained on test diet for the possible production of a second litter and should be killed and

examined at some time before the end of the experiment.

For parental (P) females, dosing should begin after at least five days of acclimatisation

and continue for at least two weeks prior to mating. Daily dosing of the P females should

continue throughout the 3-week mating period, pregnancy and up to the weaning of the F1

offspring. Consideration should be given to modifications in the dosing schedule based on

available information on the test substance, such as induction of its metabolism or

bioaccumulation.

Mating procedure

Either 1:1 (one male to one female) or 1:2 (one male to two females) matings may be

used in this study.

Based on 1: 1 mating, one female should be placed with the same male until pregnancy

occurs or three weeks have elapsed. Each morning the females should be examined for presence

of sperm or vaginal plugs. Day 0 of pregnancy is defined as the day a vaginal plug or sperm

are found.

Those pairs that fail to mate should be evaluated to determine the cause of the apparent

infertility. This may involve such procedures as additional opportunities to mate with other

proven sires or dams, microscopic examination of the reproductive organs, and examination of

the oestrous cycle or spermeogenesis.

Litter size

Animals dosed during the fertility study are allowed to litter normally and rear their

progency to the stage of weaning without standardisation. If standardisation is carried out, the

following procedure is suggested: on day 4 after birth, the size of each litter may be adjusted
415

page 5 "One-Generation Reproduction Toxicity Study"

by eliminating extra pups by selection to yield, as nearly as possible, four males and four

females per litter. Elimination of runts only is not appropriate. Whenever the number of male

or female pups prevents having four of each sex per litter, partial adjustment (for example, five

males and three females) is acceptable. Adjustments are not applicable for litters of less than

eight pups.

• O b s e r v a t i o n s

Throughout the test period, each animal should be observed at least once daily. Pertinent

behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity,

including mortality, should be recorded. During pre-mating and mating periods, food

consumption should be measured weekly. Optionally, during pregnancy food consumption may

be measured daily. After parturition, and during lactation, food consumption measurements

should be made on the same day as the litters are weighed. P males and females should be

weighed on the first day of dosing and weekly thereafter. These observations should be reported

individually for each adult animal.

The duration of gestation should be calculated from day 0 of pregnancy. Each litter

should be examined as soon as possible after delivery to establish the number and sex of pups,

stillbirths, live births and the presence of gross anomalies. Dead pups and pups killed at day 4

should be preserved and studied for possible defects.

Live pups should be counted and litters weighed on the morning after birth and on days

4 and 7 and weekly thereafter until termination of the study, when animals should be weighed

individually. Physical or behavioural abnormalities observed in the dams or offspring should be

recorded.

• P a t h o l o g y

Gross necropsy

At the time of sacrifice or death during the study the animals of the P generation should

be examined macroscopically for any structural abnormalities or pathological chances, with

special attention paid to the organs of the reproductive system. Dead or moribund pups should

be examined for defects.
415

page 6 "One-Generation Reproduction Toxicity Study"

Histopathology

The ovaries, uterus, cervix, vagina, testes, epididymides, seminal vesicles, prostate,

coagulating gland, pituitary gland and target organ(s) of all P animals should be preserved for

microscopic examination, if necessary. In the event that these organs have not been examined

in other mutliple-dose studies, they should be microscopically examined in all high-dose and

control animals and in animals which die during the study, where practicable. Organs showing

abnormalities in these animals should then be examined in all other P animals. In these

instances microscopic examination should be made of all tissues showing gross pathological

changes. As suggested under Mating procedure, above, reproductive organs of animals suspected

of infertility may be subjected to microscopic examination.

3. D A T A A N D R E P O R T I N G

• T r e a t m e n t o f r e s u l t s

Data may be summarised in tabular form, showing for each test group the number of

animals at the start of the test, the number of fertile males, the number of pregnant females, the

types of changes and the percentage of animals displaying each type of change.

When possible, numerical results should be evaluated by an appropriate statistical

method. A generally accepted statistical method should be used; the statistical methods should

be selected as a part the design of the study.

• E v a l u a t i o n o f r e s u l t s

The findings of a reproduction toxicity study should be evaluated in terms of the

observed effects, necropsy and microscopic findings. The evaluation will include the relationship

between the dose of the test substance and the presence or absence, the incidence and severity,

of abnormalities, including fertility, clinical abnormalities, body weight changes, effects on

mortality and any other toxic effects. A properly conducted reproduction test should provide a

satisfactory estimation of a no-effect level and an understanding of adverse effects on

reproduction, parturition, lactation and postnatal growth.
415

page 7 "One-Generation Reproduction Toxicity Study"

• T e s t r e p o r t

The test report should also include the following information:

– species/strain used;

– toxic response data by sex and dose, including fertility, gestation, and viability indices;

– time of death during the study or whether animals survived to termination;

– table presenting the weights of each litter, the mean pup weights and the individual weights

of the pups at termination;

– toxic or other effects on reproduction, offspring, postnatal growth, etc.;

– the day of observation of each abnormal sign and its subsequent course;

– body weight data for P animals;

– necropsy findings;

– a detailed description of microscopic findings, when performed; and

– statistical treatment of results, where appropriate.

• I n t e r p r e t a t i o n o f r e s u l t s

A reproduction toxicity study will provide information on the effects of repeated oral

exposure to a substance. The results of the study should be interpreted in conjunction with the

findings of subchronic, teratogenic and other studies. Extrapolation of the results of the study

to man is valid to a limited degree, although it can provide useful information on no-effect

levels and permissible human exposure.

4. L I T E R A T U R E

01. E.I. Goldenthal, Guidelines for Reproduction Studies for S afety Evaluation of Drugs for

Hum an Use. Drug Review Branch, Division of Toxicological Evaluation, Bureau of

Science, Federal Drug Administration, Washington, D.C. (1966).

02. T. Hasegawa, M. Hayashi, F.J.G. Ebling and I.W. Henderson, Fertility and Sterility.

Elsevier, New York (1973).
415

page 8 "One-Generation Reproduction Toxicity Study"

03. E.F. Oakberg, A m. J. A nat. 99, 507-516 (1956).

04. E.C. Roosen-Runge, Biol. Rev. 37, 343-377 (1962).

05. R.D. Yates and M. Gordon, M ale Reproductive System. Masson, New York (1977).

06. National Academy of Sciences, Principles and Procedures for Evaluating the T oxicity of

Household S ubstances (Washington, 1977).

07. A.K. Palmer, in Handbook of Teratology, Vol. 4 (edited by J.G. Wilson and F. Clarke

Graser) Plenum Press, New York (1978).

08. A.K. Palmer, in Developmental T oxicology (edited by C.A. Kimmel and J. Buelke-Sam)

Raven Press, New York (1981).