Users of this Test Guideline should consult the Preface,
in particular paragraphs 3, 4, 7 and 8. 

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Adopted: 26 May 1983 OECD GUIDELINE FOR TESTING OF CHEMICALS



### "One-Generation Reprod uction 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, liqu id, gaseous or vapour test su bstance

– Chemical identification of test su bstance – Purity (impu rities) of test su bstance – Solubility characteristics – Melting po int/boiling point (w here appro priate) – pH (where appro priate)

• S t a n d a r d d o c u m e n t s

There are no relevant internatio nal standards.

2. M E T H O D



### A. INT RODU CT ION, P U RP OS E, S COP E, RELEV ANCE,

AP P LICAT ION AND LIMIT S OF T ES T

This Test Guideline for reprodu ction testing is designed to provide general inform ation concerning the effects of a test substance on male and female reproductive perform ance, such as gonadal fu nction, oestrous cy cle, mating behaviour, conception, partu rition, lactation and weaning. It is not designed to determine specific cause and effects in all cases and w ill require modifications to study su bstances administered by the inhalation route. The study may also provide prelim inary information about developmental to xic effects of the test su bstance, such as neonatal morbidity, mortality, behaviour and terato genesis and to serve as a gu ide 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 spermato genic cycle (approxim ately 56 days in the mouse and 70 days in the rat) in order to elicit any adverse effects on spermeogenesis by the test substance.

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### "One-Generation Reprod uction Toxicity Study"

Females of the P generation should be do sed for at least two complete o estrous cy cles

in order to elicit any adverse effects on o estrus 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 du ration of the nursing period.



### B. DES CRIP T ION OF T HE T ES T P ROCEDU RE

• P r e p a r a t i o n s

Healthy young adult anim als are randomised and assigned to the treatment grou ps. The animals are kept in cages for at least five days to allow for acclimatisation. It is recomm ended that the test substance be administered in the diet or drinking water. Other routes of administration are also acceptable. All animals should be do sed by the same m ethod during the appropriate experimental period. If a vehicle or other additives are used to facilitate do sing, they should be known not to produce toxic effects. Dosing should be on a seven-day per w eek 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 w ith the rat or m ouse. If o ther species are u sed, appropriate modifications w ill be necessary. Strains with low fecu ndity should not be used. Healthy anim als, not subjected to previous experimental procedures, should be u sed. The test animals should be characterised as to species, strain, sex, weight and/or age.

Number and sex

Each test and co ntrol group should co ntain 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 o bjective is to produce enough pregnancies and o ffspring to assure a meaningful evalu ation of the potential of the substance to affect fertility, pregnancy and m aternal behaviour in P generation animals and suckling, growth and developm ent of the F 1 offspring from conception to weaning.

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### "One-Generation Reprod uction Toxicity Study"

Housing and feeding conditions

The tem perature 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 su pply of drinking water. Pregnant females should be caged individu ally 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 co ntrol group should be u sed. If a vehicle is used in administering the test substance, the co ntrol group should receive the vehicle in the highest

volume used. If a test su bstance causes redu ced dietary intake or u tilisation, then the use of a paired-fed co ntrol group may be co nsidered necessary. Ideally, u nless lim ited by the phy sicalchem ical nature or biological effects of the test su bstance, 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 adm inistered by gavage or capsule the dose given each animal should be based

on the individual anim al's body weight and adjusted w eekly for changes in body w eight. 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 perform ance, studies at other dose levels may not be considered necessary. If a prelim inary study at the high dose level, w ith definite evidence of maternal to xicity, shows no adverse effects on fertility, studies at other dose levels may not be considered necessary.

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### "One-Generation Reprod uction Toxicity Study"

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

Experime ntal schedules

Daily dosing of the parental (P) males should begin w hen they are about five to nine weeks old, after they have been weaned and acclim atised 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 produ ction 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 acclim atisation

and co ntinue for at least two w eeks prior to m ating. D aily dosing of the P fem ales should continue throughout the 3-week mating period, pregnancy and up to the weaning of the F 1 offspring. Consideration should be given to mo difications in the dosing schedule based on

available information on the test substance, such as indu ction of its m etabolism or

bioaccumu lation.

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 m ale until pregnancy occurs or three weeks have elapsed. Each morning the fem ales 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 additio nal opportunities to mate with other proven sires or dams, m icrosco pic exam ination of the reproductive o rgans, and exam ination of

the o estrous cy cle or spermeogenesis.

Litter size

Anim als do sed during the fertility study are allowed to litter norm ally and rear their

progency to the stage of weaning w ithout standardisation. If standardisation is carried out, the following procedure is suggested: on day 4 after birth, the size of each litter may be adju sted

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### "One-Generation Reprod uction Toxicity Study"

by eliminating extra pups by selection to yield, as nearly as po ssible, four males and four females per litter. Elimination of ru nts only is not appropriate. Whenever the number of male or female pups prevents having four of each sex per litter, partial adjustment (for exam ple, 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 o nce daily. P ertinent

behavioural changes, signs of difficult or prolo nged partu rition and all signs of to xicity,

including mortality, should be reco rded. During pre-mating and m ating perio ds, food consumption should be measu red w eekly. O ptio nally, during pregnancy food consum ption may

be measu red daily. After partu rition, and du ring lactation, food consumption measurem ents should be made on the same day as the litters are weighed. P males and fem ales should be weighed on the first day of do sing and w eekly thereafter. These o bservations should be reported

individu ally for each adult animal.

The du ration of gestation should be calcu lated 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 po ssible defects.

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

4 and 7 and w eekly thereafter until termination of the study, w hen animals should be weighed

individu ally. Phy sical or behavioural abnormalities o bserved in the dams or offspring should be

reco rded.

• P a t h o l o g y

Gross necropsy

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

be examined m acrosco pically for any structu ral abnormalities or pathological chances, with special attention paid to the organs of the reprodu ctive system. Dead or mo ribund pups should

be examined for defects.

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### "One-Generation Reprod uction Toxicity Study"

Histopathology

The o varies, u terus, cervix, vagina, testes, epididym ides, seminal vesicles, prostate, coagulating gland, pituitary gland and target organ(s) of all P animals should be preserved for

microsco pic exam ination, if necessary. In the event that these organs have not been examined in other mutliple-dose stu dies, they should be m icrosco pically 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 m icrosco pic exam ination should be m ade of all tissues show ing gross pathological changes. As suggested under Mating procedure, abo ve, reproductive o rgans of animals suspected of infertility may be subjected to m icrosco pic exam ination.

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, show ing for each test group the num ber 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 po ssible, numerical results should be evalu ated by an appropriate statistical

method. A generally accepted statistical method should be u sed; 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 reprodu ction to xicity study should be evaluated in terms of the observed effects, necropsy and m icrosco pic findings. The evalu ation will include the relatio nship 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 o ther toxic effects. A pro perly condu cted reprodu ction test should provide a satisfactory estim ation of a no -effect level and an understanding of adverse effects on reprodu ction, partu rition, lactation and po stnatal growth.

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### "One-Generation Reprod uction Toxicity Study"

• T e s t r e p o r t

The test report should also include the following information:

– species/strain u sed;

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

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

– table presenting the weights of each litter, the mean pup weights and the individual weights of the pups at term ination;

– toxic or other effects on reprodu ction, o ffspring, postnatal growth, etc.;

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

– body weight data for P animals;

– necropsy findings;

– a detailed description of m icrosco pic findings, w hen performed; and

– statistical treatm ent of results, w here 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 reprodu ction to xicity study w ill pro vide information on the effects of repeated oral exposure to a substance. The resu lts of the study should be interpreted in conjunction with the findings of subchronic, terato genic and o ther studies. Extrapo lation of the resu lts of the study to man is valid to a limited degree, although it can provide u seful information on no -effect

levels and perm issible human exposure.

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

0 1. E.I. Goldenthal, Guidelines for Repr oduction Stud ies for S afety Evaluation of Drugs for

Hum an Use . Drug Review Branch, D ivision of Toxicolo gical Evalu ation, Bureau of Science, Federal Drug Administration, Washington, D.C. (1 966).

0 2. T. Hasegawa, M. Hayashi, F.J.G. Ebling and I.W. H enderson, Fertility and Ster ility

. Elsevier, New York (1 973).

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### "One-Generation Reprod uction Toxicity Study"

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

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

0 5. R.D. Yates and M. Gordon, M ale Reproductive System. Masson, New York (1 977).

0 6. Natio nal Academy of Sciences, Principles and Procedures for Evaluating the T oxicity of

Household S ubstances (Washington, 1977).

0 7. A.K. Palmer, in Handbook of Teratology , Vol. 4 (edited by J.G. Wilson and F. C larke Graser) Plenum Press, New York (1 978).

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

Raven P ress, New York (1 981).