Section

[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

From the U.S. Government Printing Office via GPO Access

[CITE: 40CFR799.9355]

[Page 366-372]

TITLE 40--PROTECTION OF ENVIRONMENT

CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)

PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE

TESTING REQUIREMENTS--Table of Contents

Subpart H_Health Effects Test Guidelines

Sec. 799.9355 TSCA reproduction/developmental toxicity screening test.

(a) Scope--(1) Applicability. This section is intended to meet

testing requirements of the Toxic Substances Control Act (TSCA) (15

U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test

guideline is the Office of Prevention, Pesticides, and Toxic Substances

(OPPTS) harmonized test guideline 870.3550 (July 2000, final

guidelines). This source is available at the address in paragraph (h) of

this section.

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(b) Purpose. (1) This guideline is designed to generate limited

information concerning the effects of a test substance on male and

female reproductive performance such as gonadal function, mating

behavior, conception, development of the conceptus, and parturition. It

is not an alternative to, nor does it replace, the existing

comprehensive test standards in Sec. Sec. 799.9370 and 799.9380.

(2) This screening test guideline can be used to provide initial

information on possible effects on reproduction and/or development,

either at an early stage of assessing the toxicological properties of

chemicals, or on chemicals of high concern. It can also be used as part

of a set of initial screening tests for existing chemicals for which

little or no toxicological information is available, as a dose range

finding study for more extensive reproduction/developmental studies, or

when otherwise considered relevant.

(3) This test does not provide complete information on all aspects

of reproduction and development. In particular, it offers only limited

means of detecting postnatal manifestations of prenatal exposure, or

effects that may be induced during postnatal exposure. Due (amongst

other reasons) to the relatively small numbers of animals in the dose

groups, the selectivity of the end points, and the short duration of the

study, this method will not provide evidence for definite claims of no

effects.

Part 792--Good Laboratory Practice Standards apply to this section. The

following definitions also apply to this section.

Dosage is a general term comprising of dose, its frequency and the

duration of dosing.

Dose is the amount of test substance administered. Dose is expressed

as weight (g, mg) as weight of test substance per unit weight of test

animal (e.g., mg/kg), or as constant dietary concentration parts per

million (ppm).

No-observed-effects level (NOEL) is the maximum dose used in a study

which produces no adverse effects. The NOEL is expressed in terms of the

weight of a test substance given daily per unit weight of test animal

(milligrams per kilograms per day).

(d) Principle of the test. (1) The test substance is administered in

graduated doses to several groups of males and females. Males should be

dosed for a minimum of four weeks and up to and including the day before

scheduled sacrifice (this includes a minimum of two weeks prior to

mating, during the mating period and, approximately, two weeks post-

mating). In view of the limited pre-mating dosing period in males,

fertility may not be a particular sensitive indicator of testicular

toxicity. Therefore, a detailed histological examination of the testes

is essential. The combination of a pre-mating dosing period of two weeks

and subsequent mating/fertility observations with an overall dosing

period of at least four weeks, followed by detailed histopathology of

the male gonads, is considered sufficient to enable detection of the

majority of effects on male fertility and spermatogenesis.

(2) Females should be dosed throughout the study. This includes two

weeks prior to mating (with the objective of covering at least two

complete oestrous cycles), the variable time to conception, the duration

of pregnancy and at least four days after delivery, up to and including

the day before scheduled sacrifice.

(3) Duration of study, following acclimatization, is dependent on

the female performance and is approximately 54 days, (at least 14 days

premating, (up to) 14 days mating, 22 days gestation, 4 days lactation).

(4) During the period of administration, the animals are observed

closely each day for signs of toxicity. Animals which die or are

sacrificed during the test period are necropsied and, at the conclusion

of the test, surviving animals are sacrificed and necropsied.

(e) Description of the method--(1) Selection of animal species. This

test standard is designed for use with the rat. If other species are

used, appropriate modifications will be necessary. Strains with low

fecundity or well-known high incidence of developmental defects should

not be used. Healthy virgin animals, not subjected to previous

experimental procedures, should be

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used. The test animals should be characterized as to species, strain,

sex, weight and/or age. At the commencement of the study the weight

variation of animals used should be minimal and not exceed 20% of the

mean weight of each sex.

(2) Housing and feeding conditions. (i) The temperature in the

experimental animal room should be 22 [deg]C (3[deg]). Although the relative humidity should be at

least 30% and preferably not exceed 70% other than during room cleaning,

the aim should be 50-60%. Lighting should be artificial, the sequence

being 12 hours light, 12 hours dark. For feeding, conventional

laboratory diets may be used with an unlimited supply of drinking water.

The choice of diet may be influenced by the need to ensure a suitable

admixture of a test substance when administered by this method.

(ii) Animals may be housed individually or be caged in small groups

of the same sex; for group caging, no more than five animals should be

housed per cage. Mating procedures should be carried out in cages

suitable for the purpose. Pregnant females should be caged individually

and provided with nesting materials.

(3) Preparation of the animals. Healthy young adult animals must be

randomly assigned to the control and treatment groups. Cages should be

arranged in such a way that possible effects due to cage placement are

minimized. The animals must be uniquely identified and kept in their

cages for at least five days prior to the start of the study to allow

for acclimatization to the laboratory conditions.

(4) Preparation of doses. (i) It is recommended that the test

substance be administered orally unless other routes of administration

are considered more appropriate. When the oral route is selected, the

test compound is usually administered by gavage; however, alternatively,

test compounds may be administered via the diet or drinking water.

(ii) Where necessary, the test substance is dissolved or suspended

in a suitable vehicle. It is recommended that, wherever possible, the

use of an aqueous solution/suspension be considered first, followed by

consideration of a solution/emulsion in oil (e.g., corn oil) and then by

possible solution in other vehicles. For vehicles other than water the

toxic characteristics of the vehicle must be known. The stability of the

test substance in the vehicle should be determined.

(f) Procedure--(1) Number and sex of animals. It is recommended that

each group be started with at least 10 animals of each sex. Except in

the case of marked toxic effects, it is expected that this will provide

at least 8 pregnant females per group which normally is the minimum

acceptable number of pregnant females per group. The objective is to

produce enough pregnancies and offspring to assure a meaningful

evaluation of the potential of the substance to affect fertility,

pregnancy, maternal and suckling behaviour, and growth and development

of the F1 offspring from conception to day 4 post-partum.

(2) Dosage. (i) Generally, at least three test groups and a control

group should be used. Dose levels may be based on information from acute

toxicity tests or on results from repeated dose studies. Except for

treatment with the test substance, animals in the control group should

be handled in an identical manner to the test group subjects. If a

vehicle is used in administering the test substance, the control group

should receive the vehicle in the highest volume used.

(ii) Dose levels should be selected taking into account any existing

toxicity and (toxico-) kinetic data available for the test compound or

related materials. The highest dose level should be chosen with the aim

of inducing toxic effects but not death or severe suffering. Thereafter,

a descending sequence of dose levels should be selected in order to

demonstrate any dose response relationships and no adverse effects at

the lowest dose level. Two to four fold intervals are frequently optimal

for setting the descending dose levels and addition of a fourth test

group is often preferable to using very large intervals (e.g., more than

a factor of 10) between dosages.

(3) Limit test. If an oral study at one dose level of at least 1000

mg/kg body weight/day or, for dietary or drinking water administration,

an equivalent percentage in the diet, or drinking

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water using the procedures described for this study, produces no

observable toxic effects and if toxicity would not be expected based

upon data from structurally related compounds, then a full study using

several dose levels may not be considered necessary. The limit test

applies except when human exposure indicates the need for a higher oral

dose level to be used. For other types of administration, such as

inhalation or dermal application, the physical chemical properties of

the test substance often may dictate the maximum attainable

concentration.

(4) Administration of doses. (i) The animals must be dosed with the

test substance daily for seven days a week. When the test substance is

administered by gavage, this should be done in a single dose to the

animals using a stomach tube or a suitable intubation cannula. The

maximum volume of liquid that can be administered at one time depends on

the size of the test animal. The volume should not exceed 1 ml/100 g

body weight, except in the case of aqueous solutions where 2 ml/100 g

body weight may be used. Except for irritating substances which will

normally reveal exacerbated effects with higher concentrations,

variability in test volume should be minimized by adjusting the

concentration to ensure a constant volume at all dose levels.

(ii) For substances administered via the diet or drinking water, it

is important to ensure that the quantities of the test substance

involved do not interfere with normal nutrition or water balance. When

the test substance is administered in the diet either a constant dietary

concentration (parts per million (ppm)) or a constant dose level in

terms of the animals' body weight may be used; the alternative used must

be specified. For a substance administered by gavage, the dose should be

given at similar times each day, and adjusted at least weekly to

maintain a constant dose level in terms of animal body weight.

(5) Experimental schedule. (i) Dosing of both sexes should begin at

least 2 weeks prior to mating, after they have been acclimatized for at

least five days. The study should be scheduled in such a way that mating

begins soon after the animals have attained full sexual maturity. This

may vary slightly for different strains of rats in different

laboratories, e.g., Sprague Dawley rats 10 weeks of age, Wistar rats

about 12 weeks of age. Dams with offspring should be sacrificed on day 4

post-partum, or shortly thereafter. The day of birth (viz. when

parturition is complete) is defined as day 0 post-partum. Females

showing no-evidence of copulation are sacrificed 24-26 days after the

last day of the mating period. Dosing is continued in both sexes during

the mating period. Males should further be dosed after the mating period

at least until the minimum total dosing period of 28 days has been

completed. They are then sacrificed, or, alternatively, are retained and

continued to be dosed for the possible conduction of a second mating if

considered appropriate.

(ii) Daily dosing of the parental females should continue throughout

pregnancy and at least up to, and including, day 3 post-partum or the

day before sacrifice. For studies where the test substance is

administered by inhalation or by the dermal route, dosing should be

continued at least up to, and including, day 19 of gestation.

(iii) The experimental schedule is given in the following figure 1.

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[GRAPHIC] [TIFF OMITTED] TR15DE00.064

(6) Mating procedure. Normally, 1:1 (one male to one female) matings

should be used in this study. Exceptions can arise in the case of

occasional deaths of males. The female should be placed with the same

male until pregnancy occurs or two weeks have elapsed. Each morning the

females should be examined for the presence of sperm or a vaginal plug.

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

found.

(7) Observations. (i) Throughout the test period, general clinical

observations should be made at least once a day, and more frequently

when signs of toxicity are observed. They should be made preferably at

the same time(s) each day, considering the peak period of anticipated

effects after dosing. Pertinent behavioural changes, signs of difficult

or prolonged parturition and all signs of toxicity, including mortality,

should be recorded. These records should include time of onset, degree

and duration of toxicity signs.

(ii) The duration of gestation should be recorded and is 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, runts (pups that are significantly smaller

than corresponding control pups) and the presence of gross

abnormalities.

(iii) Live pups should be counted and sexed and litters weighed

within 24 hours of parturition (day 1) and on day 4 post-partum. In

addition to the observations on parent animals, described by paragraph

(f)(7) of this section, any abnormal behaviour of the offspring should

be recorded.

(8) Body weight and food/water consumption. (i) Males and females

should be individually weighed on the first day of dosing, at least

weekly thereafter, and at termination. During pregnancy, females should

be weighed on days 0, 7, 14 and 20 and within 24 hours of parturition

(day 1) and day 4 post-partum.

(ii) During pre-mating, pregnancy and lactation, food consumption

should be measured at least weekly. The measurement of food consumption

during mating is optional. Water consumption during these periods should

also be

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measured when the test substance is administered via drinking water.

(9) Pathology--(i) Gross necropsy. (A) At the time of sacrifice or

death during the study, the adult animals should be examined

macroscopically for any abnormalities or pathological changes. Special

attention should be paid to the organs of the reproductive system. The

number of implantation sites should be recorded. Corpora lutea should be

counted.

(B) The testes and epididymides of all male adult animals should be

weighed.

thereafter, should, at least, be carefully examined externally for gross

abnormalities.

(D) The ovaries, testes, epididymides, accessory sex organs and all

organs showing macroscopic lesions of all adult animals should be

preserved. Formalin fixation is not recommended for routine examination

of testes and epididymides. An acceptable method is the use of Bouin's

fixative for these tissues.

(ii) Histopathology. (A) Detailed histological examination should be

performed on the ovaries, testes and epididymides of the animals of the

highest dose group and the control group. The other preserved organs may

be examined when necessary. Examinations should be extended to the

animals of other dosage groups when changes are seen in the highest dose

group.

(B) Detailed testicular histopathological examination (e.g., using

Bouin's fixative, paraffin embedding and transverse sections of 4-5

m thickness) should be conducted with special

emphasis on stages of spermatogenesis and histopathology interstitial

testicular cell structure. The evaluation should identify treatment-

related effects such as retained spermatids, missing germ cell layers or

types, multinucleated giant cells or sloughing of spermatogenic cells

into the lumen (the specifications for the evaluation are discussed in

paragraph (g)(2) of this section). Examination of the intact epididymis

should include the caput, corpus, and cauda, which can be accomplished

by evaluation of a longitudinal section. The epididymis should be

evaluated for leukocyte infiltration, change in prevalence of cell

types, aberrant cell types, and phagocytosis of sperm. PAS and

hematoxylin staining may be used for examination of the male

reproductive organs. Histopathological examination of the ovary should

detect qualitative depletion of the primordial follicle population.

(g) Data and reporting--(1) Data. Individual animal data should be

provided. Additionally, all data should be summarised in tabular form,

showing for each test group the number of animals at the start of the

test, the number of animals found dead during the test or sacrificed for

humane reasons, the time of any death or humane sacrifice, the number of

fertile animals, the number of pregnant females, the number of animals

showing signs of toxicity, a description of the signs of toxicity

observed, including time of onset, duration, and severity of any toxic

effects, the types of histopathological changes, and all relevant litter

data.

(2) Evaluation of results. (i) The findings of this toxicity study

should be evaluated in terms of the observed effects, necropsy and

microscopic findings. This evaluation must include the relationship

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

incidence and severity of abnormalities, including gross lesions,

identified target organs, infertility, clinical abnormalities, affected

reproductive and litter performance, body weight changes, effects on

mortality and any other toxic effects.

(ii) Because of the short period of treatment of the male, the

histopathology of the testis and epididymus must be considered along

with the fertility data, when assessing male reproductive effects.

(iii) Due to the limited dimensions of the study, statistical

analysis in the form of tests for ``significance'' are of limited value

for many endpoints, especially reproductive endpoints. If statistical

analyses are used then the method chosen should be appropriate for the

distribution of the variable examined, and be selected prior to the

start of the study. Because of the small group size, the use of historic

control data (e.g.,

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for litter size), where available, may also be useful as an aid to the

interpretation of the study.

(3) Test report. The test report must include the following

information:

(i) Test substance:

(A) Physical nature and, where relevant, physicochemical properties.

(B) Identification data.

(ii) Vehicle (if appropriate): Justification for choice of vehicle

if other than water.

(iii) Test animals:

(A) Species/strain used.

(B) Number, age and sex of animals.

(D) Individual weights of animals at the start of the test.

(iv) Test conditions:

(A) Rationale for dose level selection.

(B) Details of test substance formulation/diet preparation, achieved

concentrations, stability and homogeneity of the preparation.

(D) Conversion from diet/drinking water test substance concentration

(parts per million (ppm)) to the actual dose (mg/kg body weight/day), if

applicable.

(E) Details of food and water quality.

(v) Results (toxic response data by sex and dose):

(A) Time of death during the study or whether animals survived to

termination.

(B) Nature, severity and duration of clinical observations (whether

reversible or not).

(D) Food consumption and water consumption, if applicable.

(E) Effects on reproduction, including information on mating/

precoital interval, fertility, fecundity and gestation duration.

(F) Effects on offspring, including number of pups born (live and

dead), sex ratio, postnatal growth (pup weights) and survival (litter

size), gross abnormalities and clinical observations during lactation.

(G) Body weight at termination and organ weight data for the

parental animals.

(H) Necropsy data, including number of implantations and number of

corpora lutea.

(I) Calculations of pre- and postimplantation loss.

(J) Detailed description of histopathological findings.

(K) Statistical treatment of results, where appropriate.

(vi) Discussion of results.

(vii) Conclusions.

(4) Interpretation of results. The study will provide evaluations of

reproduction/developmental toxicity associated with administration of

repeated doses. It could provide an indication of the need to conduct

further investigations and provides guidance in the design of subsequent

studies.

(h) References. For additional background information on this test

guideline, the following references should be consulted. These

references are available for inspection at the TSCA Nonconfidential

Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M

St., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,

except legal holidays.

(1) OECD (1995). Reproduction/Developmental Toxicity Screening Test,

OECD 421, OECD Guidelines for Testing of Chemicals.

(2) [Reserved]

[65 FR 78789, Dec. 15, 2000]