[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
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.
    (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR 
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.
    (C) Dead pups and pups sacrificed at day 4 post-partum, or shortly 
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.
    (C) Source, housing conditions, diet, etc.
    (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.
    (C) Details of the administration of the test substance.
    (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).
    (C) Body weight/body weight change data.
    (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]