[Code of Federal Regulations]
[Title 40, Volume 30]
[Revised as of July 1, 2004]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.4700]
[Page 177-180]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart E_Specific Organ/Tissue Toxicity
Sec. 798.4700 Reproduction and fertility effects.
(a) Purpose. This guideline for two-generation reproduction testing
is designed to provide general information concerning the effects of a
test substance on gonadal function, conception, parturition, and the
growth and development of the offspring. The study may also provide
information about the effects of the test substance on neonatal
morbidity, mortality, and preliminary data on teratogenesis and serve as
a guide for subsequent tests.
(b) Principle of the test method. The test substance is administered
to parental (P) animals prior to their mating, during the resultant
pregnancies, and through the weaning of their F1 offspring.
The substance is then administered to selected F1 offspring
during their growth into adulthood, mating, and production of an
F2 generation, up until the F2 generation is
weaned.
(c) Test procedures--(1) Animal selection--(i) Species and strain.
The rat is the preferred species. If another mammalian species is used,
the tester shall provide justification/reasoning for its
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selection. Strains with low fecundity shall not be used.
(ii) Age. Parental (P) animals shall be about 5 to 8 weeks old at
the start of dosing.
(iii) Sex. (A) For an adequate assessment of fertility, both males
and females shall be studied.
(B) The females shall be nulliparous and non-pregnant.
(iv) Number of animals. Each test and control group shall contain at
least 20 males and a sufficient number of females to yield at least 20
pregnant females at or near term.
(2) Control groups. (i) A concurrent control group shall be used.
This group shall be an untreated or sham treated control group or if a
vehicle is used in administering the test substance, a vehicle control
group.
(ii) If a vehicle is used in administering the test substance, the
control group shall receive the vehicle in the highest volume used.
(iii) If a vehicle or other additive is used to facilitate dosing,
it shall not interfere significantly with absorption of the test
substance or produce toxic effects.
(3) Dose levels and dose selection. (i) At least three dose levels
and a concurrent control shall be used.
(ii) The highest dose level should induce toxicity but not high
levels of mortality in the parental (P) animals.
(iii) The lowest dose level should not produce any grossly
observable evidence of toxicity.
(iv) Ideally the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used,
dose levels should be spaced to produce a gradation of toxic effects.
(4) Exposure conditions. The animals should be dosed with the test
substance, ideally, on a 7 days per week basis.
(i) Dosing, mating, delivery, and sacrifice schedule.
(A) Daily dosing of the parental (P) males and females shall begin
when they are 5 to 8 weeks old. For both sexes, dosing shall be
continued for at least 10 weeks before the mating period.
(B) Dosing of P males shall continue through the 3 week mating
period. At the end of the mating period, P males may be sacrificed and
examined, or may be retained for possible production of a second litter.
If these animals are retained for a second litter, dosing shall be
continued. Dosing of the F1 males saved for mating shall
continue from the time they are weaned through the period they are mated
with the F1 females (11 weeks). F1 males may be
sacrificed after the F1 mating period.
(C) Daily dosing of the P females shall continue through the three
week mating period, pregnancy, and to the weaning of the F1
offspring. Dosing of the F1 females saved for mating shall
continue from the time they are weaned, through the period they are
mated with the F1 males (11 weeks from the time of weaning)
pregnancy, and to the weaning of the F2 offspring.
(ii) All animals are sacrificed as scheduled.
(A) All P males should be sacrificed at the end of the 3-week mating
period, or may be retained for possible production of a second litter.
If these animals are retained for a second litter, dosing shall be
continued.
(B) F1 males selected for mating should be sacrificed at
the end of the three week period of the F1 mating.
(C) F1 males and females not selected for mating should
be sacrified when weaned.
(D) The P females should be sacrificed upon weaning of their
F1 offspring.
(E) F1 dams and their F2 offspring are
sacrificed when the offspring are weaned.
(5) Administration of the test substance--(i) Oral studies. (A) It
is recommended that the test substance be administered in the diet or
drinking water.
(B) If administered by gavage or capsule, the dosage administered to
each animal prior to mating shall be based on the individual animal's
body weight and adjusted weekly. During pregnancy the dosage shall be
based on the body weight at day 0 and 6 of pregnancy.
(ii) If another route of administration is used, the tester should
provide justification and reasoning for its selection.
(6) Mating procedure--(i) Parental. (A) For each mating, each female
shall be
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placed with a single male from the same dose level until pregnancy
occurs or 1 week has elapsed. If mating has not occurred after 1 week,
the female shall be placed with a different male. Paired matings should
be clearly identified.
(B) 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 proven fertile males or
females, histological examination of the reproductive organs, and
examination of the estrus or spermatogenic cycles.
(C) Each day, the females shall be examined for presence of sperm or
vaginal plugs. Day 0 of pregnancy is defined as the day vaginal plugs or
sperm are found.
(ii) F1 cross. (A) For mating the F1 offspring, one male
and one female are randomly selected at weaning from each litter for
cross mating with another pup of the same dose level but different
litter, to produce the F2 generation.
(B) F1 males and females not selected for mating are
sacrificed upon weaning.
(iii) Special housing. After evidence of copulation, pregnant
animals shall be caged separately in delivery or maternity cages.
Pregnant animals shall be provided with nesting materials when
parturition is near.
(iv) Standardization of litter sizes. (A) On day 4 after birth, the
size of each litter should be adjusted by eliminating extra pups by
random selection to yield, as nearly as possible, 4 males and 4 females
per litter.
(B) Whenever the number of male or female pups prevents having 4 of
each sex per litter, partial adjustment (for example, 5 males and 3
females) is permitted. Adjustments are not appropriate for litters of
less than 8 pups.
(C) Elimination of runts only is not appropriate.
(D) Adjustments of the F2 litters is conducted in the
same manner.
(7) Observation of animals. (i) A gross examination shall be made at
least once each day. Pertinent behavioral changes, signs of difficult or
prolonged parturition, and all signs of toxicity, including mortality,
shall be recorded. These observations shall be reported for each
individual animal. Food consumption for all animals shall be monitored
weekly except during the mating period.
(ii) The duration of gestation shall be calculated from day 0 of
pregnancy.
(iii) Each litter should be examined as soon as possible after
delivery for the number of pups, stillbirths, live births, sex, and the
presence of gross anomalies. Live pups should be counted and litters
weighed at birth or soon thereafter, and on days 4, 7, 14, and 21 after
parturition.
(iv) Physical or behavioral abnormalities observed in the dams of
offspring shall be recorded.
(v) P males and females shall be weighed on the first day of dosing
and weekly thereafter. F1 litters shall be weighed at birth,
or soon thereafter, and on days 4, 7, 14, and 21. In all cases, litter
weights shall be calculated from the weights of the individual pups.
(8) Gross necropsy. (i) A complete gross examination shall be
performed on all adult animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) Special attention shall be directed to the organs of the
reproductive system.
(iii) The following organs and tissues, or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: Vagina; uterus; ovaries; testes;
epididymides; seminal vesicles; prostate, pituitary gland; and, target
organ(s) when previously identified of all P and F1 animals
selected for mating.
(9) Histopathology. Except if carried out in other studies of
comparable duration and dose levels the following histopathology shall
be performed:
(i) Full histopathology on the organs listed above for all high
dose, and control P1 and F1 animals selected for
mating.
(ii) Organs demonstrating pathology in these animals shall then be
examined in animals from the other dose groups.
(iii) Microscopic examination shall be made of all tissues showing
gross pathological changes.
(d) Data and reporting--(1) Treatment of results. Data shall be
summarized in
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tabular form, showing for each test group the number of animals at the
start of the test, the number of animals pregnant, the types of change
and the percentage of animals displaying each type of change.
(2) Evaluation of study results. (i) An evaluation of test results,
including the statistical analysis, based on the clinical findings, the
gross necropsy findings, and the microscopic results shall be made and
supplied. This should include an evaluation of the relationship, or lack
thereof, between the animals' exposure to the test substance and the
incidence and severity of all abnormalities.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered.
(3) Test report. In addition to the reporting requirements as
specified under 40 CFR part 792, subpart J the following specific
information shall be reported:
(i) Toxic response data by sex and dose, including fertility,
gestation, viability and lactation indices, and length of gestation.
(ii) Species and strain.
(iii) Date of death during the study or whether animals survived to
termination.
(iv) Toxic or other effects on reproduction, offspring, or postnatal
growth.
(v) Date of observation of each abnormal sign and its subsequent
course.
(vi) Body weight data for P, F1, and F2
animals.
(vii) Necropsy findings.
(viii) Detailed description of all histopathological findings.
(ix) Statistical treatment of results where appropriate.
(e) References. For additional background information on this test
guideline the following references should be consulted:
(1) Clermont, Y., Perry, B. ``Quantitative Study of the Cell
Population of the Seminiferous Tubules in Immature Rats,'' American
Journal of Anatomy. 100:241-267 (1957).
(2) Goldenthal, E.I. Guidelines for Reproduction Studies for Safety
Evaluation of Drugs for Human Use. Drug Review Branch, Division of
Toxicological Evaluation, Bureau of Science, Food and Drug
Administration, Washington, DC (1966).
(3) Hasegawa, T., Hayashi, M., Ebling, F.J.G., Henderson, I.W.
Fertility and Sterility. (New York: American Elsevier Publishing Co.,
Inc., 1973).
(4) Oakberg, E.F. ``Duration of Spermatogenesis in the Mouse and
Timing of Stages of the Cycle of the Seminiferous Epithelium,'' American
Journal of Anatomy. 9:507-516 (1956).
(5) Roosen-Runge, E.C. ``The Process of Spermatogenesis in
Mammals,'' Biological Review. 37:343-377 (1962).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19077, May 20, 1987]