[Code of Federal Regulations]
[Title 40, Volume 14]
[Revised as of July 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR79.63]

[Page 555-560]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 79--REGISTRATION OF FUELS AND FUEL ADDITIVES--Table of Contents
 
            Subpart F--Testing Requirements for Registration
 
Sec. 79.63  Fertility assessment/teratology.

    (a) Purpose. Fertility assessment/teratology is an in vivo study 
designed to provide information on potential health hazards to the fetus 
arising from the mother's repeated inhalation exposure to vehicle/engine 
emissions before and during her pregnancy. By including a mating of test 
animals, the study provides preliminary data on the effects of repeated 
vehicle/engine emissions exposure on gonadal function, conception, and 
fertility. Since this is a one-generation test that ends with 
examination of full-term fetuses, but not of live pups, it is not 
capable of determining effects on reproductive development which would 
only be detected in viable offspring of treated parents.
    (b) Definitions. For the purposes of this section, the following 
definitions apply:
    Developmental toxicity means the ability of an agent to induce in 
utero death, structural or functional abnormalities, or growth 
retardation after contact with the pregnant animal.
    Estrous cycle means the periodic recurrence of the biological phases 
of the female reproductive system which prepare the animal for 
conception and the development of offspring. The phases of the estrous 
cycle for a particular animal can be characterized by the general 
condition of the cells present in the vagina and the presence or absence 
of various cell types.

[[Page 556]]

    Vaginal cytology evaluation means the use of wet vaginal cell smears 
to determine the phase of a test animal's estrous cycle and the 
potential for adverse exposure effects on the regularity of the animal's 
cycle. In the rat, common cell types found in the smears correlate well 
with the various stages of the estrous cycle and to changes occurring in 
the reproductive tract.
    (c) Principle of the test method. (1) For a two week period before 
exposures start, daily vaginal cell smears are examined from a surplus 
of female test animals to identify and cull those females which are 
acyclic. After culling, testers shall randomly assign at each exposure 
concentration (including unexposed) a minimum of twenty-five females for 
breeding and fifteen non-bred females for later histologic evaluation. 
Test animals shall be exposed by inhalation to graduated concentrations 
of the test atmosphere for a minimum of six hours per day over the next 
13 weeks. Males and females in both test and control groups are mated 
after nine weeks of exposure. Exposures for pregnant females continue 
through gestation day 15, while exposures for males and all non-pregnant 
females shall continue for the full exposure period.
    (2) Beginning two weeks before the start of the mating period, daily 
vaginal smears resume for all to-be-bred females to characterize their 
estrous cycles. This will continue for four weeks or until a rat's 
pregnancy is confirmed, i.e., day 0, by the presence of sperm in the 
cell smear. On pregnancy day 20, shortly before the expected date of 
delivery, each pregnant female is sacrificed, her uterus removed, and 
the contents examined for embryonic or fetal deaths, and live fetuses. 
At the end of the exposure period, males and all non-pregnant females 
shall be weighed, and various organs and tissues, as appropriate, shall 
be removed and weighed, fixed with stain, and sectioned for viewing 
under a light microscope.
    (3) This assay may be done separately or in combination with the 
subchronic toxicity study, pursuant to the provisions in Sec. 79.62.
    (d) Limit test. If a test at one dose level of the highest 
concentration that can be achieved while maintaining a particle size 
distribution with a mass median aerodynamic diameter (MMAD) of 4 
micrometers ([mu]m) or less, using the procedures described in section 
79.60 of this part produces no observable toxic effects and if toxicity 
would not be expected based upon data of structurally related compounds, 
then a full study using three dose levels might not be necessary. 
Expected human exposure though may indicate the need for a higher dose 
level.
    (e) Test procedures--(1) Animal selection--(i) Species and strain. 
The rat is the preferred species. Strains with low fecundity shall not 
be used and the candidate species shall be characterized for its 
sensitivity to developmental toxins. If another rodent species is used, 
the tester shall provide justification for its selection.
    (ii) Animals shall be a minimum of 10 weeks old at the start of the 
exposure period.
    (iii) Number and sex. Each test and control group shall have a 
minimum of 25 males and 40 females. In order to ensure that sufficient 
pups are produced to permit meaningful evaluation of the potential 
developmental toxicity of the test substance, twenty pregnant test 
animals are required for each exposure and control level.
    (2) Observation period. The observation period shall be 13 weeks, at 
a minimum.
    (3) Concentration levels and concentration selection. (i) To select 
the appropriate concentration levels, a pilot or trial study may be 
advisable. Since pregnant animals have an increased minute ventilation 
as compared to non-pregnant animals, it is recommended that the trial 
study be conducted in pregnant animals. Similarly, since presumably the 
minute ventilation will vary with progression of pregnancy, the animals 
should be exposed during the same period of gestation as in the main 
study. It is not always necessary, though, to carry out a trial study in 
pregnant animals. Comparisons between the results of a trial study in 
non-pregnant animals, and the main study in pregnant animals will 
demonstrate whether or not the test substance is more toxic in pregnant

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animals. In the trial study, the concentration producing embryonic or 
fetal lethalities or maternal toxicity should be determined.
    (ii) The highest concentration level shall induce some overt 
maternal toxicity such as reduced body weight or body weight gain, but 
not more than 10 percent maternal deaths.
    (iii) The lowest concentration level shall not produce any grossly 
observable evidence of either maternal or developmental toxicity.
    (4) Inhalation exposure. (i) All data developed within this study 
shall be in accordance with good laboratory practice provisions under 
Sec. 79.60.
    (ii) The general conduct of this study shall be in accordance with 
the vehicle emissions inhalation exposure guideline in Sec. 79.61.
    (iii) Pregnant females shall be exposed to the test atmosphere on 
each and every day between (and including) the first and fifteenth day 
of gestation.
    (f) Test performance--(1) Study conduct. Directions specific to this 
study are:
    (i) The duration of exposure shall be at least six hours daily, 
allowing appropriate additional time for chamber equilibrium.
    (ii) Where an exposure chamber is used, its design shall minimize 
crowding of the test animals. This is best accomplished by individual 
caging.
    (iii) Pregnant animals shall not be subjected to beyond the minimum 
amount of stress. Since whole-body exposure appears to be the least 
stressful mode of exposure, it is the preferred method. In general 
oronasal or head-only exposure, which is sometimes used to avoid 
concurrent exposure by the dermal or oral routes, is not recommended 
because of the associated stress accompanying the restraining of the 
animals. However, there may be specific instances where it may be more 
appropriate than whole-body exposure. The tester shall provide 
justification/reasoning for its selection.
    (iv) Measurements shall be made at least every other day of food 
consumption for all animals in the study. Males and females shall be 
weighed on the first day of exposure and 2-3 times per week thereafter, 
except for pregnant dams.
    (v) The test animal housing, mating, and exposure chambers shall be 
operated on a twenty-four hour lighting schedule, with twelve hours of 
light and twelve hours of darkness. Test animal exposure shall only 
occur during the light portion of the cycle.
    (vi) Signs of toxicity shall be recorded as they are observed 
including the time of onset, degree, and duration.
    (vii) Females showing signs of abortion or premature delivery shall 
be sacrificed and subjected to a thorough macroscopic examination.
    (viii) Animals that die or are euthanized because of morbidity will 
be necropsied promptly.
    (2) Vaginal cytology. (i) For a two week period before the mating 
period starts, each female in the to-be-bred population shall undergo a 
daily saline vaginal lavage. Two wet cell smears from this lavage shall 
be examined daily for each subject to determine a baseline pattern of 
estrus. Testers shall avoid excessive handling and roughness in 
obtaining the vaginal cell samples, as this may induce a condition of 
pseudo-pregnancy in the test animals.
    (ii) This will continue for four weeks or until day 0 of a rat's 
pregnancy is confirmed by the presence of sperm in the cell smear.
    (3) Mating and fertility assessment. (i) Beginning nine weeks after 
the start of exposure, each exposed and control group female (exclusive 
of the histology group females) shall be paired during non-exposure 
hours with a male from the same exposure concentration group. Matings 
shall continue for a period of two weeks, or until all mated females are 
determined to be pregnant. Mating pairs shall be clearly identified.
    (ii) Each morning, including weekends, cages shall be examined for 
the presence of a sperm plug. When found, this shall mark gestation day 
0 and pregnancy shall be confirmed by the presence of sperm in the day's 
wet vaginal cell smears.
    (iii) Two weeks after mating is begun, or as females are determined 
to be pregnant, bred animals are returned to pre-mating housing. Daily 
exposures continues through gestation day 15 for all pregnant females or 
through the balance of the exposure period for non-pregnant females and 
all males.

[[Page 558]]

    (iv) Those pairs which fail to mate shall be evaluated in the course 
of the study to determine the cause of the apparent infertility. This 
may involve such procedures as additional opportunities to mate with a 
proven fertile partner, histological examination of the reproductive 
organs, and, in males, examination of the spermatogenic cycles. The 
stage of estrus for each non-pregnant female in the breeding group will 
be determined at the end of the exposure period.
    (4) All animals in the histology group shall be subject to 
histopathologic examination at the end of the study's exposure period.
    (g) Treatment of results. (1) All observed results, quantitative and 
incidental, shall be evaluated by an appropriate statistical method. The 
specific methods, including consideration of statistical power, shall be 
selected during the design of the study.
    (2) Data and reporting. In addition to the reporting requirements 
specified under Secs. 79.60 and 79.61, the final test report must 
include the following information:
    (i) Gross necropsy. (A) All animals shall be subjected to a full 
necropsy which includes examination of the external surface of the body, 
all orifices, and the cranial, thoracic, and abdominal cavities and 
their contents. Special attention shall be directed to the organs of the 
reproductive system.
    (B) The liver, kidneys, adrenals, pituitary, uterus, vagina, 
ovaries, testes, epididymides and seminal vesicles (with coagulating 
glands), and prostate shall be weighed wet, as soon as possible after 
dissection, to avoid drying.
    (i) At the time of sacrifice on gestation day 20 or at death during 
the study, each dam shall be examined macroscopically for any structural 
abnormalities or pathological changes which may have influenced the 
pregnancy.
    (ii) The contents of the uterus shall be examined for embryonic or 
fetal deaths and the number of viable fetuses. Gravid uterine weights 
need not be obtained from dead animals where decomposition has occurred. 
The degree of resorption shall be described in order to help estimate 
the relative time of death.
    (iii) The number of corpora lutea shall be determined in each 
pregnant dam.
    (iv) Each fetus shall be weighed, all weights recorded, and mean 
fetal weights determined.
    (v) Each fetus shall be examined externally and the sex determined.
    (vi) One-half of the rat fetuses in each litter shall be examined 
for skeletal anomalies, and the remaining half shall be examined for 
soft tissue anomalies, using appropriate methods.
    (ii) Histopathology. (A) Histopathology on vagina, uterus, ovaries, 
testes, epididymides, seminal vesicles, and prostate as appropriate for 
all males and histology group females in the control and high 
concentration groups and for all animals that died or were euthanized 
during the study. If abnormalities or equivocal results are seen in any 
of these organs/tissues, the same organ/tissue from test animals in 
lower concentration groups shall be examined.

    Note: Testes, seminal vesicles, epididymides, and ovaries, at a 
minimum, shall be examined in perfusion-fixed (pressure or gravity 
method) test subjects, when available.

    (B) All gross lesions in all study animals shall be examined.
    (C) As noted under mating procedures, reproductive organs of animals 
suspected of infertility shall be subject to microscopic examination.
    (D) The following organs and tissues, or representative samples 
thereof, shall be preserved in a suitable medium for future 
histopathological examination: all gross lesions; vagina; uterus; 
ovaries; testes; epididymides; seminal vesicles; prostate; liver; and 
kidneys/adrenals.
    (3) Evaluation of results. (i) The findings of a developmental 
toxicity study shall be evaluated in terms of the observed effects and 
the exposure levels producing effects. It is necessary to consider the 
historical developmental toxicity data on the species/strain tested.
    (ii) There are several criteria for determining a positive result 
for reproductive/teratologic effects; a statistically significant dose-
related decrease in the weight of the testes for treated subjects over 
control subjects,

[[Page 559]]

a decrease in neonatal viability, a significant change in the presence 
of soft tissue or skeletal abnormalities, or an increased rate of 
embryonic or fetal resorption or death. Other criteria, e.g., 
lengthening of the estrous cycle or the time spent in any one stage of 
estrus, changes in the proportion of viable male vs female fetuses or 
offspring, the number and type of cells in vaginal smears, or pathologic 
changes found during gross or microscopic examination of male or female 
reproductive organs may be based upon detection of a reproducible and 
statistically significant positive response for that evaluation 
parameter. A positive result indicates that, under the test conditions, 
the test substance does induce reproductive organ or fetal toxicity in 
the test species.
    (iii) A test substance which does not produce either a statistically 
significant dose-related change in the reproductive organs or cycle or a 
statistically significant and reproducible positive response at any one 
of the test points may not induce reproductive organ toxicity in this 
test species, but further investigation , e.g., to establish absorption 
and bioavailability of the test substance, should be considered.
    (h) Test report. In addition to the reporting requirements as 
specified under 40 CFR 79.60 and the vehicle emissions inhalation 
toxicity guideline as published in 40 CFR 79.61, the following specific 
information shall be reported:
    (1) Individual animal data. (i) Time of death during the study or 
whether animals survived to termination.
    (ii) Date of onset and duration of each abnormal sign and its 
subsequent course.
    (iii) Feed and body weight data.
    (iv) Necropsy findings.
    (v) Male test subjects.
    (A) Testicle weight, and body weight: testicle weight ratio.
    (B) Detailed description of all histopathological findings, 
especially for the testes and the epididymides.
    (vi) Female test subjects.
    (A) Uterine weight data.
    (B) Beginning and ending collection dates for vaginal cell smears.
    (C) Estrous cycle length compared within and between groups 
including mean cycle length for groups.
    (D) Percentage of time spent in each stage of cycle.
    (E) Stage of estrus at time of mating/sacrifice and proportion of 
females in estrus between concentration groups.
    (F) Detailed description of all histopathological findings, 
especially for uterine/ovary samples.
    (vii) Pregnancy and litter data. Toxic response data by exposure 
level, including but not limited to, indices of fertility and time-to-
mating, including the number of days until mating and the number of full 
or partial estrous cycles until mating.
    (A) Number of pregnant animals,
    (B) Number and percentage of live fetuses, resorptions.
    (viii) Fetal data. (A) Numbers of each sex.
    (B) Number of fetuses with any soft tissue or skeletal 
abnormalities.
    (2) Type of stain/fixative and procedures used in preparing tissue 
samples.
    (3) Statistical treatment of the study results.
    (i) References. For additional background information on this test 
guideline, the following references should be consulted.
    (1) 40 CFR 798.2675, Oral Toxicity with Satellite Reproduction and 
Fertility Study.
    (2) 40 CFR 798.4350, Inhalation Developmental Toxicity Study.
    (3) Chapin, R.E. and J.J. Heindel (1993) Methods in Toxicology, Vol. 
3, Parts A and B: Reproductive Toxicology, Academic Press, Orlando, FL.
    (4) Gray, L.E., et al. (1989) ``A Dose-Response Analysis of 
Methoxychlor-Induced Alterations of Reproductive Development and 
Function in the Rat'' Fund. App. Tox. 12, 92-108.
    (5) Leblond, C.P. and Y. Clermont (1952) ``Definition of the Stages 
of the Cycle of the Seminiferous Epithelium of the Rat.'' Ann. N. Y. 
Acad. Sci. 55:548-73.
    (6) Morrissey, R.E., et al. (1988) ``Evaluation of Rodent Sperm, 
Vaginal Cytology, and Reproductive Organ Weight Data from National 
Toxicology Program 13-week Studies.'' Fundam. Appl. Toxicol. 11:343-358.
    (7) Russell, L.D., Ettlin, R.A., Sinhattikim, A.P., and Clegg, E.D

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(1990) Histological and Histopathological Evaluation of the Testes, 
Cache River Press, Clearwater, FL.

[59 FR 33093, June 27, 1994, as amended at 61 FR 36513, July 11, 1996]