United States Prevention, Pesticides EPA712-C-96-208
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.3800
Reproduction and
Fertility Effects
"Public Draft'
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the
OPP Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-1530 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
-------
INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-1530 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 870.3800 Reproduction and fertility effects.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.} and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPPT guideline under 40 CFR
798.4700, OPP guideline 83-4, and OECD guideline 416.
(b) Purpose. This guideline for two-generation reproduction testing
is designed to provide general information concerning the effects of a test
substance on the integrity and performance of the male and female repro-
ductive systems, including gonadal function, the estrous cycle, mating be-
havior, conception, gestation, parturition, lactation, and weaning, and on
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 prenatal and postnatal developmental
toxicity and serve as a guide for subsequent tests. For further information
on functional deficiencies and developmental effects, additional study seg-
ments can be incorporated into the protocol, utilizing the guidelines for
developmental toxicity or developmental neurotoxicity.
(c) Good laboratory practice standards. The study should be con-
ducted in accordance with the laboratory practices stipulated in 40 CFR
Part 160 (FIFRA) and 40 CFR Part 792 (TSCA)—Good Laboratory Prac-
tice Standards.
(d) Principle of the test method. The test substance is administered
to parental (P) animals prior to and during their mating, during the result-
ant pregnancies, and through the weaning of their Fl offspring. The sub-
stance is then administered to selected Fl offspring during their growth
into adulthood, mating, and production of an F2 generation, until the F2
generation is weaned.
(e) Test procedures—(1) Animal selection—(i) Species and strain.
The rat is the preferred species for testing. If another mammalian species
is used, the tester should provide justification/reasoning for its selection,
and appropriate modifications will be necessary. Healthy parental animals,
which have been acclimated to laboratory conditions for at least 5 days
and have not been subjected to previous experimental procedures, should
be used. Strains of low fecundity should not be used.
(ii) Age. Parental (P) animals should be 5 to 9 weeks old at the start
of dosing. The animals of all test groups should be of uniform weight,
age, and parity as nearly as practicable, and should be representative of
the species and strain under study.
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(iii) Sex. (A) For an adequate assessment of fertility, both males and
females should be studied.
(B) The females should be nulliparous and nonpregnant.
(iv) Animal care. Animal care and housing should be in accordance
with the recommendations contained in DHHS/PHS NIH Publication No.
86-23, 1985, Guidelines for the Care and Use of Laboratory Animals,
or other appropriate guidelines.
(v) Number of animals. Each control group should contain a suffi-
cient number of mating pairs to yield at least 20 pregnant females. Each
test group should contain a similar number of mating pairs.
(vi) Identification of test system. Each animal should be assigned
a unique identification number. For the P generation, this should be done
before dosing starts. For the Fl generation, this should be done for animals
selected for mating; in addition, records indicating the litter of origin
should be maintained for all selected Fl animals.
(2) Administration of test and control substances—(i) Dose levels
and dose selection. (A) At least three dose levels and a concurrent control
should be used. Healthy animals should be randomly assigned to the con-
trol and treatment groups. The dose levels should be spaced to produce
a gradation of toxic effects. Unless limited by the physical-chemical nature
or biological effects of the test substance, the highest dose level should
induce toxicity but not more than approximately 10 percent mortality in
the parental (P) animals. The intermediate dose levels should produce
minimal observable toxic effects. The lowest dose level should not produce
any evidence of toxicity.
(B) It is desirable that additional information on metabolism and
pharmacokinetics of the test substance be available to demonstrate the ade-
quacy of the dosing regimen. This information should be available prior
to testing.
(C) The highest dose tested should not exceed 1,000 mg/kg/day (or
20,000 ppm in the diet), unless potential human exposure data indicate
the need for higher doses.
(ii) Control groups. (A) A concurrent control group should be used.
This group should be an untreated or sham treated group or a vehicle-
control group if a vehicle is used in administering the test substance.
(B) If a vehicle is used in administering the test substance, the control
group should receive the vehicle in the highest volume used.
(C) If a vehicle or other additive is used to facilitate dosing, consider-
ation should be given to the following characteristics: Effects on the ab-
sorption, distribution, metabolism, or retention of the test substance; effects
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on the chemical properties of the test substance which may alter its toxic
characteristics; and effects on the food or water consumption or the nutri-
tional status of the animals.
(D) If a test substance is administered in the diet and causes reduced
dietary intake or utilization, the use of a pair-fed control group may be
considered necessary.
(iii) Route of administration. (A) The oral route of administration
(diet, drinking water, or gavage) is preferred.
(B) If administered by gavage, the dosage administered to each ani-
mal prior to mating and during gestation and lactation should be based
on the individual animal body weight and adjusted weekly at a minimum.
(C) If another route of administration is used, the tester should pro-
vide justification and reasoning for its selection, and appropriate modifica-
tions may be necessary.
(D) All animals should be dosed by the same method during the ap-
propriate experimental period.
(iv) Dosing schedule. (A) The animals should be dosed with the test
substance on a 7-days-a-week basis.
(B) Daily dosing of the parental (P) males and females should begin
when they are 5 to 9 weeks old. Daily dosing of the Fl males and females
should begin at weaning. For both sexes (P and Fl), dosing should be
continued for at least 10 weeks before the mating period.
(C) Daily dosing of the P and Fl males and females should continue
until termination.
(3) Mating procedure—(i) Parental. (A) For each mating, each fe-
male should be placed with a single randomly selected male from the same
dose level (1:1 mating) until pregnancy occurs or either 3 estrous periods
or 2 weeks has elapsed. Animals should be separated as soon as possible
after evidence of copulation is observed. If mating has not occurred after
2 weeks or 3 estrous periods, the animals should be separated without
further opportunity for mating. Mating pairs should be clearly identified
in the data.
(B) Vaginal smears should be collected daily and examined for all
females during mating, until evidence of copulation is observed.
(C) Each day, the females 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.
(ii) Fl mating. For mating the Fl offspring, at least one male and
one female should be randomly selected from each litter for mating with
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another pup of the same dose level but different litter, to produce the F2
generation.
(iii) Second mating. In certain instances, such as poor reproductive
performance in the controls, or in the event of treatment-related alterations
in litter size, the adults may be remated to produce an Fib or F2b litter.
If production of a second litter is deemed necessary in either generation,
the dams should be remated approximately 1-2 weeks following weaning
of the last Fla or F2a litter.
(iv) Special housing. After evidence of copulation, pregnant animals
should be caged separately in delivery or maternity cages. Pregnant ani-
mals should be provided with nesting materials when parturition is near.
(v) Standardization of litter sizes. (A) Animals should be allowed
to litter normally and rear their offspring to weaning. Standardization of
litter sizes is optional.
(B) If standardization is performed, the following procedure should
be used. On day 4 after birth, the size of each litter may be adjusted by
eliminating extra pups by random selection to yield, as nearly as possible,
four males and four females per litter or five males and five females per
litter. Selective elimination of pups, i.e. based upon body weight, is not
appropriate. Whenever the number of male or female pups prevents having
four (or five) of each sex per litter, partial adjustment (for example, five
males and three females, or four males and six females) is acceptable.
Adjustments are not appropriate for litters of eight pups or less.
(4) Observation of Animals—(i) Parental. (A) Throughout the test
period, each animal should be observed at least once daily. Mortality,
moribundity, pertinent behavioral changes, signs of difficult or prolonged
parturition, and all signs of overt toxicity should be recorded at this
cageside examination. In addition, thorough physical examinations should
be conducted weekly on each animal.
(B) Parental animals (P and Fl) should be weighed on the first day
of dosing and weekly thereafter. Parental females (P and Fl) should be
weighed at a minimum on gestation days 0, 7, 14, and 21, and during
lactation on the same days as the weighing of litters.
(C) During the premating and gestation periods, food consumption
should be measured weekly at a minimum. Water consumption should be
measured weekly at a minimum if the test substance is administered in
the water.
(D) Estrous cycle length and normality should be evaluated by vaginal
smears for all P and Fl females during a minimum of 3 weeks prior to
mating and throughout cohabitation; care should be taken to prevent the
induction of pseudopregnancy.
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(E) For all P and Fl males at termination, sperm from the testis and
epididymis should be collected for enumeration of homogenization-resist-
ant spermatids and cauda epididymal sperm reserves, respectively. In addi-
tion, sperm from the cauda epididymis (or vas deferens) should be col-
lected for evaluation of sperm motility and sperm morphology.
(7) The total number of homogenization-resistant testicular sperm and
cauda epididymal sperm should be enumerated (see paragraphs (g)(2) and
(g)(12) of this guideline). Cauda sperm reserves can be derived from the
concentration and volume of sperm in the suspension used to complete
the qualitative evaluations, and the number of sperm recovered by subse-
quent mincing and/or homogenizing of the remaining cauda tissue. Enu-
meration in only control and high-dose P and Fl males may be performed
unless treatment-related effects are observed; in that case, the lower dose
groups should also be evaluated.
(2) An evaluation of epididymal (or vas deferens) sperm motility
should be performed. Sperm should be recovered while minimizing dam-
age (refer to paragraph (g)(5) of this guideline), and the percentage of
progressively motile sperm should be determined either subjectively or ob-
jectively. For objective evaluations, an acceptable counting chamber of
sufficient depth can be used to effectively combine the assessment of mo-
tility with sperm count and sperm morphology. When computer-assisted
motion analysis is performed (refer to paragraphs (g)(l), (g)(6), (g)(15),
and (g)(18) of this guideline), the derivation of progressive motility relies
on user-defined thresholds for average path velocity and straightness or
linear index. If samples are videotaped, or images otherwise recorded, at
the time of necropsy, subsequent analysis of only control and high-dose
P and Fl males may be performed unless treatment-related effects are ob-
served; in that case, the lower dose groups should also be evaluated. In
the absence of a video or digital image, all samples in all treatment groups
should be analyzed at necropsy.
(3) A morphological evaluation of an epididymal (or vas deferens)
sperm sample should be performed. Sperm (at least 200) should be exam-
ined as fixed, wet preparations (refer to paragraph (g)(8) of this guideline)
and classified as either normal (both head and midpiece/tail appear normal)
or abnormal. Examples of morphologic sperm abnormalities would include
fusion, isolated heads, and misshapen heads and/or tails. Evaluation of
only control and high-dose P and Fl males may be performed unless treat-
ment-related effects are observed; in that case, the lower dose groups
should also be evaluated.
(ii) Offspring. (A) Each litter should be examined as soon as possible
after delivery (lactation day 0) to establish the number and sex of pups,
stillbirths, live births, and the presence of gross anomalies. Pups found
dead on day 0 should be preserved and examined for possible defects and
cause of death.
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(B) Live pups should be counted, sexed, and weighed individually
at birth, or soon thereafter, and on days 4, 7, 14, and 21 of lactation.
(C) The age of vaginal opening and preputial separation should be
determined for Fl weanlings selected for mating. If there is a treatment-
related effect in Fl sex ratio or sexual maturation, anogenital distance
should be measured on day 0 for all F2 pups.
(5) Termination schedule, (i) All P and Fl adult males and females
should be terminated when they are no longer needed for assessment of
reproductive effects.
(ii) Fl offspring not selected for mating and all F2 offspring should
be terminated after weaning.
(6) Gross necropsy, (i) At the time of termination or death during
the study, all parental animals (P and Fl) and at least three pups per sex
per litter from the unselected Fl weanlings and the F2 weanlings should
be examined macroscopically for any structural abnormalities or patholog-
ical changes. Special attention should be paid to the organs of the repro-
ductive system.
(ii) Dead pups or pups that are terminated in a moribund condition
should be preserved and examined for possible defects and/or cause of
death.
(iii) The uteri of all cohabited females should be examined, in a man-
ner which does not compromise histopathological evaluation, for the pres-
ence and number of implantation sites.
(7) Organ weights, (i) At the time of termination, the following or-
gans of all P and Fl parental animals should be weighed:
(A) Uterus, ovaries.
(B) Testes, epididymides (total and cauda), seminal vesicles (with co-
agulating glands and their fluids), and prostate.
(C) Brain, liver, kidneys, adrenal glands, spleen, and known target
organs.
(ii) For Fl and F2 weanlings that are examined macroscopically, the
following organs should be weighed:
(A) Brain.
(B) Spleen and thymus.
(8) Tissue preservation. The following organs and tissues, or rep-
resentative samples thereof, should be fixed and stored in a suitable me-
dium for histopathological examination.
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(i) For the parental (P and Fl) animals:
(A) Vagina, uterus with cervix, and ovaries with oviducts.
(B) Testis (preserved in Bouins fixative or comparable preservative),
one epididymis, seminal vesicles, prostate, and coagulating gland.
(C) Target organs, when previously identified, from all P and Fl ani-
mals selected for mating.
(D) Grossly abnormal tissue.
(ii) For Fl and F2 weanlings selected for macroscopic examination:
Grossly abnormal tissue and target organs, when known.
(9) Histopathology—(i) Parental animals. Full histopathology of the
organs listed in paragraph (e)(8)(i) of this guideline should be performed
for all high dose and control P and Fl animals selected for mating. Organs
demonstrating treatment-related changes should also be examined in the
low- and mid-dose groups. Additionally, reproductive organs of the low-
and mid-dose animals suspected of reduced fertility, e.g., those that failed
to mate, conceive, sire, or deliver healthy offspring, or for which estrous
cyclicity or sperm number, motility, or morphology were affected, should
be subjected to histopathological evaluation. Besides gross lesions such
as atrophy or tumors, testicular histopathological examination should be
conducted in order to to 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 (refer to paragraph
(g)(13) of this guideline). Examination of the intact epididymis should in-
clude the caput, corpus, and cauda, which can be accomplished by evalua-
tion of a longitudinal section, and should be conducted in order to identify
such lesions as sperm granulomas, leukocytic infiltration (inflammation),
aberrant cell types within the lumen, or the absence of clear cells in the
cauda epididymal epithelium. The postlactational ovary should contain pri-
mordial and growing follicles as well as the large corpora lutea of lacta-
tion. Histopathological examination should detect depletion of the pri-
mordial follicle population. Five ovarian sections should be taken at least
100 (im apart from the inner third of each ovary. Examination should in-
clude enumeration of the total number of primordial follicles from these
10 sections (see paragraph (g)(4) of this guideline) for comparison with
control ovaries. Examination should also confirm the presence or absence
of growing follicles and corpora lutea in comparison with control ovaries.
(ii) Weanlings. For Fl and F2 weanlings, histopathological character-
ization of all developmental anomalies noted at macroscopic examination
should be performed, with emphasis on the organs of the reproductive sys-
tem.
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(f) Data and reporting—(1) Treatment of results. Data should be
reported individually and summarized in tabular form, showing for each
test group the types of change and the number of animals displaying each
type of change.
(2) Evaluation of study results, (i) An evaluation of test results, in-
cluding the statistical analysis, should be provided. This should include
an evaluation of the relationship, or lack thereof, between the exposure
of the animals to the test substance and the incidence and severity of all
abnormalities.
(ii) Statistical analysis of the study findings should include sufficient
information on the method of analysis, so that an independent reviewer/
statistician can reevaluate and reconstruct the analysis.
(iii) In any study which demonstrates an absence of toxic effects, fur-
ther investigation to establish absorption and bioavailability of the test sub-
stance should be considered.
(3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J and 40 CFR, part 160, subpart J, the
following specific information should be reported. Both individual and
summary data should be presented.
(i) Species and strain.
(ii) Toxic response data by sex and dose, including indices of mating,
fertility, gestation, birth, viability, and lactation; offspring sex ratio;
precoital interval, including the number of days until mating and the num-
ber of estrous periods until mating; and duration of gestation calculated
from day 0 of pregnancy. The report should provide the numbers used
in calculating all indices.
(iii) Day (week) of death during the study or whether animals sur-
vived to termination.
(iv) Toxic or other effects on reproduction, offspring, or postnatal
growth.
(v) Developmental milestone data (mean age of vaginal opening and
preputial separation, and mean anogenital distance, when measured).
(vi) Number of P and Fl females cycling normally and mean estrous
cycle length.
(vii) Day (week) of observation of each abnormal sign and its subse-
quent course.
(viii) Body weight and body weight change data by sex for P, Fl,
and F2 animals.
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(ix) Food (and water, if applicable) consumption, food efficiency
(body weight gain per gram of food consumed), and test material con-
sumption for P and F1 animals, except for the period of cohabitation.
(x) Total cauda epididymal sperm number, number and percent of
progressively motile sperm, number and percent of morphologically nor-
mal sperm, and number and percent of sperm with each identified anom-
aly.
(xi) Stage of estrous at the time of termination for P and Fl parental
females.
(xii) Necropsy findings.
(xiii) Implantation data and postimplantation loss calculations for P
and Fl parental females.
(xiv) Absolute and relative organ weight data.
(xv) Detailed description of all histopathological findings.
(xvi) Adequate statistical treatment of results.
(xvii) A copy of the study protocol and any amendments should be
included.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline:
(1) Chapin, R.E. et al. Methods for assessing rat sperm motility. Re-
productive Toxicology 6:267-273 (1992).
(2) Gray, L.E. et al. A dose-response analysis of methoxychlor-in-
duced alterations of reproductive development and function in the rat. Fun-
damental and Applied Toxicology 12:92-108 (1989).
(3) Heindel, J.J. and R.E. Chapin, (eds.). Part B. Female Reproductive
Systems, Methods in Toxicology, Academic, Orlando, FL (1993).
(4) Heindel, J.J. et al. Histological assessment of ovarian follicle num-
ber in mice as a screen of ovarian toxicity. In: Growth Factors and the
Ovary, A.N. Hirshfield (ed.), Plenum, NY, pp. 421-426 (1989).
(5) Klinefelter, G.R. et al. The method of sperm collection signifi-
cantly influences sperm motion parameters following ethane
dimethanesulfonate administration in the rat. Reproductive Toxicology
5:39-44 (1991).
(6) Klinefelter, G.R. et al. Direct effects of ethane dimethane
sulphonate on epididymal function in adult rats: an in vitro demonstration.
Journal of Andrology 13:409-421 (1992).
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(7) Korenbrot, C.C. et al. Preputial separation as an external sign of
pubertal development in the male rat. Biology of Reproduction 17:298-
303 (1977).
(8) Linder, R.E. et al. Endpoints of spermatoxicity in the rat after
short duration exposures to fourteen reproductive toxicants. Reproductive
Toxicology 6:491-505 (1992).
(9) Manson, J.M. and Y.J. Kang. Test methods for assessing female
reproductive and developmental toxicology. In: Principles and Methods
of Toxicology, A.W. Hayes (ed.), Raven, New York (1989).
(10) Organisation for Economic Co-Operation and Development, No.
416: Two Generation Reproduction Toxicity Study, Guidelines for Testing
of Chemicals. [C(83)44 (Final)] (1983).
(11) Pederson, T. and H. Peters. Proposal for classification of oocytes
and follicles in the mouse ovary. Journal of Reproduction and Fertility
17:555-557 (1988).
(12) Robb, G.W. et al. Daily sperm production and epididymal sperm
reserves of pubertal and adult rats. Journal of Reproduction and Fertility
54:103-107(1978).
(13) Russell, L.D. et al. Histological and Histopathological Evalua-
tion of the Testis, Cache River, Clearwater, FL (1990).
(14) Sadleir, R.M.F.S. Cycles and seasons, In: Reproduction in Mam-
mals: I. Germ Cells and Fertilization, C.R. Auston and R.V. Short (eds.),
Cambridge, NY (1979).
(15) Slott, V.L. et al. Rat sperm motility analysis: methodologic con-
siderations. Reproductive Toxicology 5:449-458 (1991).
(16) Smith, B.J. et al. Comparison of random and serial sections in
assessment of ovarian toxicity. Reproductive Toxicology 5:379-383 (1991).
(17) Thomas, J.A. Toxic responses of the reproductive system. In:
Casarett and Doull's Toxicology, M.O. Amdur, J. Doull, and C.D.
Klaassen (eds.), Pergamon, NY (1991).
(18) Toth, G.P. et al. The automated analysis of rat sperm motility
following subchronic epichlorhydrin administration: Methodologic and sta-
tistical considerations. Journal ofAndrology 10:401-415 (1989).
(19) U.S. Environmental Protection Agency. OPP Guideline 83-4:
Reproductive and Fertility Effects. Pesticide Assessment Guidelines, Sub-
division F, Hazard Evaluation: Human and Domestic Animals. Office of
Pesticides and Toxic Substances, Washington, DC, EPA-540/9-82-025
(1982).
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(20) U.S. Environmental Protection Agency. Subpart E—Specific
Organ/Tissue Toxicity, 40 CFR 798.4700: Reproduction and Fertility Ef-
fects.
(21) Working, P.K. and M. Hurtt. Computerized videomicrographic
analysis of rat sperm motility. Journal of Andrology 8:330-337 (1987).
(22) Zenick, H. and E.D. Clegg. Assessment of male reproductive
toxicity: a risk assessment approach. In: Principles and Methods of Toxi-
cology, A.W. Hayes (ed.), Raven, NY (1989).
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