United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–208

February 1996

Health Effects Test

Guidelines

OPPTS 870.3800

Reproduction and

Fertility Effects

‘‘Public Draft’’
i

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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.’’
i

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin 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.’’
1

OPPTS 870.3800 Reproduction and fertility effects.

(a) Scope—(1) Applicability. This guideline is intended to meet testing 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 harmonized 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 reproductive systems, including gonadal function, the estrous cycle, mating behavior, 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 segments can be incorporated into the protocol, utilizing the guidelines for

developmental toxicity or developmental neurotoxicity.

(c) Good laboratory practice standards. The study should be conducted in accordance with the laboratory practices stipulated in 40 CFR

Part 160 (FIFRA) and 40 CFR Part 792 (TSCA)—Good Laboratory Practice Standards.

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

to parental (P) animals prior to and during 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, 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 sufficient 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 F1 generation, this should be done for animals

selected for mating; in addition, records indicating the litter of origin

should be maintained for all selected F1 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 control 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 adequacy 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 vehiclecontrol 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, consideration should be given to the following characteristics: Effects on the absorption, 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 nutritional 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 animal 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 provide justification and reasoning for its selection, and appropriate modifications may be necessary.

(D) All animals should be dosed by the same method during the appropriate 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 F1 males and females

should begin at weaning. For both sexes (P and F1), dosing should be

continued for at least 10 weeks before the mating period.

(C) Daily dosing of the P and F1 males and females should continue

until termination.

(3) Mating procedure—(i) Parental. (A) For each mating, each female 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) F1 mating. For mating the F1 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 F1b 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 F1a or F2a litter.

(iv) Special housing. After evidence of copulation, pregnant animals

should be caged separately in delivery or maternity cages. Pregnant animals 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 F1) should be weighed on the first day

of dosing and weekly thereafter. Parental females (P and F1) 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 F1 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 F1 males at termination, sperm from the testis and

epididymis should be collected for enumeration of homogenization-resistant spermatids and cauda epididymal sperm reserves, respectively. In addition, sperm from the cauda epididymis (or vas deferens) should be collected for evaluation of sperm motility and sperm morphology.

(1) 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 subsequent mincing and/or homogenizing of the remaining cauda tissue. Enumeration in only control and high-dose P and F1 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 damage (refer to paragraph (g)(5) of this guideline), and the percentage of

progressively motile sperm should be determined either subjectively or objectively. For objective evaluations, an acceptable counting chamber of

sufficient depth can be used to effectively combine the assessment of motility with sperm count and sperm morphology. When computer-assisted

motion analysis is performed (refer to paragraphs (g)(1), (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 F1 males may be performed unless treatment-related effects are observed; 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 examined 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 F1 males may be performed unless treatment-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 F1 weanlings selected for mating. If there is a treatmentrelated effect in F1 sex ratio or sexual maturation, anogenital distance

should be measured on day 0 for all F2 pups.

(5) Termination schedule. (i) All P and F1 adult males and females

should be terminated when they are no longer needed for assessment of

reproductive effects.

(ii) F1 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 F1) and at least three pups per sex

per litter from the unselected F1 weanlings and the F2 weanlings should

be examined macroscopically for any structural abnormalities or pathological changes. Special attention should be paid to the organs of the reproductive 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 manner which does not compromise histopathological evaluation, for the presence and number of implantation sites.

(7) Organ weights. (i) At the time of termination, the following organs of all P and F1 parental animals should be weighed:

(A) Uterus, ovaries.

(B) Testes, epididymides (total and cauda), seminal vesicles (with coagulating glands and their fluids), and prostate.

(C) Brain, liver, kidneys, adrenal glands, spleen, and known target

organs.

(ii) For F1 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 representative samples thereof, should be fixed and stored in a suitable medium for histopathological examination.
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(i) For the parental (P and F1) 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 F1 animals selected for mating.

(D) Grossly abnormal tissue.

(ii) For F1 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 F1 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 lowand 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 include the caput, corpus, and cauda, which can be accomplished by evaluation 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 primordial and growing follicles as well as the large corpora lutea of lactation. Histopathological examination should detect depletion of the primordial follicle population. Five ovarian sections should be taken at least

100 μm apart from the inner third of each ovary. Examination should include 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 F1 and F2 weanlings, histopathological characterization of all developmental anomalies noted at macroscopic examination

should be performed, with emphasis on the organs of the reproductive system.
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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, including 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, 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 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 number 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 survived 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 F1 females cycling normally and mean estrous

cycle length.

(vii) Day (week) of observation of each abnormal sign and its subsequent course.

(viii) Body weight and body weight change data by sex for P, F1,

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 consumption 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 normal sperm, and number and percent of sperm with each identified anomaly.

(xi) Stage of estrous at the time of termination for P and F1 parental

females.

(xii) Necropsy findings.

(xiii) Implantation data and postimplantation loss calculations for P

and F1 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 additional background information on this test guideline:

(1) Chapin, R.E. et al. Methods for assessing rat sperm motility. Reproductive Toxicology 6:267–273 (1992).

(2) Gray, L.E. et al. A dose-response analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundamental 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 number 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 significantly 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 Evaluation of the Testis, Cache River, Clearwater, FL (1990).

(14) Sadleir, R.M.F.S. Cycles and seasons, In: Reproduction in Mammals: 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 considerations. 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 statistical considerations. Journal of Andrology 10:401–415 (1989).

(19) U.S. Environmental Protection Agency. OPP Guideline 83–4:

Reproductive and Fertility Effects. Pesticide Assessment Guidelines, Subdivision 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 Effects.

(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 Toxicology, A.W. Hayes (ed.), Raven, NY (1989).