United States Prevention, Pesticides EPA712-C-96-324
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Microbial Pesticide
Test Guidelines
OPPTS 885.3650
Reproductive/Fertility
Effects
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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.).
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), internet: http://
fedbbs.access.gpo.gov, or call 202-512-0132 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 885.3650 Reproductive/fertility effects.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP guideline 152A-30.
(b) Purpose. The reproductive/fertility effects study is designed to
provide and estimate of potential human hazard from an MPCA if:
(1) Significant infectivity of the MPCA is observed in test animals
in the subchronic Tier II study (OPPTS 885.3600), and in which no signs
of toxicity or pathogenicity were observed.
(2) The MPCA is a virus which can persist or replicate in mammalian
cell culture lines (OPPTS 885.3500).
(3) The MPCA is not amenable to thorough taxonomic classification,
but is related to organisms known to be parasitic for mammalian cells.
(4) The MPCA preparation is not sufficiently well purified, but it
is indicated that the preparation may contain contaminants which are para-
sitic for mammals.
(c) General design. This guideline for a one-generation reproductive/
fertility study is designed to provide information on the effects of an
MPCA on fertility and on embryo/fetal development of test animals. Ef-
fects of the MPCA on fertility and fetal development to be evaluated in-
clude the number of nonpregnant females, numbers that gave normal birth,
number of resorptions, litter size, delayed birth, embryolethality, and off-
spring body weight. Transmittal of the MPCA from parent to offspring
also is evaluated.
(d) Principle of the test method. The MPCA is administered to male
and female parents prior to their mating, and to maternal parents during
the resultant pregnancies.
(e) Substance to be tested. Testing shall be performed with the tech-
nical grade of each active ingredient.
(f) Test procedures—(1) Animal selection—(i) Species and strain.
The mouse or the rat are the preferred species. Commonly used laboratory
strains should be employed. If another species is used, justification/reason-
ing for the alternate selection should be provided. All test animals should
be free of parasites or pathogens. Strains with low fecundity should not
be used.
(ii) Age. Test animals should be between 6 and 8 weeks old prior
to the first dosing.
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(iii) Sex. (A) Both males and females are to be studied for an ade-
quate assessment of the MPCA on fertility.
(iv) Numbers. Each test and control group should 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 untreated control group is re-
quired.
(ii) A separate vehicle control group is not required except when the
toxicity of the vehicle is unknown.
(iii) A control group dosed with inactivated MPCA is recommended.
(3) Dosing—(i) Dose level. One dose level of at least 108 units of
the MPCA per test animal should be used. Justification/reasoning must
be provided if a dose level of at least 108 units per test animal is not
used. Quantification of the units of the MPCA should be done concurrently
with dosing.
(ii) Dose route. Administration of the MPCA usually will be by the
oral route. If persistence or infectivity of the MPCA in the Tier I studies
was observed only after same other route of dosing (e.g. intravenous), this
route must be used.
(iii) Dose frequency. The frequency of dosing with at least 108 units
per test animal should be such that a significant level of MPCA is main-
tained in the parents prior to and during the mating period, and in the
female parents during pregnancy.
(4) Observation period. Duration of observation should be from the
initial dosing with the MPCA to sacrifice of the offspring.
(5) Observation of animals, (i) A careful clinical examination should
be made on each test animal at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study, e.g. necropsy of,
and MPCA enumeration from those animals found dead, and isolation of
weak or moribund animals.
(iii) Cageside observations should include, but not be limited to,
changes in:
(A) The skin and fur.
(B) Eyes and mucous membranes.
(C) Respiratory system.
(D) Circulatory system.
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(E) Autonomic and central nervous system.
(F) Somatomotor activity.
(G) Behavior pattern.
(H) Particular attention should be directed to observations of tremors,
convulsions, diarrhea, lethargy, salivation, sleep, and coma.
(iv) Individual weights of animals should be determined shortly be-
fore the test material is administered, weekly thereafter, and at death or
at sacrifice.
(v) The time of death should be recorded as precisely as possible.
(6) Mating procedure—(i) Parental. (A) For each mating, each fe-
male should be placed with a single, randomly selected male until preg-
nancy occurs or 3 weeks have elapsed. Mixed matings with other males
should be avoided.
(B) Those pairs that fail to mate successfully should be evaluated
to determine the cause of the apparent infertility. This may involve such
procedures as additional opportunities to mate with other sires or dams,
examination of the reproductive organs, and examination of the estrus or
spermatogenic cycles.
(C) Each morning, the female should 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) Special housing. Near parturition, pregnant animals should be
caged separately in delivery or maternity cages and provided with nesting
materials. The cages and materials should be free from contamination by
the MPCA. Dosing should cease prior to isolation of the pregnant females.
(7) Observation of pregnant females, (i) Food consumption and pro-
longed parturition should be recorded, in addition to the above clinical
observations (paragraphs (f)(5)(i) through (v) of this guideline).
(ii) The duration of gestation should 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, and the presence of physical
and behavioral abnormalities.
(iv) Litters should be weighed at birth or soon thereafter.
(8) MPCA enumeration in parents and offspring, (i) Infectivity
or persistence should be assessed by using sensitive techniques to deter-
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mine, as quantitatively as possible, the presence of the MPCA in test ani-
mals.
(ii) Organs, tissues, and body fluids of each male parent should be
assayed at the time when it is confirmed that the female of the mated
pair is determined to be pregnant.
(iii) Organs, tissues, and body fluids of each female parent should
be assayed as soon as possible after birth of the litter.
(iv) Quantitative determinations of the MPCA in the pups from each
litter should be made on postpartum day 1.
(g) Data and reporting—(1) Evaluation of study results, (i) An
evaluation of test results shall include a reporting of any and all effects
of the MPCA on the test animals, all observations made, statistical analy-
ses, MPCA quantification in the dosing preparations and in the parents
and offspring, evidence that a significant level of the MPCA was main-
tained in the parents, dosing schedule, and animal weights. This should
include an evaluation of the relationship, or lack thereof, between carriage
of the MPCA by parents and abnormal effects an reproduction and fertility.
(2) Test report. In addition to the information recommended by
OPPTS 885.0001, the test report shall include the following information:
(i) Fertility indices and length of gestation.
(ii) Species and strain.
(iii) Time of death during the study or whether animals survived to
termination.
(iv) Effects on reproduction and on offspring.
(v) Time of observation of each abnormal sign, including pathogenic-
ity, and its subsequent course.
(vi) Body weight data for parents and offspring.
(vii) Necropsy findings.
(viii) MPCA enumerations.
(ix) Statistical treatment of results, where appropriate.
(h) Tier progression. Any further testing that is to be required will
be determined after consultation with the Agency.
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