United States Prevention, Pesticides EPA712-C-96-205
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.3500
Preliminary
Developmental Toxicity
Screen
'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 202-512-0132 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 870.3500 Preliminary developmental toxicity screen.
(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 40 CFR 798.4420 Preliminary Devel-
opmental Toxicity Screen.
(b) Purpose. The in vivo developmental toxicity assay is designed
to assess the potential of agents to induce toxic effects in the conceptus
after administration to the mother during pregnancy. This test should be
used only to prioritize environmental agents for testing by more rigorous
standard protocols.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792—Good Laboratory Practice Standards (GLP) apply to this test
guideline. The following definition also applies to this test guideline.
Developmental toxicity is the capability of an agent to induce in utero
death, structural or functional abnormalities, or growth retardation after
contact with the pregnant animal.
(d) Principle of the test method. The test substance is administered
to pregnant animals during a significant portion of the major period of
organogenesis. A single dose level is administered. This dose level should
be high enough to elicit significant maternal toxicity. The dams are al-
lowed to give birth and the neonates are counted and weighed on
day-1 and day-3 postpartum (day-1 is the day after birth). The underlying
hypothesis for this assay is that most prenatal insults will manifest them-
selves as reduced viability and/or growth during the postnatal period.
(e) Test procedures—(1) Animal selection—(i) Species and strain.
Testing must be performed in a mammalian species and strain which will
allow human handling of newborn pups without cannibalization or aban-
donment. The preferred species would be either rat or mouse, and of neces-
sity, a strain that does not exhibit the behavior referred to above. The
strain should be commonly used and should not have low fecundity.
(ii) Age. Young adult animals (nulliparous females) should be used.
(iii) Sex. Pregnant female animals should be used.
(iv) Number of animals. At least 30 bred animals should be used
for each compound. The objective is to ensure that sufficient litters are
produced to permit meaningful evaluation of the potential developmental
toxicity of the test substance.
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(2) Control group. A concurrent control group should be used. This
group should be an untreated or sham treated control group, or, if a vehicle
is used in administering the test substance, a vehicle control group. Except
for treatment with the test substance, animals in the control group(s)
should be handled in an identical manner to test group animals.
(3) Dose levels and dose selection, (i) A single dose level with a
concurrent control and, when appropriate, a vehicle control, should be
used.
(ii) The vehicle should be neither developmentally toxic nor have ef-
fects on reproduction.
(iii) To select the appropriate dose levels, a pilot or trial study may
be advisable. It is not always necessary to carry out a trial study in preg-
nant animals. Comparison of the results from a trial study in non-pregnant,
and the main study in pregnant animals will demonstrate whether or not
the test substance is more toxic in pregnant animals.
(iv) Unless limited by the physical/chemical nature or biological prop-
erties of the substance, the dose level used should be high enough to cause
overt maternal toxicity as evidenced by significant death, weight loss or
other adverse clinical manifestations, or 1 gm/kg, if lower dose levels fail
to induce maternal toxicity.
(4) Observation period. Day-0 in the test is the day in which a vagi-
nal plug and/or sperm are observed. The dose period should encompass
a significant portion of the period of major organogenesis. This may be
taken as day-7 to day-11 for rat and mouse. When there is evidence of
rapid clearance it may be advisable to extend the dosing period for 2 days
to cover the critical period of palatal closure.
(5) Administration of test substance. The test substance or vehicle
is usually administered orally, by intubation unless the chemical or phys-
ical characteristics of the test substance or pattern of human exposure sug-
gest a more appropriate route of administration. The test substance should
be administered at approximately the same time each day.
(6) Exposure conditions. The female test animals are treated with
the test substance daily throughout the appropriate treatment period. When
given by gavage, the dose may be based on the weight of the females
at the start of substance administration, or, alternatively, in view of the
rapid weight gain which takes place during pregnancy, the animals may
be weighed periodically and the dosage based on the most recent weight
determination.
(7) Observation of pregnant animals, (i) A gross examination of
the dams should be made at least once prior to parturition.
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(ii) Pregnant animals should be weighed the day prior to the begin-
ning of treatment, and that day on which treatment ends.
(iii) Cage-side observations should include, but not be limited to:
change in skin and fur, eye and mucous membranes, as well as respiratory,
autonomic and central nervous systems, somatomotor activity and behav-
ioral pattern.
(iv) Signs of toxicity should be recorded as they are observed, includ-
ing the time of onset, the degree and duration.
(v) During the dosing period females that die or are sacrificed because
they are moribund should be examined for signs of pregnancy and details
of the conditions of the uterus and/or its contents recorded. Animals that
have not delivered 2 days after expected date of parturition should be sac-
rificed and similar examinations made.
(8) Observation of dams after birth. Dams should be observed for
signs of overt toxicity during the postpartum period at the same time
neonatal examinations are being made.
(9) Neonatal examinations, (i) Dams are allowed to give birth and
the litters are examined for gross anomalies and presence of milk, counted,
and weighed on postpartum day-1 and day-3.
(ii) Dead pups should be subjected to a thorough external examination
and gross soft tissue abnormalities noted.
(iii) For those compounds that induce only neonatal growth reduction
it may be advisable to normalize litter size on postpartum day-3 (to ap-
proximately 4 females and 4 males) and leave them with the dam through
weaning. This procedure will determine if the growth reduction is transient
or if it represents a permanent functional alteration.
(f) Data and reporting—(1) Treatment of results. Data should be
summarized in tabular form, showing for each test group: the number of
animals at the start of the test, the number of pregnant animals, the mater-
nal weight during the treatment period, the average number of live neo-
nates on day-1 and day-3, the average neonatal weight on day-1 and
day-3, and the average weight gained during that period.
(2) Evaluation of results. The findings of this bioassay should be
evaluated in terms of the types of effects noted. All data analyses should
compare treatment groups and their concurrent controls. Statistical treat-
ment of the results should involve analysis of variance, and the number
of live pups on day-1 and day-3 should be used as a covariate in the
analyses of postnatal body weight so as to correct for differences in pup
weights due to litter size. The number of animals going to term must be
sufficiently large to allow for a reasonable detection of compound-induced
deficiencies. Conditions which significantly reduce the number of dams
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going to term (e.g. lack of pregnancy or compound-induced maternal
death) should lead to a repeat of the study.
(3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J the following specific information should
be reported:
(i) Toxic response data.
(ii) Species and strain.
(iii) Date of maternal death during the study or whether animals sur-
vived to termination.
(iv) Date of onset and duration of each abnormal sign and its subse-
quent course.
(v) Pregnancy data.
(vi) Litter data including number live and dead; and average litter
weight on day-1 and day-3 postpartum.
(vii) Necropsy data on dead pups.
(g) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Chernoff, N. and Kavlock, R. An in vivo teratology screen utiliz-
ing pregnant mice, Journal of Toxicology and Environmental Health
10:541-550 (1982).
(2) Doe, J. E. et al. Comparative aspects of the reproductive toxi-
cology by inhalation in rats of ethylene glycol monomethyl ether and pro-
pylene glycol monomethyl ether. Toxicology and Applied Pharmacology
69:43-47 (1983).
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