United States Prevention, Pesticides EPA712-C-96-207
Environmental Protection and Toxic Substances February 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.3700
Prenatal Developmental
Toxicity Study
"Public Draft'
-------
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."
-------
OPPTS 870.3700 Prenatal developmental toxicity study.
(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
798.4900, OPP guideline 83-3, and OECD guideline 414.
(b) Purpose. This guideline for developmental toxicity testing is de-
signed to provide general information concerning the effects of exposure
of the pregnant test animal on the developing organism; this may include
death, structural abnormalities, or altered growth. For information on test-
ing for functional deficiencies and other postnatal effects, the guidelines
for the two-generation reproductive toxicity study and the developmental
neurotoxicity study should be consulted.
(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 pregnant animals at least from implantation to the end of gestation.
Shortly before the expected date of delivery, the pregnant females are ter-
minated, the uterine contents are examined, and the fetuses are processed
for visceral and skeletal evaluation.
(e) Test procedures—(1) Animal selection—(i) Species and strain.
It is recommended that testing be performed in the most relevant species,
and that laboratory species and strains which are commonly used in pre-
natal developmental toxicity testing be employed.
(ii) Age. Young adult animals should be used.
(iii) Sex. Nulliparous female animals should be used at each dose
level. Animals should be mated with males of the same species and strain,
avoiding the mating of siblings, if parentage is known. Day 0 in the test
is the day on which a vaginal plug and/or sperm are observed.
(iv) Animal care. Animal care and housing should be in accordance
with the recommendations contained in the DHHS/PHS NIH Publication
No. 86-23, 1985, Guidelines for the Care and Housing of Laboratory Ani-
mals, or other appropriate guidelines.
(v) Number of animals. Each test and control group should contain
at least 20 animals with implantation sites at necropsy.
-------
(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, using body weight-dependent procedures. The
dose levels should be spaced to produce a gradation of toxic effects. Un-
less limited by the physical/chemical nature or biological properties of the
substance, the highest dose should induce some developmental and/or ma-
ternal toxicity but not more than approximately 10 percent maternal mor-
tality. The intermediate dose levels should produce minimal observable
toxic effects. The lowest dose level should not produce any evidence of
either maternal or developmental 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 need not exceed 1,000 mg/kg/day by oral
or dermal administration, or 2 mg/L (or the maximum attainable concentra-
tion) by inhalation, unless potential human exposure data indicates the
need for higher doses.
(ii) Control group. (A) A concurrent control group should be used.
This group should be a sham-treated control group or a vehicle-control
group if a vehicle is used in administering the test substance.
(B) The vehicle control group should receive the vehicle in the high-
est 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
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.
(iii) Route of administration. (A) The test substance or vehicle is
usually administered orally by intubation.
(B) If another route of administration is used, for example, when the
route of administration is based upon the principle route of potential
human exposure, the tester should provide justification and reasoning for
its selection, and appropriate modifications may be necessary. Further in-
formation on dermal or inhalation exposure is provided under paragraphs
(h)(6) and (h)(17) of this guideline. The test substance should be adminis-
tered at approximately the same time each day.
-------
(C) When administered by gavage or dermal application, the dose
to each animal should be based on the most recent individual body weight
determination.
(iv) Dosing schedule. At minimum, the test substance should be ad-
ministered daily from implantation to cesarean section on the day prior
to parturition. Alternatively, if preliminary studies do not indicate a high
potential for preimplantation loss, treatment may be extended to include
the entire period of gestation, from fertilization to approximately 1 day
before term.
(f) Observation of animals—(1) Maternal, (i) Each animal should
be observed at least once daily. Mortality, moribundity, pertinent behav-
ioral changes, and all signs of overt toxicity should be recorded at this
cageside observation. In addition, thorough physical examinations should
be conducted at the same time maternal body weights are recorded.
(ii) Animals should be weighed on day 0, at termination, and at least
at 3-day intervals during the dosing period.
(iii) Food consumption should be recorded on the same days that body
weights are recorded.
(iv) Termination schedule. (A) Females should be terminated imme-
diately prior to the expected day of delivery.
(B) Females showing signs of abortion or premature delivery prior
to scheduled termination should be killed and subjected to a thorough mac-
roscopic examination.
(v) Gross necropsy. At the time of termination or death during the
study, the dam should be examined macroscopically for any structural ab-
normalities or pathological changes which may have influenced the preg-
nancy. Evaluation of the dams during cesarean section and subsequent fetal
analyses should be conducted without knowledge of treatment group in
order to minimize bias.
(vi) Examination of uterine contents. (A) Immediately after termi-
nation or as soon as possible after death, the uteri should be removed
and the pregnancy status of the animals ascertained. Uteri that appear
nongravid should be further examined (e.g. by ammonium sulfide staining)
to confirm the nonpregnant status.
(B) Gravid uteri should be weighed. Gravid uterine weights should
not be obtained from dead animals if auto lysis or decomposition has oc-
curred.
(C) The number of corpora lutea should be determined for pregnant
animals.
-------
(D) The uterine contents should be examined for embryonic or fetal
deaths and the number of viable fetuses. The degree of resorption should
be described in order to help estimate the relative time of death of the
conceptus.
(2) Fetal, (i) The sex and body weight of each fetus should be deter-
mined.
(ii) Each fetus should be examined for external anomalies.
(iii) Fetuses should be examined for skeletal and soft tissue anomalies
(e.g. variations and malformations or other categories of anomalies as de-
fined by the performing laboratory).
(A) For rodents, approximately one-half of each litter should be pre-
pared and examined for skeletal (bone and cartilage) alterations and the
remainder should be prepared and examined for soft tissue anomalies,
using appropriate methods. It is also acceptable to examine all fetuses by
careful dissection for soft tissue anomalies followed by an examination
for skeletal anomalies.
(B) For rabbits, all fetuses should be examined for both soft tissue
and skeletal alterations. The heads of one-half of the fetuses should be
removed and processed for evaluation of soft tissue alterations, using
standard serial sectioning methods. The heads of the remaining fetuses
should be sectioned by a single midcoronal incision for evaluation of the
internal structure of the brain, then processed for evaluation of skeletal
alterations.
(g) 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 dams, fetuses, and litters
displaying each type of change.
(2) Evaluation of study results. The following should be provided:
(i) Maternal and fetal test results, including an evaluation of the rela-
tionship, or lack thereof, between the exposure of the animals to the test
substance and the incidence and severity of all findings.
(ii) Criteria used for categorizing fetal external, soft tissue, and skele-
tal anomalies.
(iii) When appropriate, historical control data to enhance interpreta-
tion of study results.
(iv) Statistical analysis of the study findings, which should include
sufficient information on the method of analysis, so that an independent
reviewer/statistician can reevaluate and reconstruct the analysis.
-------
(v) 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:
(i) Species and strain.
(ii) Maternal toxic response data by dose, including but not limited
to:
(A) The number of animals at the start of the test, the number of
animals surviving, the number pregnant, and the number aborting.
(B) Day of death during the study or whether animals survived to
termination.
(C) Day of observation of each abnormal clinical sign and its subse-
quent course.
(D) Body weight and body weight change data, including body weight
change corrected for gravid uterine weight.
(E) Food consumption and, if applicable, water consumption data.
(F) Necropsy findings, including uterine weight.
(iii) Developmental endpoints by dose for litters with implants, in-
cluding:
(A) Corpora lutea counts.
(B) Implantation data, number and percent of live and dead fetuses,
and resorptions.
(C) Pre- and postimplantation loss calculations.
(iv) Developmental endpoints by dose for litters with live fetuses,
including:
(A) Number and percent of live offspring.
(B) Sex ratio.
(C) Fetal body weight data, preferably by sex and with sexes com-
bined.
(D) External, soft tissue, and skeletal (bone and cartilage) malforma-
tion and variation data. The total number and percent of fetuses and litters
with any external, soft tissue, or skeletal alteration, as well as the types
and incidences of individual anomalies, should be reported.
-------
(v) The numbers used in calculating all percentages or indices.
(vi) Adequate statistical treatment of results.
(vii) A copy of the study protocol and any amendments should be
included.
(h) References. The following references should be consulted for ad-
ditional background information on this test guideline:
(1) Barrow, M.V. and W.J. Taylor. A rapid method for detecting mal-
formations in rat fetuses. Journal of Morphology 127:291-306 (1969).
(2) Edwards, J.A. The external development of the rabbit and rat em-
bryo. In Advances in Teratology (ed. D.H.M. Woolam) Vol. 3. Academic,
NY (1968).
(3) Fritz, H. Prenatal ossification in rabbits as indicative of fetal matu-
rity. Teratology 11:313-320 (1974).
(4) Gibson, J.P. et al. Use of the rabbit in teratogenicity studies. Toxi-
cology and Applied Pharmacology 9:398-408 (1966).
(5) Inouye, M. Differential staining of cartilage and bone in fetal
mouse skeleton by alcian blue and alizarin red S. Congenital Anomalies
16(3): 171-173 (1976).
(6) Igarashi, E. et al. Frequence of spontaneous axial skeletal vari-
ations detected by the double staining technique for ossified and cartilagi-
nous skeleton in rat fetuses. Congenital Anomalies 32:381-391 (1992).
(7) Kimmel, C.A. et al. Skeletal development following heat exposure
in the rat. Teratology 47:229-242 (1993).
(8) Kimmel, C.A. and E.Z. Francis. Proceedings of the workshop on
the acceptability and interpretation of dermal developmental toxicity stud-
ies. Fundamental and Applied Toxicology 14:386-398 (1990).
(9) Kimmel, C.A. and J.G. Wilson. Skeletal deviation in rats: mal-
formations or variations? Teratology 8:309-316 (1973).
(10) Marr, M.C. et al. Comparison of single and double staining for
evaluation of skeletal development: the effects of ethylene glycol (EG)
in CD rats. Teratology 37:476 (1988).
(11) Marr, M.C. et al. Developmental stages of the CD (Sprague-
Dawley) rat skeleton after maternal exposure to ethylene glycol. Teratol-
ogy 46:169-181 (1992).
(12) Monie, I.W. et al. Dissection procedures for rat fetuses permit-
ting alizarin red staining of skeleton and histological study of viscera. Sup-
plement to Teratology Workshop Manual, pp. 163-173 (1965).
-------
(13) Organisation for Economic Co-Operation and Development, No.
414: Teratogenicity, Guideline for Testing of Chemicals. [C(83)44 (Final)]
(1983).
(14) Salewski (Koeln), V.E. Faerbermethode zum makroskopischen
nachweis von implantations stellen am uterus der ratte. Naunyn-
Schmeidebergs Archiv fur Pharmakologie und Experimentelle Pathologic
247:367 (1964).
(15) Spark, C. and A.B. Dawson. The order and time of appearance
of centers of ossification in the fore and hind limbs of the albino rat, with
special reference to the possible influence of the sex factor. American
Journal of Anatomy 41:411-445 (1928).
(16) Staples, R.E. and V.L. Schnell. Refinements in rapid clearing
technique in the KOH—alizarin red S method for fetal bone. Stain Tech-
nology 39:61-63 (1964).
(17) Strong, R.M. The order time and rate of ossification of the albino
rat (mus norvegicus albinus) skeleton. American Journal of Anatomy 36:
313-355(1928).
(18) Stuckhardt, J.L. and S.M. Poppe. Fresh visceral examination of
rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Car-
cinogenesis, andMutagenesis 4:181-188 (1984).
(19) U.S. Environmental Protection Agency. Guideline 83-3:
Teratogencity Study. 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).
(20) U.S. Environmental Protection Agency. Subpart E—Specific
Organ/Tissue Toxicity, 40 CFR 798.4350: Inhalation Developmental Tox-
icity Study.
(21) U.S. Environmental Protection Agency. Subpart E—Specific
Organ/Tissue Toxicity, 40 CFR 798.4900: Developmental Toxicity Study.
(22) U.S. Environmental Protection Agency Guidelines for Devel-
opmental Toxicity Risk Assessment. FEDERAL REGISTER 56:63798-63826
(1991).
(23) Van Julsingha, E.B. and C.G. Bennett. A dissecting procedure
for the detection of anomalies in the rabbit foetal head. In: Methods in
Prenatal Toxicology (eds. D. Neubert, H.J. Merker, and T.E. Kwasigroch).
University of Chicago, Chicago, IL, pp. 126-144 (1977).
(24) Walker, D.G. and Z.T. Wirtschafter. The Genesis of the Rat Skel-
eton. Thomas, Springfield, IL (1957).
-------
(25) Wilson, J.G. Embryological considerations in teratology. In
"Teratology: Principles and Techniques" (ed. J.G. Wilson and J.
Warkany). University of Chicago, Chicago, IL, pp 251-277 (1965).
(26) Wilson, J.G. and F.C. Fraser, ed. Handbook of Teratology, Vol.
4. Plenum, NY (1977).
8
------- |