United States       Prevention, Pesticides     EPA712-C-98-207
          Environmental Protection    and Toxic Substances     August 1998
          Agency        (7101)
&EPA   Health Effects Test
          Guidelines
          OPPTS 870.3700
          Prenatal Developmental
          Toxicity Study

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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 disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in ASCII and PDF  (portable  document  format) from EPA's World Wide
Web  site (http://www.epa.gov/epahome/research.htm) under the heading
"Researchers and Scientists/Test Methods  and Guidelines/OPPTS Har-
monized Test Guidelines."

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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.\ as amended by the Food
Quality Protection Act (FQPA)(Pub. L. 104-170),  and the Toxic  Sub-
stances 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 and an assessment of ma-
ternal  effects.  For information  on testing for functional deficiencies and
other postnatal effects, the guidelines for the two-generation reproductive
toxicity study  and the  developmental neurotoxicity study should be  con-
sulted.

     (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 one day prior to the
expected day of parturition. Shortly before the expected date of delivery,
the pregnant females are  terminated, 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. The preferred rodent
species is the rat and the preferred non-rodent species is the rabbit.

     (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 in the rodent
or that insemination is performed or observed in the rabbit.

     (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.

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     (v) Number of animals. Each test and control group should contain
a sufficient number of animals to yield  approximately 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, in a manner which results in comparable mean
body weight values among all groups. The dose levels should be spaced
to produce  a gradation of toxic effects. Unless  limited by the physical/
chemical nature or biological properties of the test substance, the highest
dose should be chosen with  the aim to induce some developmental and/
or maternal toxicity but not death or severe suffering. In the case of mater-
nal mortality, this should not be more than approximately 10 percent. 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  (i.e.,  the  no-observed-adverse-effect level,
NOAEL) or should be at or  near the limit of detection for the most sen-
sitive endpoint. Two- or four-fold intervals are frequently optimal for spac-
ing the dose levels, and the  addition of a fourth test group is often pref-
erable to using very large intervals (e.g., more than a factor of 10) between
dosages.

     (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 indicate the need
for higher  doses.  If a test performed at  the  limit  dose  level,  using the
procedures described for this study, produces no observable toxicity and
if an effect would not be expected based upon data  from structurally relat-
ed compounds, then a full study using three-dose levels may not be consid-
ered necessary.

     (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

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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 principal 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)(12), (h)(28), and (h)(29) of this  guideline.  Care should be taken to
minimize  stress on the maternal animals. For materials administered by
inhalation, whole-body exposure is preferable to nose-only exposure due
to the stress of restraint required for nose-only exposure.

     (C) The test  substance should be administered at approximately the
same time each day.

     (D)  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 the day before cesarean section on
the  day prior to the expected day of parturition.  Alternatively, if prelimi-
nary studies do not indicate  a high potential for preimplantation loss, treat-
ment may be extended to include the entire period of gestation, from fer-
tilization to approximately 1 day prior to the expected day of termination.

     (f) Observation of animals—(1) Maternal, (i) Each animal should
be observed at least once daily, considering the peak period of anticipated
effects after dosing. Mortality, moribundity, pertinent behavioral changes,
and all signs of overt toxicity should be recorded at this cageside observa-
tion. 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 at least 3-day intervals,
preferably on days when 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.

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     (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 non-
gravid should be further examined (e.g. by ammonium sulfide staining)
to confirm the nonpregnant status.

     (B) Each gravid uterus (with cervix) should be weighed. Gravid uter-
ine  weights should not be  obtained from dead animals  if autolysis or de-
composition has  occurred.

     (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 by  standard techniques  and examined for skeletal alterations, pref-
erably bone and  cartilage. The  remainder should be prepared and examined
for  soft tissue anomalies, using appropriate  serial sectioning or gross dis-
section techniques. 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 bodies of these fetuses should be evaluated
by careful dissection for soft-tissue anomalies, followed by preparation and
examination for  skeletal anomalies. An adequate evaluation of the  internal
structures of the  head,  including  the  eyes,  brain, nasal passages,  and
tongue, should be conducted for at least half  of the fetuses.

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     (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. Historical data (on litter incidence and fetal incidence
within litter), when used,  should be compiled, presented, and  analyzed in
an appropriate and relevant manner. In order to justify its  use as an analyt-
ical  tool, information such as the  dates of study conduct, the strain and
source of the animals, and the vehicle and route of administration should
be included.

     (iv) 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.  In the evaluation
of study data, the litter should  be considered the basic  unit of 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.  Both individual and
summary data should be presented.

     (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.

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     (D) Body weight and body weight change data, including body weight
change adjusted for gravid uterine weight.
     (E) Food consumption and, if applicable, water consumption data.
     (F) Necropsy findings, including gravid 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 (early and late).
     (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 malformation 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 individ-
ual 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) Aliverti, V.L. et al. The extent of fetal  ossification as an index
of delayed development in teratogenicity studies in the rat.  Teratology
20:237-242 (1979).
     (2) Barrow, M.V. and W.J. Taylor. A rapid method for detecting mal-
formations in rat fetuses. Journal of Morphology 127:291-306 (1969).
     (3) Burdi,  A.R. Toluidine blue-alizarin red S staining of cartilage and
bone in whole-mount  skeltons in vitro.  Stain  Technolology 40:45-48
(1965).

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     (4) 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).

     (5) Fritz, H. Prenatal ossification in rabbits as indicative of fetal matu-
rity.  Teratology 11:313-320 (1974).

     (6) Fritz, H.  and R. Hess. Ossification of the rat and mouse skeleton
in the perinatal period. Teratology 3:331-338 (1970).

     (7) Gibson, J.P. et al. Use of the rabbit in teratogenicity studies. Toxi-
cology and Applied Pharmacology 9:398-408 (1966).

     (8) 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).

     (9) 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).

     (10) Kaufman, M. (ed.) The Atlas of Mouse Development.  Academic
Press, London (1993).

     (11) Kimmel, C.A. et al. Skeletal development following heat expo-
sure  in the rat. Teratology 47:229-242 (1993).

     (12) Kimmel,  C.A. and E.Z. Francis. Proceedings of the  workshop
on the acceptability and interpretation of dermal developmental  toxicity
studies. Fundamental and Applied Toxicology 14:386-398 (1990).

     (13) Kimmel, C.A. and C.  Trammell. A rapid procedure for routine
double staining of  cartilage and bone in fetal and adult animals. Stain
Technology 56:271-273 (1981).

     (14) Kimmel, C.A. and J.G. Wilson. Skeletal deviation in rats: mal-
formations or variations? Teratology 8:309-316 (1973).

     (15) 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).

     (16) 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).

     (17) McLeod,  M.J.  Differential staining of cartilage and bone  in
whole mouse fetuses by Alcian blue and alizarin red S. Teratology 22:299-
301 (1980).

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     (18) 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).

     (19) Organization  for Economic Cooperation and Development, No.
414:  Teratogenicity, Guidelines  for Testing of  Chemicals.  [C(83)44
(Final)] (1983).

     (20) Salewski (Koeln),  V.E.  Faerbermethode zum makroskopischen
nachweis  von implantations   stellen  am  uterus  der ratte.  Naunyn-
Schmeidebergs Archiv fur Pharmakologie  und Experimented Pathologic
247:367 (1964).

     (21) 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).

     (22) Staples, R.E. Detection of visceral alterations in mammalian
fetuses. Teratology 9(3):A37-A38 (1974).

     (23) 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).

     (24) 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).

     (25) 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).

     (26)  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).

     (27) U.S. Environmental Protection  Agency.  Subpart E—Specific
Organ/Tissue Toxicity,  40 CFR 798.4900:  Developmental Toxicity Study.

     (28) U.S. Environmental  Protection  Agency.  Health Effects Test
Guidelines, OPPTS 870.3200, 21/28-Day Dermal Toxicity, July 1998.

     (29) U.S. Environmental  Protection  Agency.  Health Effects Test
Guidelines, OPPTS 870.3465, 90-Day Inhalation Toxicity, July 1998.

     (30) U.S.  Environmental  Protection  Agency Guidelines for Devel-
opmental Toxicity Risk Assessment.  FEDERAL REGISTER (56 FR 63798-
63826, December  5, 1991).

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     (31) 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).

     (32) Walker, D.G. and Z.T. Wirtschafter. The Genesis of the Rat Skel-
eton. Thomas, Springfield, IL (1957).

     (33) Whitaker, J. and D.M. Dix. Double-staining for rat foetus skele-
tons in teratological studies. Laboratory Animals 13:309-310 (1979).

     (34) 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).

     (35) Wilson, J.G. and F.C. Fraser, ed. Handbook of Teratology,  Vol.
4. Plenum, NY (1977).

     (36) Yasuda, M. and T. Yuki. Color Atlas of Fetal Skeleton of the
Mouse, Rat,  and Rabbit.  Ace Art Co., Osaka, Japan (1996).

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