.... United States Environmental Protection Agency Prevention, Pesticides and Toxic Substances (7101) EPA 712–C–96–207 February 1996 

# Health Effects Test Guidelines OPPTS 870.3700 Prenatal Developmental Toxicity Study

‘‘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 theNational 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 proceduresthat 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 theEPA 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.’’

### 1 OPPTS 870.3700 Prenatal developmental toxicity study. (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 798.4900, OPP guideline 83–3, and OECD guideline 414. (b) Purpose. This guideline for developmental toxicity testing is designed to provide general information concerning the effects of exposure of the pregnant test animal on the developing organism; this may includedeath, structural abnormalities, or altered growth. 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 consulted. (c) Good laboratory practice standards. The study should be conducted in accordance with the laboratory practices stipulated in 40 CFRPart 160 (FIFRA) and 40 CFR Part 792 (TSCA)—Good Laboratory Practice 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 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 prenatal 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 Animals , 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 (2) Administration of test and control substances—(i) Dose levels and dose selection. (A) At least three dose levels and a concurrent controlshould be used. Healthy animals should be randomly assigned to the control and treatment groups, using body weight-dependent procedures. Thedose levels should be spaced to produce a gradation of toxic effects. Unless limited by the physical/chemical nature or biological properties of thesubstance, the highest dose should induce some developmental and/or maternal toxicity but not more than approximately 10 percent maternal mortality. 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 andpharmacokinetics 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 need not exceed 1,000 mg/kg/day by oralor dermal administration, or 2 mg/L (or the maximum attainable concentration) 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 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 on the chemical properties of the test substance which may alter its toxiccharacteristics; and effects on the food or water consumption or the nutritional 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 forits selection, and appropriate modifications may be necessary. Further information on dermal or inhalation exposure is provided under paragraphs(h)(6) and (h)(17) of this guideline. The test substance should be administered at approximately the same time each day.

### 3 (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 administered 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 shouldbe observed at least once daily. Mortality, moribundity, pertinent behavioral 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 immediately prior to the expected day of delivery. (B) Females showing signs of abortion or premature delivery priorto scheduled termination should be killed and subjected to a thorough macroscopic examination. (v) Gross necropsy. At the time of termination or death during thestudy, the dam should be examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. 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 termination 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 shouldnot be obtained from dead animals if autolysis or decomposition has occurred. (C) The number of corpora lutea should be determined for pregnant animals.

### 4 (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 determined. (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 defined by the performing laboratory).(A) For rodents, approximately one-half of each litter should be prepared 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 relationship, 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 skeletal anomalies.(iii) When appropriate, historical control data to enhance interpretation 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.

### 5(v) 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: (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 subsequent 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, including: (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 combined.(D) External, soft tissue, and skeletal (bone and cartilage) 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 individual anomalies, should be reported.

### 6 (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 additional background information on this test guideline:(1) Barrow, M.V. and W.J. Taylor. A rapid method for detecting malformations in rat fetuses. Journal of Morphology 127:291–306 (1969).(2) Edwards, J.A. The external development of the rabbit and rat embryo. 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 maturity. Teratology 11:313–320 (1974). (4) Gibson, J.P. et al. Use of the rabbit in teratogenicity studies. Toxicology 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 variations detected by the double staining technique for ossified and cartilaginous 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 onthe acceptability and interpretation of dermal developmental toxicity studies. Fundamental and Applied Toxicology 14:386–398 (1990).(9) Kimmel, C.A. and J.G. Wilson. Skeletal deviation in rats: malformations 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 (SpragueDawley) rat skeleton after maternal exposure to ethylene glycol. Teratology 46:169–181 (1992). (12) Monie, I.W. et al. Dissection procedures for rat fetuses permitting alizarin red staining of skeleton and histological study of viscera. Supplement to Teratology Workshop Manual, pp. 163–173 (1965).

### 7 (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. NaunynSchmeidebergs Archiv f u  ̈ r Pharmakologie und Experimentelle Pathologie 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 Technology 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, Carcinogenesis, and Mutagenesis 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—SpecificOrgan/Tissue Toxicity, 40 CFR 798.4350: Inhalation Developmental Toxicity 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 Developmental 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 Skeleton. Thomas, Springfield, IL (1957).

### 8 (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).