In Vivo Assessment of Prenatal Developmental Toxicity in Rodents

Michael G. Narotsky1, Robert J. Kavlock1

1 U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 13.5
DOI:  10.1002/0471140856.tx1305s16
Online Posting Date:  August, 2003
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Abstract

This unit presents two basic protocols for testing prenatal developmental toxicity in rodents. The standard protocol for assessing prenatal developmental toxicity, often referred to as a Segment II study, describes the methods used for performing cesarean sections and examining cesarean‚Äźdelivered fetuses for external, skeletal, and visceral alterations. The protocol for the Chernoff/Kavlock assay, a developmental toxicity screen, describes methods for examining parturition, and postnatal growth and viability of prenatally exposed litters.

Keywords: prenatal developmental toxicity; teratology; pregnancy; Segment II; Chernoff/Kavlock assay

     
 
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Table of Contents

  • Basic Protocol 1: Segment II Study
  • Basic Protocol 2: Chernoff/Kavlock Assay
  • Support Protocol 1: Cesarean Sections
  • Support Protocol 2: Examination of Apparently Nongravid Uteri
  • Support Protocol 3: Examination of Fetal Visceral Specimens
  • Support Protocol 4: Preparation of Fetal Skeletal Specimens
  • Support Protocol 5: Examination of Fetal Skeletal Specimens
  • Support Protocol 6: Analysis and Interpretation of Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Segment II Study

  Materials
  • Young adult (e.g., 60‐ to 90‐day‐old) timed‐pregnant rats or mice of any strain with high fecundity
  • Test substance
  • Bodian's solution (see recipe) or Bouin's solution (see recipe)
  • 4% (w/v) NaCl
  • Additional reagents and equipment for care and handling of rodents (e.g., Coligan et al., ), cesarean section ( protocol 3), examination of apparently nongravid uteri ( protocol 4), examination of fetal visceral specimens ( protocol 5), preparation of fetal skeletal specimens ( protocol 6), and examination of fetal skeletal specimens ( protocol 7), and data analysis and interpretation ( protocol 8)

Basic Protocol 2: Chernoff/Kavlock Assay

  Materials
  • Young adult (e.g., 60‐ to 90‐day‐old) timed‐pregnant rats or mice of any strain with high fecundity
  • Test substance
  • Additional reagents and equipment for care and handling of rodents (e.g., Coligan et al., ) and examination of apparently nongravid uteri ( protocol 4)

Support Protocol 1: Cesarean Sections

  Materials
  • Mouse or rat from Segment II study (see protocol 1)
  • Physiological saline: 0.9% (w/v) NaCl
  • 4% (w/v) NaCl
  • Bodian's solution (see recipe)
  • Surgical instruments
  • Dissecting microscope
  • Weigh boats (or petri dishes)
  • 24‐ or 48‐well plates
  • Absorbent paper and/or cardboard trays
  • 18‐well polystyrene trays (Flambeau Products Corp.)
  • Dry‐erase marking pen (or equivalent)
  • Solvent‐resistant specimen jars
  • Dissecting microscope
  • Additional reagents and equipment for care and handling of rodents (e.g., Coligan et al., ) and examination of apparently nongravid uteri ( protocol 4)

Support Protocol 2: Examination of Apparently Nongravid Uteri

  Materials
  • Uteri (see Basic Protocols protocol 11 and protocol 22 and protocol 3)
  • 2% (w/v) ammonium sulfide (see recipe)
  • Surgical instruments
  • Petri dishes
  • 6‐well tissue culture plate (or similar container)
  • Sealable specimen bags

Support Protocol 3: Examination of Fetal Visceral Specimens

  Materials
  • Fetal specimens (see protocol 1 and protocol 3) in Bodian's solution
  • Bodian's solution (see recipe) or Bouin's solution (see recipe)
  • Thymol crystals
  • Dissecting microscope
  • Dissection instruments including:
    • Forceps
    • Iris scissors
    • Ultramicrodissection scissors
    • Razor blades (or scalpels)
  • Air‐tight vials (e.g., scintillation vials)

Support Protocol 4: Preparation of Fetal Skeletal Specimens

  Materials
  • Fetuses (see protocol 1 and protocol 3)
  • 4% NaCl
  • 95% (or 100%) and 70% ethanol
  • 150 mg/liter Alcian blue (see recipe)
  • 1% (w/v) potassium hydroxide (KOH; see recipe)
  • 25 mg/liter Alizarin red S (see recipe)
  • 1:1 (v/v) glycerol/70% ethanol solution (see recipe)
  • 2:2:1 (v/v/v) glycerol/70% ethanol/benzyl alcohol solution (see recipe)
  • Dissecting instruments including forceps and iris scissors
  • 18‐well polystyrene trays (Flambeau Products Corp.)
  • Flexible screen
  • Colored tape and marking pen

Support Protocol 5: Examination of Fetal Skeletal Specimens

  Materials
  • Stained fetal specimen in 1:1 glycerol/70% ethanol ( protocol 6)
  • 1:1 (v/v) glycerol/70% ethanol solution (see recipe)
  • Glycerol
  • Shallow transparent dish (e.g., ashtray or petri dish)
  • Forceps
  • Dissecting microscope with base illumination
  • Specimen containers (e.g., jars, trays, and vials)

Support Protocol 6: Analysis and Interpretation of Data

  Materials
  • Statistical software (e.g., SAS, GraphPad InStat, BenchMark Dose Software)
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Figures

Videos

Literature Cited

   American Veterinary Medical Association (AVMA). 2001. 2000 Report of the AVMA Panel on Euthanasia. J. Am. Vet. Med. Assoc. 218:669‐696.
   Barrow, M.V. and Taylor, W.J. 1969. A rapid method for detecting malformations in rat fetuses. J. Morphol. 127:291‐305.
   Burg, J.R. and Hardin, B.D. 1987. Recommendations for the statistical analysis of Chernoff/Kavlock test data. Teratog. Carcinog. Mutagen. 7:49‐54.
   Chernoff, N. and Kavlock, R.J. 1982. An in vivo teratology screen utilizing pregnant mice. J. Toxicol. Environ. Health 10:541‐550.
   Christ, S.A., Randall, J.L., Read, E.J., and Smith, M.K. 1991. Comparison of fetal rat examination techniques (fixed vs. fresh) in the detection of cardiac malformations, specifically levocardia. Teratology 43:435.
   Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Strober, W. 2003. Current Protocols in Immunology. John Wiley & Sons, New York.
   Environmental Protection Agency (EPA). 1991. Guidelines for developmental toxicity risk assessment. Fed. Regist. 56:63798‐63826.
   Environmental Protection Agency (EPA), Office of Research and Development. 1995. The use of the benchmark dose approach in health risk assessment. EPA/630/R‐94/007. EPA, Washington, D.C.
   Environmental Protection Agency (EPA). 1996. Health Effects Test Guidelines OPPTS 870.3500 Preliminary Developmental Toxicity Screen “Public Draft”. EPA‐712‐C‐96‐205. EPA, Washington, D.C.
   Environmental Protection Agency (EPA). 1998a. Health Effects Test Guidelines OPPTS 870.6300 Developmental Neurotoxicity Study. EPA 712‐C‐98‐239. EPA, Washington, D.C.
   Environmental Protection Agency (EPA). 1998b. Health Effects Test Guidelines, OPPTS 870.3700, Prenatal Developmental Toxicity Study. EPA, Washington, D.C.
   Environmental Protection Agency (EPA). 1998c. Health Effects Test Guidelines, OPPTS 870.3800, Reproduction and Fertility Effects. EPA, Washington, D.C.
   Environmental Protection Agency (EPA), National Center for Exposure Assessment. 2000. Draft Benchmark Dose Technical Guidance Document. EPA/630/R‐00/001. EPA, Washington, D.C.
   Food and Drug Administration (FDA). 1966. Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use. FDA, Washington, D.C.
   Food and Drug Administration (FDA). 1997. Good Laboratory Practice Regulations for Nonclinical Laboratory Studies. U.S. Code of Federal Regulations. Title 21 Part 8. FDA, Washington, D.C.
   Gad, S.C. and Weil, C.S. 1989. Statistics for toxicologists. In Principles and Methods of Toxicology (A.W. Hayes, ed.) pp. 435‐483. Raven Press, New York.
   Hardin, B.D. 1987. A recommended protocol for the Chernoff/Kavlock preliminary developmental toxicity test and a proposed method for assigning priority scores based on results of that test. Teratog. Carcinog. Mutagen. 7:85‐94.
   Hardin, B.D., Becker, R.A., Kavlock, R.J., Seidenberg, J.M., and Chernoff, N. 1987a. Workshop on the Chernoff/Kavlock preliminary developmental toxicity test. Teratog. Carcinog. Mutagen. 7:119‐127.
   Hardin, B.D., Schuler, R.L., Burg, J.R., Booth, G.M., Hazelden, K.P., MacKenzie, K.M., Piccirillo, V.J., and Smith, K.N. 1987b. Evaluation of 60 chemicals in a preliminary developmental toxicity test. Teratog. Carcinog. Mutagen. 7:29‐48.
   Haseman, J.K., Huff, J., and Boorman, G.A. 1984. Use of historical control data in carcinogenicity studies in rodents. Toxicol. Pathol. 12:126‐135.
   International Conference on Harmonisation (ICH). 1994. Harmonised tripartite guideline: Detection of toxicity to reproduction for medicinal products. In Proceedings of the Second International Conference on International Harmonisation (P. D'Arcy and D. Harron, eds.) pp. 557‐577. Queens University Press, Belfast, U.K.
   Institute for Laboratory Animal Research (ILAR). 1996. Guidelines for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C.
   Kavlock, R.J., Short, R.D., Jr., and Chernoff, N. 1987. Further evaluation of an in vivo teratology screen. Teratog. Carcinog. Mutagen. 7:7‐16.
   Kimmel, C.A. and Francis, E.Z. 1990. Proceedings of the workshop on the acceptability and interpretation of dermal developmental toxicity studies. Fundam. Appl. Toxicol. 14:386‐398.
   Kimmel, C.A. and Price, C.J. 1990. Developmental toxicity studies. In Handbook of In Vivo Toxicity Testing (D.L. Arnold, H.C. Grice, and D.R. Grewski, eds.) pp. 271‐301. Academic Press, San Diego.
   Lau, C., Rogers, J., Hanson, R., Barbee, B., Narotsky, M., Schmid, J., and Richards, J. 2001. Developmental toxicity of perfluorooctane sulfonate (PFOS) in the rat and mouse. Teratology 63:290.
   Menegola, E., Broccia, M.L., and Giavini, E. 2001. Atlas of rat fetal skeleton double stained for bone and cartilage. Teratology 64:125‐133.
   Moore, J.A., Daston, G.P., Faustman, E., Golub, M.S., Hart, W.L., Hughes, C., Jr., Kimmel, C.A., Lamb, J.C.T., Schwetz, B.A., and Sciallim, A.R. 1995. An evaluative process for assessing human reproductive and developmental toxicity of agents. Reprod. Toxicol. 9:61‐95.
   Narotsky, M.G. and Kavlock, R.J. 1995. A multidisciplinary approach to toxicological screening: II. Developmental toxicity. J. Toxicol. Environ. Health 45:145‐171.
   Narotsky, M.G., Francis, E.Z., and Kavlock, R.J. 1994. Developmental toxicity and structure‐activity relationships of aliphatic acids, including dose‐response assessment of valproic acid in mice and rats. Fundam. Appl. Toxicol. 22:251‐265.
   Narotsky, M.G., Weller, E.A., Chinchilli, V.M., and Kavlock, R.J. 1995. Nonadditive developmental toxicity in mixtures of trichloroethylene, Di(2‐ethylhexyl) phthalate, and heptachlor in a 5 × 5 × 5 design. Fundam. Appl. Toxicol. 27:203‐216.
   Narotsky, M.G., Brownie, C.F., and Kavlock, R.J. 1997. Critical period of carbon tetrachloride‐induced pregnancy loss in Fischer‐344 rats, with insights into the detection of resorption sites by ammonium sulfide staining. Teratology 56:252‐261.
   Narotsky, M.G., Best, D.S., Guidici, D.L., and Cooper, R.L. 2001. Strain comparisons of atrazine‐induced pregnancy loss in the rat. Reprod. Toxicol. 15:61‐69.
   Nishimura, K. 1974. A microdissection method for detecting thoracic visceral malformations in mouse and rat fetuses. Cong. Anom. 14:23‐40.
   Organisation for Economic Co‐operation and Development (OECD). 2000. OECD Guideline for the Testing of Chemicals, Proposal for Updating Guideline 414, Prental Developmental Toxicity Study. OECD, Paris.
   Rands, P.L., White, R.D., Carter, M.W., Allen, S.D., and Bradshaw, W.S. 1982. Indicators of developmental toxicity following prenatal administration of hormonally active compounds in the rat. I. Gestational length. Teratology 25:37‐43.
   Seidenberg, J.M. and Becker, R.A. 1987. A summary of the results of 55 chemicals screened for developmental toxicity in mice. Teratog. Carcinog. Mutagen. 7:17‐28.
   Staples, R.E. 1977. Detection of visceral alterations in mammalian fetuses. Teratology 9:A37‐A38.
   Stuckhardt, J.L. and Poppe, S.M. 1984. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratog Carcinog. Mutagen. 4:181‐188.
   Taylor, P. 1986. Practical Teratology. Academic Press, New York.
   Weber, M. and Weber, E. 1991. Die Rasierklingen‐Schnittechnik in der teratologischen Forschung [The razor blade sectioning technique in teratological research]. Anat. Anz. 173:291‐297.
   Wickramaratne, G.A. 1987. The Chernoff‐Kavlock assay: Its validation and application in rats. Teratog. Carcinog. Mutagen. 7:73‐83.
   Wier, P.J., Lewis, S.C., and Traul, K.A. 1987. A comparison of developmental toxicity evident at term to postnatal growth and survival using ethylene glycol monoethyl ether, ethylene glycol monobutyl ether and ethanol. Teratog. Carcinog. Mutagen. 7:55‐64.
   Wilson, J.G. 1965. Methods for administering agents and detecting malformations in experimental animals. In Teratology: Principles and Techniques (J.G. Wilson and J. Warkany, ed.) pp. 262‐277. University of Chicago Press, Chicago.
   Wilson, J.G. 1973. Environment and birth defects. Academic Press, New York.
   Wise, L.D., Beck, S.L., Beltrame, D., Beyer, B.K., Chahoud, I., Clark, R.L., Clark, R., Druga, A.M., Feuston, M.H., Guittin, P., Henwood, S.M., Kimmel, C.A., Lindstrom, P., Palmer, A.K., Petrere, J.A., Solomon, H.M., Yasuda, M., and York, R.G. 1997. Terminology of developmental abnormalities in common laboratory mammals (version 1). Teratology 55:249‐292.
   Yasuda, M. and Yuki, T. 1996. Color Atlas of Fetal Skeleton of the Mouse, Rat, and Rabbit. Ace Art Co., Osaka, Japan.
Key References
   Gad and Weil, 1989. See above.
  A book chapter providing statistical guidance for toxicologists.
   Hood, 1997. Handbook of Developmental Toxicology. CRC Press, Boca Raton, Fla.
  A practical guide for developmental toxicity testing. Encompasses areas such as study design, data presentation, data interpretation, regulatory concerns, and risk assessment.
   Taylor, 1986. See above.
  A handbook providing technical advice for conducting developmental toxicity studies in rats, mice, and rabbits.
   Wise et al., 1997. See above.
  An internationally developed glossary of terms for describing structural developmental abnormalities in common laboratory animals.
Internet Resources
   http://teratology.org
  Web site of the Teratology Society.
   http://www.etsoc.com
  Web site of the European Teratology Society.
   http://www.midwest-teratology.org
  Web site of the Midwest Teratology Association.
   http://nbts.org
  Web site of the Neurobehavioral Teratology Society.
   http://www.interscience.wiley.com/jpages/1542-0752
  Web site of Birth Defects Research, parts A, B, and C, the official publications of the Teratology Society.
   http://hcd.org
  Historical control database of preclinical developmental teratology and reproductive toxicity parameters, a joint project of the Midwest Teratology Association and the Middle Atlantic Reproduction and Teratology Association. Includes a searchable database allowing the retrieval of historical control data from contributing laboratories.
   http://www.ifts-atlas.org
  Atlas of developmental abnormalities in common laboratory mammals, provided by the International Federation of Teratology Societies. Includes a searchable database of images of both rare and common observations in fetuses and neonates from common laboratory mammals. Glossaries of common terms and syndromes are also included.
   http://www.ich.org/pdfICH/s5a.pdf
  ICH Harmonized Tripartite Guideline, Detection of Toxicity to Reproduction for Medicinal Products.
   http://www.oecd.org/ehs/test/health.htm
  Listing of OECD guidelines for the testing of chemicals. Text of draft guidelines are available online.
   http://www.epa.gov/docs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/870-3700.pdf
  U.S. EPA Health Effects Test Guidelines, OPPTS 870.3700, Prenatal Developmental Toxicity Study.
   http://www.epa.gov/docs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Drafts/870-3500.pdf
  Health Effects Test Guidelines OPPTS 870.3500 Preliminary developmental toxicity screen “Public Draft.” U.S. EPA guidelines for the Chernoff/Kavlock assay.
   http://www.nal.usda.gov/awic/legislat/21cfr97.htm
  Good Laboratory Practice for Nonclinical Laboratory Studies.
   http://www.access.gpo.gov/nara/cfr/cfrhtml_00/Title_40/40cfr799_nav_00.html
  Electronic Code of Federal Regulations, Title 40, Chapter I, Part 799. Click on “799.9370” for the TSCA prenatal developmental toxicity guidelines.
   http://www.epa.gov/ncea/raf/pdfs/devtox.pdf
  Guidelines for Developmental Toxicity Risk Assessment (EPA, ). Provides guidance and discussion on interpreting data from developmental toxicity studies, and applying these data towards the assessment of risk to human health.
   http://cfpub.epa.gov/ncea/cfm/bmds.cfm?ActType=default
  BenchMark Dose Software, developed by the U.S. Environmental Protection Agency, can be downloaded from this Web site.
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