1. Susan Makris USEPA, National Center for Environmental Assessment Washington, DC

2. Disclosures The views expressed in this presentation are those of the speaker and do not necessarily represent the views or policies of the U.S. EPA. I have no financial or other interests which pose a conflict of interest. No animals have been harmed in the making of this slide presentation.

3. Definition: Developmental Toxicity Adverse effects on the developing organism that may result from exposure prior to conception (to either parent), during prenatal development, or postnatally to the time of sexual maturation. Adverse developmental effects may be detected at any point in the life span of the organism. The major manifestations of developmental toxicity include: Death Altered growth Structural abnormalities Functional deficits USEPA, 1991, Developmental Toxicity Risk Assessment Guideline OECD, 2008, GD 43: Reproductive Toxicity Testing and Assessment

4. Prenatal Developmental Toxicity Study OPPTS 870.3700; OECD 414  Exposure of dams during major period of fetal organogenesis or during entire duration of gestation  Laparohysterectomy conducted immediately prior to expected day of parturition  Maternal evaluation: o Clinical observations o Body weight, food consumption, and/or water consumption o Necropsy findings  Macroscopic pathology  Ovarian corpora lutea counts  Non-reproductive organ weights are optional o Evaluation of gravid uterus  Gravid uterine weight  Implantation status Counts (live, dead, early and late resorptions, empty implantation sites) Placement in uterine horns  Examination of placental and amniotic fluid  Fetal evaluation: o Fetal sex o External examination o Visceral (soft tissue) examination o Skeletal examination

5. EPA Data Requirements for Pesticides Acute testing Acute oral, dermal & inhalation Primary eye & dermal irritation Dermal sensitization Acute neurotoxicity Subchronic testing 90-day oral, dermal, &/or inhalation 21/28-day dermal 90-day neurotoxicity Chronic testing Chronic oral Carcinogenicity Developmental toxicity and reproduction Prenatal developmental toxicity (Rodent & NonRodent ) Reproduction and fertility effects Developmental neurotoxicity Mutagenicity testing Bacterial reverse mutation assay In vitro mammalian cell assay In vivo cytogenetics Special testing Metabolism and pharmacokinetics Dermal penetration Immunotoxicity 40 CFR Part 158 (Subpart F – Toxicology) Federal Register, Vol. 72, No. 207, Oct. 26, 2007, pp.60975-76. Why are two species required for developmental toxicity testing? Thalidomide

6. Purpose of Testing - - Use of Information Broad screening of environmental agents for data that can be used in hazard evaluation and human health risk assessment Basic Components of the Risk Assessment Framework (NRC, 1983, 2009) Reference Value Derivation: RfV = NOAEL (BMD) / UF Uncertainty factors: Human variability Animal-to-human variability LOAEL to NOAEL Duration (subchronic to chronic) Data base Other, e.g., FQPA 10X Hazard Labeling: Classification and Labeling (OECD) Prop 65 (CalEPA) Critical studies for chronic RfV derivation are: Chronic Reproduction Developmental toxicity Examples

7. Basic Assumptions on Relevance of Developmental Effects to Human Health Risk for Environmental Agents An agent that produces an adverse developmental effect in experimental animals can potentially pose a developmental hazard to humans. All manifestations of developmental toxicity are of concern (i.e., death, structural abnormalities, growth alterations, functional deficits). The types of developmental effects seen in animal studies are not necessarily the same as those that may be produced in humans. The most appropriate and/or most sensitive species is used to estimate human risk. A threshold is assumed for the dose-response curve. These assumptions are: Based upon empirical data on human developmental toxicants (EPA, 1991) Considered to be plausibly conservative and protective of public health

8. Determining the Most Appropriate Species to Screen Environmental Agents for Developmental Toxicity Information needed for chemical-specific decisions on species selection: Mode/mechanism of action or adverse outcome pathway information Toxicokinetic data (maternal and fetal); internal dose The reality: These data are seldom available for environmental agents. The result: The default position is generally applied for high-exposure substances (i.e., both rodent and non-rodent studies are required). For one or more species

9. Moving Away from the Default Position The use of validated high through-put, in vitro, and alternative species data Stem cell assays, mouse limb bud assay, whole embryo culture, zebra fish, transcriptomics, in silico models, etc. Predominant current uses: Testing prioritization Weight of evidence discussions Mode of action considerations Unlikely to be a complete replacement for animal testing May help inform future decisions on the approach to testing or the most appropriate species

10. Learning from Past Experience Retrospective reviews of prenatal developmental toxicity studies conducted in multiple species for the same environmental agents. Important considerations in a cross-species retrospective review: Database used Agents tested Number of studies available Quality/adequacy of studies and reports Comparable protocols used for both rodents and non-rodents Consideration of both developmental and maternal toxicity Common quantitative metric of outcomes (e.g., LOAELs in mg/kg/day) Consideration of qualitative severity of effects

11. RIVM Analysis of EU Classified Developmental Toxicants Survey of published literature: ~60 classified developmental toxicants - - 22 chemicals with OECD 414 compliant studies (11 rat, 3 mouse, 7 rabbit, 1 macaque) Minimal Parameter Set Detecting All Compounds: Rorije et al., 2012 Conclusion: At the LOAEL, the most sensitive effects = maternal & fetal weight, fetal survival, skeletal malformations Limitation: No single chemical in this group was studied in both rodents and non-rodents

12. Rat vs. Rabbit – Environmental Chemicals Conclusions: No overall cross-species difference in sensitivity was seen for detection of developmental NOAELs. Rat and rabbit NOAELs would differ less than a factor of 14 for 95% of the substances. Limitation: NOAELs (not LOAELs) were used as the toxicity metric. Review: 54 chemicals with GHS hazard classification for developmental toxicity, each with both rat and rabbit OECD 414 type studies, derived from peer reviewed North American and European regulatory data sources. Janer et al., 2008

13. EPA ToxRef DataBase Data Extraction: 383 rat and 368 rabbit prenatal devtox studies on 387 chemicals (mostly pesticides) Conclusions: 53 of 283 chemicals (18.7%) had critical developmental effects that were either specific (i.e., with no maternal toxicity) or sensitive (dLEL<mLEL) in one or the other species. For the majority of studies developmental (and maternal) effects in rats and rabbits were within a 10-fold range. Knudsen et al., 2009

14. EPA ToxRef DataBase (cont.) Data Extraction: 383 rat and 368 rabbit prenatal devtox studies on 387 chemicals (mostly pesticides) Knudsen et al., 2009 Conclusions (cont.): Species differences in developmental response: In the rat – greater incidence of cleft palate, urogenital and somatic body wall defects, and fetal weight reduction ( ) In the rabbit – greater incidence of neurosensory, cardiovascular, and visceral body wall defects, and of pregnancy loss ( ).

15. Survey of EPA Integrated Risk Information System (IRIS) Database IRIS Data Set (www.epa.gov/IRIS/): >550 assessments (79 w/ Toxicological Review files, 69 of these w/ comparative devtox data) Life Stage Susceptibility analysis: 1)Developmental toxicity LOAEL < maternal toxicity LOAEL, and/or 2)Developmental outcome > severity compared to maternal outcome (e.g., fetal death vs. decreased maternal body weight gain) Cross-Species Sensitivity analysis: 1)Developmental toxicity LOAEL for one species < developmental LOAEL for the other, and/or 2)Developmental outcomes > severity in one species compared to the other (e.g., decreased fetal weight or structural variations vs. death or structural malformations) Conclusion: neither rodents nor non-rodents were more sensitive than the other in detecting developmental toxicity or in identifying life stage susceptibility Limitation: relatively small data set Makris and Iyer, 2012 – SOT poster

16. Species-Specific DevTox Predictive Model Sipes et al., 2011 Conclusion: while there are cross-species similarities, there are also species differences in the correlation of targets from the HTS in vitro assays with in vivo developmental toxicity. Neither the rat nor the rabbit is predictive of the other. ToxRef DataBase developmental toxicants (subset of 251 rat and 234 rabbit studies): Evaluated the correlation between in vivo toxicity and results of in vitro HTP screening data. Predictive models scored and ranked the chemicals based on in vitro assay activity.

17. Summary for Environmental Agents Prenatal developmental toxicity studies are important in screening for hazard characterization and risk assessment An evaluation in 2 species (rodent and non-rodent) is required or recommended for high exposure chemicals Sufficient data are seldom available to move away from this default position Several retrospective analyses of rat vs. rabbit studies indicate that the majority of developmental outcomes fall within a ~10-fold dose range, but species-specific responses and outliers are evident More research is needed to provide the predictive tools to depart from the default and conduct information-based testing Replace animals with alternative Reduce the number of animals used Refine the testing Research is needed to accomplish this goal 3 Rs: 4 th R:

18. Minnie Jessica Current status: For the thousands of untested industrial/environmental chemicals, alternative assays are being utilized to help prioritize testing For chemicals with a high potential for human exposure, a health- protective approach (often utilizing in vivo testing in 2 species) is retained when screening for developmental toxicity Thanks! Friends