1. June 2010 LANDSIEDEL 1 Chemical Industries Role in Tomorrows Toxicity Testing Robert Landsiedel, Susanne Kolle, Tzutzuy Ramirez, Hennicke Kamp and Ben van Ravenzwaay The Future of Chemical Toxicity Testing in the United States and Beyond June 21 st 2010 National Press Club, Washington DC Holeman Lounge, 13 th Floor

2. Endocrine Disruption fog Organ Toxicity sand bar Genotoxicity fog Developmental Toxicity shoal Neurotoxicit y sand bar

3. June 2010 LANDSIEDEL 3 Animal Welfare Testing Acceptance Validity Time Costs test substance use Producer’s Responsibility REACH Cosmetics Directive C&L non-EU legislation Alternative Methods Optimizing Toxicity Testing

4. June 2010 LANDSIEDEL 4 Acute Oral Toxicity Cytotoxicity vs. Rat Acute Oral Toxicity Assay: Balb/c 3T3 mouse cell line Viability assay using Neutral Red Uptake Prediction model based on correlation of acute oral rat LD 50 and cytotoxicity IC 50 Data set: 203 pairs of cytotoxicity data and acute oral data (screening or OECD 423) agrochemical active ingredients, agrochemical formulations, chemicals

5. June 2010 LANDSIEDEL 5 300 - 2000 mg/kg (cat. 4) Acute Oral Toxicity Cytotoxicity vs. Rat Acute Oral Toxicity Periodically assess the performance of methods in routine testing.

6. June 2010 LANDSIEDEL 6 CPRG CPR yeast ß-gal estrogenic activity antiestrogenic activity Yeast Estrogen/ Androgen Screening Assays (YES/YAS) Recombinant yeast strains (human receptors and reporter gene) Agonistic and antagonistic effects measureable Determination of effect by reporter assay Incubation of yeast cells with test substances Measurement of hormonal activity and cytotoxicity Reproductive Toxicity Endocrine Disruption

7. June 2010 LANDSIEDEL 7 YES/YAS negative further development positive alternative structures positive further development, but develop alternative structures focus in vivo testing  extended 28 day study  Hershberger assay  Uterotrophic assay  early Reprotox testing Reproductive Toxicity Screening Strategy negative

8. June 2010 LANDSIEDEL 8 Mode of Action Metabolomics Test substance 28 day Rat studies Metabolite profile in plasma MetaMap ® TOX  identification of relevant metabolites  correlation analysis against reference profiles  comparison against specific patterns for toxicity Identification of  target organs  modes of action  substance classes

9. June 2010 LANDSIEDEL 9 Three patterns of liver effects: rats (females left and males right) treated with liver enzyme inducers, liver toxic compounds and peroxisome proliferators relative to untreated controls Metabolomics MoA Identification

10. June 2010 LANDSIEDEL 10 Metabolomics Example: 2- and 4-Acetylaminofluorene 2-Acetylaminofluorene -strong liver enzyme inducer -liver carcinogen -immune suppressant -bladder carcinogen 4-Acetylaminofluorene -slight liver enzyme inducer -no liver carcinogen -lipid accumulation in liver -immune suppressant

11. June 2010 LANDSIEDEL 11 Metabolite profile compared to other liver enzyme inducers 2-AAF 4-AAF Metabolomics Example: 2- and 4-Acetylaminofluorene Extract more information from (regulatory) studies, to provide a sound scientific basis for grouping and read across of chemicals: from QSAR to QBAR

12. June 2010 LANDSIEDEL 12 Acute dermal toxicity: Total number of selective (relevant) classifications based on LD 50 dermal in ECB data base: < 1% Mouse cancer bioassay: The mouse does not add significantly to conclusions on cancer risk assessment (compared to rat study) 90 day and 12 month dog studies: LOEL in 90 day dog is lower than that in rats for ~50% of compounds LOEL in 90 day dog study is more than 3-fold compared to 12 month study for about 90% of compounds Redundancy in Animal Testing of Chemicals HESI ACSA Task Force

13. June 2010 LANDSIEDEL 13 New paradigmCurrent paradigmAnimals 7681272Total 4872Dogs 0520Mice 720680Rats Potential reduction in Animal Testing for Systemic Toxicity HESI ACSA Task Force Periodically assess the regulatory impact of studies, and delete those that are not used.

14. June 2010 LANDSIEDEL 14 Endocrine Disruption Organ Toxicity Genotoxicity Developmental Toxicity Neurotoxicit y Vielen Dank. Thank you.