Current activities on Alternative Research in Japan. Hajime Kojima, Ph.D., JaCVAM, NIHS

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1 Current activities on Alternative Research in Japan Hajime Kojima, Ph.D., JaCVAM, NIHS

2 Current validation and peer review in Japan Test method Photoxicity Skin sensitization Corrocivity Skin irritation Endocrine disrupter Mutagenicity Material Yeast-RBC LLNA-DA LLNA-BrdU h-clat Culture model Culture model Lumi-cell, CER-estrogen reporter assay Comet assay (in vivo or in vitro) Current activities Peer Review in progress Validation in progress Validation in progress Pre-validation in progress Peer Review in progress Planning on Peer Review Planning on Validation Planning on Validation

3 Peer Review

4 Human skin models for skin corrosivity tests

5 Peer Review by ICCAM

6 Materials Cultured skin model Kit Name Components (Maker) EpiDerm TM 24well/kit (EPI-200 +culture medium :KURABO) EpiDerm TM Vitrolife-Skin TM (Gunze) 23well/kit +culture medium Methods Vitrolife-Skin TM Test protocol used was the blind trial of phase III in ECVAM validation*. The pre-test and main trial obtained cut-off percentage cell viability values (viability after 3 minute or 1 hour exposure). *Ref.:Liebsch, M., et al., ATLA 28, , 2000

7 Test chemicals No. Chemical 1 Potassium hydroxide:koh(10%) 2 Sulfuric acid (10%) 3 Tetrachloroethylene 4 Octanoic (Caprylic) acid 5 Potassium hydroxide:koh (5%) 6 Sodium hydroxide(4.88%) 7 4-Amino-1,2,4-triazole 8 Phosphoric acid 9 L-Lactic acid(lactic acid) 10 Isopropanol (2-propanol) 11 Phenol 12 Sodium perborate 13 Chromium trioxide *C: corrosive, NC: non-corrosive Ref.:Fentem, J. H., et al., Toxicol. In Vitro, , 1998, Liebsch, M., et al., ATLA 28, , 2000 C/NC* C C NC C NC C NC C NC NC C NC C

8 Pergormance of EpiDerm TM and Vitrolife-Skin TM EpiDerm TM Vitrolife- Skin TM EpiDerm (ECVAM) EPISKIN (ECVAM) Number of che micals Overall sensitivity 100% (12/12) 100% (12/12) 92% 82% Overall specificity 66.7% (4/6) 66.7% (4/6) 83% 84% Overall accuracy 83.3% (10/12) 83.3% (10/12) 92% 83% False positive rate 16.7% (2/12) 16.7% (2/12) 17% 16% False negative rate 0% (0/12) 0% (0/12) 8% 18%

9 Summary Reliability of these two models was similar to the results obtained on ECVAM validation. Though peer review of these models is not yet completed, the ad hoc.committee of toxicology at MHLW in Japan has approved the utilization of these models to evaluate the corrosivity of a chemical.

10 Battery System for Prediction of Phototoxicity: the Yeast Growth Inhibition Phototoxicity Assay and the Red Blood Cell Photohemolysis Assay

11 Battery System for Prediction of Phototoxicity Yeast Growth Inhibition Phototoxicity Assay UV absorption + - Positive Not testing Non phototoxicant Red Blood Cell Photohemolysis Assay Negative Positive Probably phototoxicant Negative Non phototoxicant

12 Yeast Growth Inhibition Phototoxicity Assay

13 Preparation of 2.5 % (v/v) RBC suspension Preparation of test chemical solutions by ten-, four- or five-fold dilution Preparation of completely hemolyzed solution Red Blood Cell Photohemolysis Assay Dispensation of 990 µl of RBC suspension or completely hemolyzed solution into 24-well plate Application 10 µl of test chemical solutions, solvents or PBS into 24-well plates +UV group 15.0 J/cm 2 irradiation with a solar simulator -UV group Unrradiation Dispensation of 100 µl of the irradiated and unirradiated RBCs suspension and complete hemolyzed solution into 96-well plates Measurement of the absorbances at both 540 nm and 525 nm or an adjacent wavelength with a microplate reader Classifications: Photohemolysis < 5 : - 5 Photohemolysis <10 : ± 10 Photohemolysis : +

14 Summary Battery system for prediction of phototoxicity: the yeast growth inhibition phototoxicity assay and the red blood cell photohemolysis assay was concluded to be a good method to predict the phototoxic potential of chemicals, though some improvements to the assay method are still required.

15 Validation

16 LLNA(Local lymph node assay) OECD guideline 429 (2002) Day1 2 3 Chemical application GPMT Buehler Day6 Apply 3 3 H-TdR in in vain of oftail Deduction Refinement Low cost& short term 5Hr 5Hrlater Extract Lymph node, measure of of 3 3 H-TdR uptake(cell growth)

17 LLNA-DA At At days apply of ofchemicals Before Expose 1 hr, hr, apply with with1%slssolution mouse CBA/JN 8~12w At At8 day, extract lymph node, Measure lymph node weight and andatp contents/mice(n=4)

18 Materials Lymph node weight (mg) crush and suspension ATP content Diluted X100 in PBS Bioruminescent (RLU) 20 sec. ATP content Measurement of ATP 20 sec.

19 Validation Plan of LLNA-DA Date:April-July/2006 Participated lab.:10, 2-3 tests/lab. Chemical used: Total 12, 4-6substances/Lab. 3 chemicals were examined by all 10 experimental laboratories while 9 chemicals were each tested by 3 different laboratories. Chemicals with the fixed 3 doses were distributed to each laboratory coded to disguise their type. The value of 3 was set as the cut-off point of the stimulation indices (SI), which summarize the ATP amount. Director: Dr. T. Ohmori (Kyoto Univ.) Organizer: Validation Committee in JSAAE Support: JaCVAM (Selection, coding & supply of chemical and materials )

20 Results and Discussion The results for the 3 chemicals examined by all laboratories and 5 of the other 9 chemicals were consistent and have small variances in the SI. There were 4 chemicals which produced inconsistent results between 3 laboratories. 2 chemicals showed the clearly dose response relationships. On the other hand, for the other 2 chemicals it seemed that the type of solvent in these chemicals caused the large variations. Sensitivity, specificity and concordance of the LLNA-DA compared to the GPMT/BT were 87.5% (7/8), 100% (3/3) and 90.9% (10/11), respectively. We conclude that, considering the published data of the LLNA, the results from this study are acceptable as a catch-up validation study, at least within the range of examined chemicals.

21 LLNA-BrdU Chemical application BrdU injection Collect lymph node

22 Validation Plan of LLNA-BrdU Date:October-December/2006 Participated lab.9, 2-3 tests/lab. Chemical used: Total 12, 4-6substances/Lab. Director: Dr. Hajime Kojima (JaCVAM) Organizer: Validation Committee in JSAAE Support: JaCVAM (Selection, coding & supply of chemical and materials )

23 Comparison with results obtained from alternative methods in GPMT Class in GP M Che mic als DEREK TOPKAT Peptideb in ding assay h-c LAT LLNA-Brd U LLNA- DA LLNA Cinna mic aldeh yde Positiv e Positive Positive Positive Positive Positiv e Positive Positive (5 c hamica ls) 2,4-Dinitro chlorob enzene Positiv e Positive Positive Positive Positive Positiv e Positive α-hexy lc in namic alde hyde Positiv e Positive Positive Positive Positive Positiv e Positive Formaldeh yde Positiv e Positive Positive Positive Positive Positiv e Positive p-phen ylened ia min e Positiv e Positive Positive Positive Positive Positiv e Positive Negativ e(3 c hemica ls) Lactic a cid Negativ e Negativ e Ne gative Nega tive Nega tiv e Nega tive Reso rc in ol Positiv e Positive Ne gative Positive Positiv e Nega tive S odium laury l sulfate Negativ e Positive Ne gative Nega tive Nega ti v e Positive

24 Comparison with various sensitization results Che mical name LLNA- DA LLNA GPMT/ BA HMT HPTA DNCB ppd A Trime litic an hydride Cin namic aldeh yde I soeu ge nol Eug en ol Ben zocain e + + /- + +/ - Abietic ac id HCA Citral I midazolidinyl u re a MB T CoCl NiSO Propyl paraben / - + Me thyl salicylate Ben zalkon ium ch loride

25 Current Validation Status of Reporter Gene Assays in Japanese METI and MHLW

26 Comparison with ER / subtypes Transient - transfection method for human ER and ER - Human ER / : HeLa & AUGp Reporter vector

27 Available Reporter Gene Assay in Japan -Stable- Receptor human ER human AR human TR Host Cell HeLa CHO-K1 Showed ca. 10 fold-induction at 1 nm of E 2 Completed data acquisition of 1457 compounds Showed favorable relationship as the result of comparison to Uterotrophic assay using 48 chemicals Pre-validated using 55 chemicals listed by ICCVAM Validated under multi-lab. validation in Japan (reported at 2 nd VMG-NA) Showed 10 fold-induction at 10 nm of DHT Completed data acquisition of 770 compounds Currently pre-validated and validated under multilab. validation in Japan Under Development Status

28 Available Reporter Gene Assay in Japan -Transient - Receptor human ER human ER human AR rat ER Host Cell HeLa HeLa CV-1 HeLa Status Showed ca. 10 fold-induction at 1 nm of E 2 Completed data acquisition of 300 compounds Pre-Validated Validated under multi-lab. validation in Japan Showed ca. 10 fold-induction at 1 nm of E 2 Completed data acquisition of 250 compounds Showed ca. 60 fold-induction at 10 nm of DHT Completing data acquisition of 250 compounds Currently pre-validated and validated under multi-lab. validation in Japan Showed ca. 20 fold-induction at 1 nm of E 2

29 HTSP of Human Estrogen Reporter Mediated Reporter Gene Assay Collaboratory of MHW METI Automation workstation Robot arm 96well Plate Estrogen receptor Response element (ERE) Co-Factors (CPB/P300, etc) reporter-gene Promoter CO 2 incubator Washer HeLa cells Droppe r Luminometer

30 LUMI-CELL TM Estrogen Receptor Assay Xenobiotic Detection Systems, Inc BG1Luc4E2 cell line (human ovarian carcinoma)

31 Proposal of International validation in Estrogen Receptor assay Collaboratory of MHW and METI JaCVAM HeLa cells Participated Lab. of Japanese Validation Lumi-cell assay Management Team Protocol Chemicals Lab. International Validation Validation Committee Peer Review Panel International guideline Proposal of OECD etc. Xenobiotic Detection Systems, Inc ICCVAM Lumi-cell Participated Lab. of USA Validation HeLa cell assay

32 Draft validation of Estrogen Receptor Assay Funding Sponsors National Institute of Environmental Health Sciences (NIEHS) The European Centre for the Validation of Alternative Methods (ECVAM) The Japanese Center for the Validation of Alternative Methods (JaCVAM) Study Management Team NICEATM ECVAM JaCVAM Consultation ICCVAM Endocrine Disruptor Working Group (EDWG) ECVAM Endocrine Disruptor Task Force (EDTF) OECD Validation Management Group for Non-Animal Testing (VMG- NA) her HeLa-9903 Agonist Assay U.S. Laboratory (To Be Determined) CERI (Lead Laboratory) E.U. Laboratory (To Be Determined) LUMI-CELL ER TA Agonist and Antagonist Assays XDS (Lead Laboratory) Japanese Laboratory (To Be Determined) E.U. Laboratory (To Be Determined)

33 International validation study of In vitro/in vivo Comet assay

34 Genotoxicity test DNA damage Point mutation In vitro Rec-Assay Comet assay Ames assay Mouse lymphoma T Kassay In vivo Unscheduled DNA synthesis Comet assay Utilization of Transgenic animal Chromosome Aberration Chromosome aberration assay using mammalian cells In vivo micronucleus test 5

35 Draft validation of Comet assay Study Management Team M.Hayashi (NIHS) L.Schectmann (FDA) R.Tice (NICEATM) T.Hurtung (ECVAM) Y.Uno (Mitsubishi) H.Kojima (JaCVAM) Consultation B. Burlinson (Huntington) Y. Sasaki (Hatinohe) T. Ohmori (Kyoto Univ.):Statician N. Nakajima OECD Validation Management Group (VMG) LOCAL MANAGEMENT TEAM M.Homma (NIHS) K.Morita (NIHS) T. Asano(Nittodenko) M.Nakajima (Biosafety Res. Ctr.) Y. Yamakage(Hatano, Inst.FDSC) Prevalidation of Comet Assays Biosafety Res. Ctr. Hatano, Inst., Food and Drug Safety Ctr. Huntington Life Sciences Merck BioReliance

36 Establishment of Management Team Researcher of USA, EU, Japan and Asian Secretary: JaCVAM Kick-off meeting Management team,lead Lab.(3-5Lab.) Protocol of pre-validation Pre-validation Lead Lab. 2-3 Samples(coded),protocol Revised protocol Management Team meeting* Validation Lead lab Lab. 20 samples Management Team meeting Proposal of test guideline OECD *Telephone meeting

37 Comet Assay Framework of International Validation Study - Schedule (unauthorized draft) - Pre-Validation Study* ( ) To pick up practical (technical) issues of Comet assay In limited laboratories (lead laboratories) Using limited positive/negative compounds (2 or 3) Definitive Validation Study ( ) To make basic data for preparing the OECD guideline In the expanded number of laboratories** (if possible) Using compounds (under a blind condition) * Studies will be done in accordance with recommendations of 3 rd IWGT (Environ. Mol. Mutagen., 2000) and 4 th International Comet Assay Workshop (Mutagenesis, 2003) **Participants should be limited in laboratories having enough experiences of Comet assays