1. 1 Analysis of published data for top concentration of cytotoxicity testing in mammalian genotoxicity testing J. Parry, E. Parry, P. Phrakonkham, R. Corvi In vitro Methods/European Centre for the Validation of Alternative Methods (ECVAM) IWGT, Basel, August 2009 NOTES 1. PLACE, DATE AND EVENT NAME 1.1. Access the slide-set place, date and event name text box beneath the JRC logo from the Slide Master. 1.2. Do not change the size nor the position of that text box. 1.3. Replace the mock-up texts for the place (Place), the date (dd Month YYYY) and the event name (Event Name) with your own texts. 1.4. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 1.5. Keep the original flush-left justification. 1.6. Keep the original font colour (white). 1.7. Keep the original font body size (7 pt) and the text on one single line. 2. SLIDE NUMBER 2.1. The slide number on the banners lower right-hand side is automatically generated. 3. SLIDES 3.1. Duplicate the first slide as needed. 3.2. Do not change the size nor the position of the slides text box. 3.3. Try not to place more text on each slide than will fit in the given text box. 3.4. Replace the mock-up heading text (Joint Research Centre (JRC)) with your own text heading. 3.5. Set it in Eurostile Bold Extended Two or in Helvetica Rounded Bold Condensed, if you own one of these typefaces. Otherwise, keep the original typeface – Arial. 3.6. Keep the original flush-left justification. 3.7. Keep the original font colour (100c 80m 0y 0k). 3.8. Keep the original font body size (28 pt) and the heading on one single line whenever possible. Reduce the font body size if needed. 3.9. Replace the mock-up text (The European Commissions Research-Based Policy Support Organisation)) with your own text. 3.10. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 3.11. Keep the original flush-left justification. 3.12. Keep the original font colour (100c 80m 0y 0k). Use black if you need a second colour. 3.13. Keep the original font body size (22 pt) or reduce it if unavoidable. 3.14. Replace the EU-27 map mock-up illustration with your own illustration(s). 3.13. Try to keep your illustration(s) right- and top- or bottom-aligned with the main text box whenever possible. NOTES 1. PLACE, DATE AND EVENT NAME 1.1. Access the slide-set place, date and event name text box beneath the JRC logo from the Slide Master. 1.2. Do not change the size nor the position of that text box. 1.3. Replace the mock-up texts for the place (Place), the date (dd Month YYYY) and the event name (Event Name) with your own texts. 1.4. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 1.5. Keep the original flush-left justification. 1.6. Keep the original font colour (white). 1.7. Keep the original font body size (7 pt) and the text on one single line. 2. SLIDE NUMBER 2.1. The slide number on the banners lower right-hand side is automatically generated. 3. SLIDES 3.1. Duplicate the first slide as needed. 3.2. Do not change the size nor the position of the slides text box. 3.3. Try not to place more text on each slide than will fit in the given text box. 3.4. Replace the mock-up heading text (Joint Research Centre (JRC)) with your own text heading. 3.5. Set it in Eurostile Bold Extended Two or in Helvetica Rounded Bold Condensed, if you own one of these typefaces. Otherwise, keep the original typeface – Arial. 3.6. Keep the original flush-left justification. 3.7. Keep the original font colour (100c 80m 0y 0k). 3.8. Keep the original font body size (28 pt) and the heading on one single line whenever possible. Reduce the font body size if needed. 3.9. Replace the mock-up text (The European Commissions Research-Based Policy Support Organisation)) with your own text. 3.10. Set it in MetaPlus Book Roman, if you own the typeface. Otherwise, keep the original typeface – Arial. 3.11. Keep the original flush-left justification. 3.12. Keep the original font colour (100c 80m 0y 0k). Use black if you need a second colour. 3.13. Keep the original font body size (22 pt) or reduce it if unavoidable. 3.14. Replace the EU-27 map mock-up illustration with your own illustration(s). 3.13. Try to keep your illustration(s) right- and top- or bottom-aligned with the main text box whenever possible.

2. 2 European Centre for the Validation of Alternative Methods ECVAM Validation Research Communication EU Policy support 7 th Amendment to Cosmetics Directive REACH

3. 3 CARCINOGENOMICS COMICS ECVAM key area genotoxicity / carcinogenicity Validation Reduction Policy support ( eg. REACH ITS) Research support Refine Reduce Replace Taskforce

4. 4 Genotoxicity testing requirements for REACH In vitro gene mutation study in bacteria 1t-10t In vitro study in mammalian cells (2 tests)> 10t In vivo somatic cell genotoxicity study if positive if positive classified as mutagen

5. 5 ECVAM Workshop Italy, April 2006 Objectives: 1.Review data from currently available systems 2.Review the performance of new test systems

6. 6 ECVAM Workshop on false positives Follow up activities Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: A follow-up to an ECVAM Workshop D. Kirkland, Mut Res, 653 (2008), 99-108 Collaboration with COLIPA and NC3Rs on improvement of current in vitro tests Analysis of published data for top concentration and upper limit of cytotoxicity in mammalian cell genotoxicity testing. (ECVAM commissioned this analysis to J. & L. Parry)

7. 7 Analysis of published data for top concentration of cytotoxicity in mammalian genotoxicity testing Objectives Review of existing data to determine whether concentration as 10 mM (or 5000 µg/ml) are needed to detect in vivo genotoxins and DNA- reactive, mutagenic carcinogens using in vitro mammalian cell tests or whether a lower level can be justified. Identified chemicals positive in mammalian cell tests at > 1mM Data from Ames tests were reviewed to see if chemicals would be detected in other parts of the standard battery if high concentration was needed to produce positive results in the mammalian cell tests.

8. 8 Screen Carcinogenicity Genotoxicity eXpert (CGX)* database of rodent carcinogens Consider chemicals with genotoxicity data Analysis of top concentration of cytotoxicity testing in mammalian genotoxicity testing Data for CA, MLA, MNT, SCE, Ames Focus on CA and MLA Ames Establish databases *Kirkland et al., Mutation Research 584 (2005) 1-256

9. 9 Content of the databases Whenever possible available original data were consulted in the original publications CA NTP DB Ishidate et al., 1988 Additional publications MLA Most of original papers reviewed because of concerns about criteria used for interpretation of data Mitchell et al., 1997 (for 2/3 chemicals original data reviewed) NTP DB Additional publications

10. 10 General Criteria used for building up the databases Follow the OECD and current Guidelines In cases were several publications available, most reliable were taken into consideration, the study that fulfilled current guideline criteria overruled the other studies For positive results lowest effective concentration was reported For negative and/or equivocal results highest concentration available was reported

11. 11 Criteria used for interpretation of CA data conservative approach Available raw data (i.e. NTP DB and additional publications) Criteria used in the NTP considered Ishidate et al., 1988 – Authors conclusions reported – Ishidate criteria for a positive call were considered quite conservative, therefore adequate for this analysis

12. 12 Criteria used for interpretation of MLA data Applied to most of the chemicals reported Available raw data For a positive call: –Exclusion of RTG data < 10% –Fulfil GEF –Dose-response or trend –Considered also datasets with negative controls with lower mutant frequency than currently accepted, in case other criteria were fulfilled

13. 13 Considerations related to this retrospective analysis For the purpose of this analysis as many positive compounds as possible were included –Included equivocal compounds, when clear positive results were available for those compounds –MLA positive results with a negative control with low frequency mutants A subset of data not checked in original papers Chemicals classified as carcinogens, classification as in vivo genotoxins lacking In the long term analysis can be improved dependent upon availability of further data

14. 14 Chemicals with genotoxicity data 404 CA 338 MLA 228 + 197 – 105 i 36 + 148 – 44 i 36 Overview of data for CA and MLA

15. 15 Distribution of concentrations which produced positive results in CA and MLA Positive concentration (mM) CA n° of chemicals (%) MLA n° of chemicals (%) 1139 (70.6%)110 (74.3%) 1 to 1046 (23.3%)32 (21.6%) >1012 (6.1%)6 (4.1%) Total n° of chemicals197148

16. 16 Ames data for CA positive at 1-10 mM Tot. n. chemicals = 46

17. 17 Chemicals positive at 1-10mM in CA equivocal/negative Ames mM Clofibrate1.03Methapyrilene hydrochloride2.51 3-Chloro-2-methylpropene Technical grade 1.32N-Methylolacrylamide2.94 Caffeic acid1.4Furfural3.12 Furan1.47Methimazole3.2 Phenylbutazone1.63Hydroquinone4.1 2-Mercaptobenzothiazole2.1Methylphenidate HCl4.63 Allyl isovalerate2.11Furosemide6 Ethionamide2.43-(p-Chlorophenyl)-1-1- dimethylurea (AKA monuron) 6.54 Styrene2.4alpha-Methylbenzyl alcohol8.15 Chlorendic acid2.5

18. 18 Ames data for MLA positive at 1-10 mM Tot. n. chemicals = 32

19. 19 Chemicals positive at 1-10mM in MLA equivocal/negative Ames mM Methapyrilene hydrochloride1.01FD & C red 1 (Ponceau 3R)3.44 Caffeic acid1.11Benzaldehyde3.77 Chlorobenzene1.11Chlorendic acid3.86 Benzofuran1.27Acetaldehyde3.98 C.I. Direct blue 151.51alpha-Methylbenzyl alcohol4.1 Furfural2.1Furosemide4.55 Toluene2.44Isophorone5.79 Allyl isovalerate2.81Acrylamide9.8

20. 20 Chemicals that require >1mM in mammalian cell tests (CA or MLA) for a +ve response 19 CA + 16 MLA + 22 CA or MLA + Ames -/e

21. 21 Compounds detected positive at >1mM in one test, but positive < 1mM in the other These compounds were removed from final list Benzaldehyde Acetaldehyde Acrylamide 2-chloro-2-methylpropene 2-mercaptobenzothiazole Hydroquinone

22. 22 CACA +ve (mM)MLA +ve (mM) Methapyrilene hydrochloride2.941.01 Caffeic acid1.41.11 Allyl isovalerate2.112.81 Chlorendic acid2.53.86 Furfural3.122.1 Furosemide64.55 alpha-Methylbenzyl alcohol8.154.1 Chlorobenzene1.11 Benzofuran1.27 Furan1.47 Phenylbutazone1.63 C.I. Direct blue 151.51 Ethionamide2.4 Styrene2.4 Clofibrate2.51 N-Methylolacrylamide1.03 Methimazole3.2 Isophorone5.79 Methylphenidate HCl4.63 3-(p-Chlorophenyl)-1-1-dimethylurea (AKA monuron)6.54 Toluene2.44 FD & C red 1 (Ponceau 3R)3.44 Chemicals that require >1mM in mammalian cell tests

23. 23 Initial evaluation on MNT (not yet checked) MNT 50 All MNT + at < 1mM MNT + 45 Ames + 26 Ames - 19 MNT - 5

24. 24 Conclusions Identified 22 (out of 404) chemicals that are Ames negative/e and require >1mM in mammalian cell tests (CA or MLA) for a +ve response Are these chemicals all relevant positive? Are they supposed to be picked up as genotoxic carcinogens? How many irrelevant chemicals would we avoided if top concentration reduced to 1mM? Work in progress, analysis can be improved in the future, but lets start discuss!!

25. 25 Aknowledgements James M. Parry Elizabeth Parry Pascal Phrakonkham, ECVAM David Kirkland, Kirkland Consulting