Analysis of published data for top concentration of cytotoxicity testing

1. Analysis of published data for top concentration of cytotoxicitytesting 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 banner’s 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 slide’s 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 Commission’s 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. European Centre for the Validation of Alternative Methods ECVAM • Validation • Research • Communication • EU Policy support • 7th Amendment to Cosmetics Directive • REACH

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

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

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

6. ECVAM Workshop on false positivesFollow 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 WorkshopD. 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. 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. Analysis of published data for top concentration of cytotoxicity in mammalian genotoxicity testing

8. Analysis of top concentration of cytotoxicitytesting in mammalian genotoxicity testing Screen Carcinogenicity Genotoxicity eXpert (CGX)* database of rodent carcinogens Consider chemicals with genotoxicity data 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. 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 Content of the databases

10. 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 General Criteria used for building up the databases

11. 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 Criteria used for interpretation of CA dataconservative approach

12. 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 Criteria used for interpretation of MLA dataApplied to most of the chemicals reported

13. 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 Considerations related to this retrospective analysis

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

15. Distribution of concentrations which produced positive results in CA and MLA

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

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

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

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

22. Chemicals that require >1mM in mammalian cell tests

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

24. Identified22 (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 let’s start discuss!! Conclusions

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