The 10 mM Debate: Further Analysis and Testing

1. The 10 mM Debate:Further Analysis and Testing Prof. David Kirkland Kirkland Consulting

2. Ames –ve carcinogens, only +ve in mammalian cells at >1 mM • 23 carcinogens (from 404 with genotox data, i.e. 5.7%) identified that are –ve or E in Ames, yet published data indicate >1 mM needed in mammalian cells (MLA or CA) for +ve response • Grouped into 4 categories: • Probable non-genotoxic (non-mutagenic) carcinogens, tumour promoters or negative for genotoxicityin vivo • Questionable carcinogens • Probable genotoxic carcinogens • Mode of carcinogenic action unknown, in vivogenotoxicity unknown or unclear • In terms of priorities, those chemicals in groups 2, 3 & 4 are considered most important for in vitro mammalian cell tests to detect

3. The 4 categories * Daminozide is +ve at just >10 mM and it’s carcinogenic mode of action is unclear ** Not included in handout

4. The 4 categories * Daminozide is +ve at just >10 mM and it’s carcinogenic mode of action is unclear ** Not included in handout

5. 2-mercaptobenzothiazole • Identified after handout sent for printing • Weak +ve in MLA (induced MF less than GEF) at <1 mM, but only +ve in CA at 2.1 mM • Some evidence of carcinogenic activity for male F344/N rats (mononuclear cell leukemia, pancreatic acinar cell adenomas, adrenal gland pheochromocytomas, and preputial gland adenomas or carcinomas) and for female F344/N rats (adrenal gland pheochromocytomas and pituitary gland adenomas). No carcinogenic activity for male B6C3Fl mice but equivocal evidence of carcinogenic activity for female B6C3Fl mice (hepatocellular adenomas or carcinomas). • -ve for DNA adducts and –ve for MN in vivo • May need to be re-tested

6. Not priorities for further evaluation • CI Direct Blue 15 (2429-74-5) –an azo-dye, and is +ve in Ames with reductive or anaerobic incubation (Zeiger, 1997). • FD&C Red 1 (3564-09-8) – an azo dye, +ve in Ames when Prival modification (FMN + hamster S9) was used (Cameron et al, 1987). • Furosemide (54-31-9) – MLA (NTP) +ve may be due to pH shift. CA +ve was associated with ppt and no concurrent cytotoxicity measures were included. • Styrene (100-42-5) - When activated either by red blood cells or Clophen-induced S9 (Norppaet al, 1985; Jantunenet al, 1986; Pohlovaet al, 1985), styrene was +ve in the range 0.1-1 mM. The metabolic activation conditions are therefore critical to its conversion to styrene oxide and its detection as a clastogen. Also the activation by P450-dependent monooxygenases needs to exceed deactivation by epoxidehydrolase (Scott and Preston, 1994). Usual induced S9 preparations possibly contain too much epoxidehydrolase and are not optimal.

7. New tests • Remaining 9 compounds designated for retesting; 8 have been tested (chlorobenzene identified too late for current programme) • Tests performed as follows: • Ally isovalerate – CHO/CA test • Benzofuran – 24 hr MLA • Caffeic acid – CHO/CA and MLA full tests • Monuron –CHO/CA full test • Daminozide – CHO/CA and MLA full tests • Furan – CHO/CA and MLA full tests • Methylolacrylamide – CHO/CA and MLA full tests • Toluene – MLA full test

8. Allylisovalerate • Studies on-going • Data not yet available

9. Benzofuran • Results so far only +S9 • Negative at 10 mM, which reduced RTG to 12%

10. Caffeic acid * 14.5% cells with CA

11. Caffeic acid, MLA 24 hr –S9 MF x 10-6 %RTG * * * mM * = exceeds GEF (126 x 10-6)

12. Monuron • Being tested in MLA • Data not yet available

13. Daminozide – non-toxic and negative The -ve NTP result for CA upt to 10 mM has been confirmed with longer treatments and later sampling times. The variable MLA results in the NTP study have not been confirmed up to 10 mM.

14. Furan * 6% cells with CA

15. Furan, CHO/CA 3 hr +S9 % cells with CA, excl gaps %Relative PD * * * mM * = statistically significant, p<0.001

16. Furan MLA 3 hr +S9 MF x 10-6 %RTG. * * * mM * = MF exceeds GEF (126 x 10-6)

17. Methylolacrylamide * 16% cells with CA ** IMF = 584 mutants/106 cells; probably clearly mutagenic between 1 and 2 mM

18. Methylolacrylamide, CHO/CA 20 hr -S9 % cells with CA, excl gaps %Relative PD * * mM * = statistically significant, p<0.001

19. Methylolacrylamide MLA, 3 hr – or + S9 MF x 10-6 %RTG. * * * * * * * * * * * mM * = Induced MF exceeds GEF

20. Methylolacrylamide MLA, 24 hr –S9 MF x 10-6 %RTG. * # mM * = Induced MF exceeds GEF # = IMF below GEF but indicative of response

21. Toluene – toxic but negative Published +ve result in MLA not confirmed even at very high levels of toxicity

22. Summary of new test results Is there any need to test above 2 mM?

23. The choices • Leave the top concentration at 10 mM • Lack of harmony with ICH • High potential for misleading positives • Reduce top concentration to 1 mM • Harmonise with ICH • Reduce frequency of misleading positives • Risk a small number of false negatives (<<0.5%, methylolacrylamide, 2-mercaptobenzothiazole, others?) • Reduce top concentration to 2 mM • Current data indicate no false negatives • Reduce frequency of misleading positives • Lack of harmony with ICH

24. Back-up slides

25. Furfural • Perhaps the most contentious of the supposed non-genotoxic chemicals • The lowest +veconc in the MLA (NTP) was 200 µg/ml +S9 and 400 µg/ml –S9 (2.1 or 4.2 mM). However, treatments were only for 3 hrs. Would this be +ve –S9 at lower concs if treated over 24 hr? • Reduction in LEC seen for several compounds in latest tests • Similarly the lowest +veconc in the CA test was 300-400 µg/ml (3-4 mM), but NTP protocols used short treatments and early sampling times. Would this be +ve at lower concs if treated for longer and sampled later?