Prof. David Kirkland Kirkland Consulting

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: 1.Probable non-genotoxic (non-mutagenic) carcinogens, tumour promoters or negative for genotoxicity in vivo 2.Questionable carcinogens 3.Probable genotoxic carcinogens 4.Mode of carcinogenic action unknown, in vivo genotoxicity 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

Probable non-genotoxic (non- mutagenic) carcinogens, tumour promoters or negative for genotoxicity in vivo Questionable carcinogens Probable genotoxic carcinogens Mode of carcinogenic action unknown, in vivo genotoxicity unknown or unclear Chlorendic acidTolueneCaffeic acidAllyl isovalerate ClofibrateFurosemide3-(p-Chlorophenyl)-1-1- dimethylurea (AKA Monuron) Benzofuran EthionamideChlorobenzeneFuranCI Direct Blue 15 FurfuralStyreneFD&C Red 1 IsophoroneMethylolacrylamide Methapyrilene HCl2-mercaptobenzothiazole** Methimazole Alpha-methylbenzyl alcohol Methylphenidate HClDaminozide* Phenylbutazone * Daminozide is +ve at just >10 mM and its carcinogenic mode of action is unclear ** Not included in handout

Probable non-genotoxic (non- mutagenic) carcinogens, tumour promoters or negative for genotoxicity in vivo Questionable carcinogens Probable genotoxic carcinogens Mode of carcinogenic action unknown, in vivo genotoxicity unknown or unclear Chlorendic acidTolueneCaffeic acidAllyl isovalerate ClofibrateFurosemide3-(p-Chlorophenyl)-1-1- dimethylurea (AKA Monuron) Benzofuran EthionamideChlorobenzeneFuranCI Direct Blue 15 FurfuralStyreneFD&C Red 1 IsophoroneMethylolacrylamide Methapyrilene HCl2-Mercaptobenzothiazole** Methimazole Alpha-methylbenzyl alcohol Methylphenidate HClDaminozide* Phenylbutazone * Daminozide is +ve at just >10 mM and its carcinogenic mode of action is unclear ** Not included in handout

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

CI Direct Blue 15 ( ) –an azo-dye, and is +ve in Ames with reductive or anaerobic incubation (Zeiger, 1997). FD&C Red 1 ( ) – an azo dye, +ve in Ames when Prival modification (FMN + hamster S9) was used (Cameron et al, 1987). Furosemide ( ) – MLA (NTP) +ve may be due to pH shift. CA +ve was associated with ppt and no concurrent cytotoxicity measures were included. Styrene ( ) - When activated either by red blood cells or Clophen-induced S9 (Norppa et al, 1985; Jantunen et al, 1986; Pohlova et al, 1985), styrene was +ve in the range 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 epoxide hydrolase (Scott and Preston, 1994). Usual induced S9 preparations possibly contain too much epoxide hydrolase and are not optimal.

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

Studies on-going Data not yet available

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

Treatment/recovery (hrs) CHO3+17 –S S920+0 –S9 +ve at 2 mM (49% RPD) +ve at 4 mM (45% RPD) +ve at 1 mM* (54% RPD) MLA3 hr –S93 hr +S924 hr –S9 +ve at 4 mM (7% RTG) -ve up to 6 mM (6% RTG) +ve 0.4 mM (28% RTG) * 14.5% cells with CA

MF x %RTG mM * * * * = exceeds GEF (126 x )

Being tested in MLA Data not yet available

Treatment/recovery (hrs) CHO3+17 –S S920+0 –S9 -ve up to 10 mM (Non-toxic) -ve up to 10 mM (Non-toxic) -ve up to 10 mM (84% RPD) MLA3 hr –S93 hr +S924 hr –S9 -ve up to 10 mM (62% RTG) -ve up to 10 mM (Non-toxic) -ve up to 10 mM (Non-toxic) 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.

Treatment/recovery (hrs) CHO3+17 –S S920+0 –S9 -ve up to 10 mM (87% RPD) +ve at 4 mM* (55% RPD) -ve up to 10 mM (93% RPD) MLA3 hr –S93 hr +S924 hr –S9 -ve up to 10 mM (72% RTG) +ve at 0.8 mM (16% RTG) -ve up to 10 mM (45% RTG) * 6% cells with CA

% cells with CA, excl gaps%Relative PD mM * * * * = statistically significant, p<0.001

MF x %RTG. mM * ** * = MF exceeds GEF (126 x )

Treatment/recovery (hrs) CHO3+17 –S S920+0 –S9 +ve at 3 mM (72% RPD) +/- at 3 mM (49% RPD) +ve at 2 mM* (67% RPD) MLA3 hr –S93 hr +S924 hr –S9 +ve at 3 mM (74% RTG) +ve at 4 mM (59% RTG) +ve at 2 mM** (5% RTG) * 16% cells with CA ** IMF = 584 mutants/10 6 cells; probably clearly mutagenic between 1 and 2 mM

% cells with CA, excl gaps%Relative PD mM * * * = statistically significant, p<0.001

MF x %RTG. mM * * * * * * * * * * * * = Induced MF exceeds GEF

MF x %RTG. mM * * = Induced MF exceeds GEF # = IMF below GEF but indicative of response #

Treatment/recovery (hrs) MLA3 hr –S93 hr +S924 hr –S9 -ve up to 2.8 mM (10% RTG) -ve up to 2.8 mM (7% RTG) -ve up to 3.2 mM (16% RTG) Published +ve result in MLA not confirmed even at very high levels of toxicity

ChemicalPrevious LECNew LEC Allyl isovalerate2.81 mM in MLAOn-going Benzofuran1.27 mM in MLANot complete; so far -ve at 10 mM in MLA (3 hr +S9) Caffeic acid1.11 mM in MLA+ve 0.4 mM in MLA (24 hr –S9) Chlorobenzene1.11 mM in MLANot yet tested Daminozide13.75 mM in CA; mM in MLA -ve up to 10 mM (CA & MLA) Furan1.47 mM in CA+ve at 0.8 mM in MLA (3 hr +S9) Methylolacrylamide2.94 mM in CA+ve at 2 mM in CA (20 hr –S9) and MLA (24 hr –S9) Toluene2.44 mM in MLA-ve up to toxic doses (10% RTG) in MLA (not tested in CA) Monuron6.54 mM in CAOn-going Is there any need to test above 2 mM?

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

Perhaps the most contentious of the supposed non-genotoxic chemicals The lowest +ve conc 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 +ve conc in the CA test was 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?