Prof. David KirklandKirkland 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 vivo2. Questionable carcinogens3. Probable genotoxic carcinogens4. 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 acid Toluene Caffeic acid Allyl isovalerate

Clofibrate Furosemide 3-(p-Chlorophenyl)-1-1-dimethylurea (AKA Monuron)

Benzofuran

Ethionamide Chlorobenzene Furan CI Direct Blue 15

Furfural Styrene FD&C Red 1

Isophorone Methylolacrylamide

Methapyrilene HCl 2-mercaptobenzothiazole**

Methimazole

Alpha-methylbenzyl alcohol

Methylphenidate HCl Daminozide*

Phenylbutazone * Daminozide is +ve at just >10 mM and it’s 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 acid Toluene Caffeic acid Allyl isovalerate

Clofibrate Furosemide 3-(p-Chlorophenyl)-1-1-dimethylurea (AKA Monuron)

Benzofuran

Ethionamide Chlorobenzene Furan CI Direct Blue 15

Furfural Styrene FD&C Red 1

Isophorone Methylolacrylamide

Methapyrilene HCl 2-Mercaptobenzothiazole**

Methimazole

Alpha-methylbenzyl alcohol

Methylphenidate HCl Daminozide*

Phenylbutazone * Daminozide is +ve at just >10 mM and it’s 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 (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 (Norppa et al, 1985; Jantunen et al, 1986; Pohlova et 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 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)

CHO 3+17 –S9 3+17 +S9 20+0 –S9

+ve at 2 mM(49% RPD)

+ve at 4 mM(45% RPD)

+ve at 1 mM*(54% RPD)

MLA 3 hr –S9 3 hr +S9 24 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

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0 0.1 0.2 0.3 0.4 0.5 0.6

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MF RTG

MF x 10-6 %RTG

mM

*

**

* = exceeds GEF (126 x 10-6)

Being tested in MLA Data not yet available

Treatment/recovery (hrs)

CHO 3+17 –S9 3+17 +S9 20+0 –S9

-ve up to 10 mM(Non-toxic)

-ve up to 10 mM(Non-toxic)

-ve up to 10 mM(84% RPD)

MLA 3 hr –S9 3 hr +S9 24 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 confirmedup to 10 mM.

Treatment/recovery (hrs)

CHO 3+17 –S9 3+17 +S9 20+0 –S9

-ve up to 10 mM(87% RPD)

+ve at 4 mM*(55% RPD)

-ve up to 10 mM(93% RPD)

MLA 3 hr –S9 3 hr +S9 24 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

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% cells withCA

Rel PD

% cells with CA, excl gaps %Relative PD

mM

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*

* = statistically significant, p<0.001

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MF

% RTG

MF x 10-6 %RTG.

mM

*

* *

* = MF exceeds GEF (126 x 10-6)

Treatment/recovery (hrs)

CHO 3+17 –S9 3+17 +S9 20+0 –S9

+ve at 3 mM(72% RPD)

+/- at 3 mM(49% RPD)

+ve at 2 mM*(67% RPD)

MLA 3 hr –S9 3 hr +S9 24 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/106 cells; probably clearly mutagenic

between 1 and 2 mM

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Rel PD

% cells with CA, excl gaps %Relative PD

mM

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* = statistically significant, p<0.001

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MF 3 hr -S9

MF 3 hr +S9

% RTG 3 hr -S9% RTG 3 hr+S9

MF x 10-6 %RTG.

mM

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* = Induced MF exceeds GEF

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MF

% RTG

MF x 10-6 %RTG.

mM

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* = Induced MF exceeds GEF# = IMF below GEF but indicative of response

#

Treatment/recovery (hrs)

MLA 3 hr –S9 3 hr +S9 24 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 highlevels of toxicity

Chemical Previous LEC New LEC

Allyl isovalerate 2.81 mM in MLA On-going

Benzofuran 1.27 mM in MLA Not complete; so far -ve at 10 mM in MLA (3 hr +S9)

Caffeic acid 1.11 mM in MLA +ve 0.4 mM in MLA (24 hr –S9)

Chlorobenzene 1.11 mM in MLA Not yet tested

Daminozide 13.75 mM in CA; 11.25 mM in MLA

-ve up to 10 mM (CA & MLA)

Furan 1.47 mM in CA +ve at 0.8 mM in MLA (3 hr +S9)

Methylolacrylamide 2.94 mM in CA +ve at 2 mM in CA (20 hr –S9) and MLA (24 hr –S9)

Toluene 2.44 mM in MLA -ve up to toxic doses (10% RTG) in MLA (not tested in

CA)

Monuron 6.54 mM in CA On-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 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?