the 10 mm debate: further analysis and testing
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The 10 mM Debate: Further Analysis and Testing. Prof. David Kirkland Kirkland Consulting. Ames – ve carcinogens, only + ve in mammalian cells at >1 mM. - PowerPoint PPT PresentationTRANSCRIPT
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
0
50
100
150
200
250
300
350
400
0 0.1 0.2 0.3 0.4 0.5 0.6
0
20
40
60
80
100
120
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
0
5
10
15
20
25
30
35
0 1 4 5 10
0
20
40
60
80
100
120
% cells withCA
Rel PD
% cells with CA, excl gaps %Relative PD
mM
* *
*
* = statistically significant, p<0.001
0
50
100
150
200
250
300
350
400
450
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
0
20
40
60
80
100
120
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
0
10
20
30
40
50
60
70
80
90
0 1 2 3 10
0
20
40
60
80
100
120
% cells withCA
Rel PD
% cells with CA, excl gaps %Relative PD
mM
*
*
* = statistically significant, p<0.001
0
200
400
600
800
1000
1200
1400
1600
0 1 2 3 4 5 6 7 8 9
0
20
40
60
80
100
120
MF 3 hr -S9
MF 3 hr +S9
% RTG 3 hr -S9% RTG 3 hr+S9
MF x 10-6 %RTG.
mM
*
*
*
*
*
*
*
*
*
**
* = Induced MF exceeds GEF
0
100
200
300
400
500
600
700
0 1 2
0
20
40
60
80
100
120
MF
% RTG
MF x 10-6 %RTG.
mM
*
* = 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?