Transcript Genotoxicity

Prof. David Kirkland
Kirkland Consulting
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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
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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 (nonmutagenic) 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)-1-1dimethylurea (AKA Monuron)
Benzofuran
Ethionamide
Chlorobenzene
Furan
CI Direct Blue 15
Styrene
FD&C Red 1
Furfural
Isophorone
Methapyrilene HCl
Methylolacrylamide
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 (nonmutagenic) 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)-1-1dimethylurea (AKA Monuron)
Benzofuran
Ethionamide
Chlorobenzene
Furan
CI Direct Blue 15
Styrene
FD&C Red 1
Furfural
Isophorone
Methapyrilene HCl
Methylolacrylamide
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
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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
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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.
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Remaining 9 compounds designated for
retesting; 8 have been tested (chlorobenzene
identified too late for current programme)
Tests performed as follows:
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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
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Studies on-going
Data not yet available
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Results so far only +S9
Negative at 10 mM, which reduced RTG to
12%
Treatment/recovery (hrs)
CHO
MLA
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)
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
MF x 10-6
%RTG
400
120
350
*
*
100
*
300
80
250
200
60
150
MF
40
100
20
50
0
0
0
0.1
0.2
0.3
mM
0.4
0.5
0.6
* = exceeds GEF (126 x 10-6)
RTG
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Being tested in MLA
Data not yet available
Treatment/recovery (hrs)
CHO
MLA
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)
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 confirmed
up to 10 mM.
Treatment/recovery (hrs)
CHO
MLA
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)
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
% cells with CA, excl gaps
%Relative PD
35
120
*
30
100
25
80
% cells with
CA
20
60
15
Rel PD
40
10
*
*
5
20
0
0
0
1
4
mM
5
10
* = statistically significant, p<0.001
MF x 10-6
%RTG.
450
120
400
*
*
350
*
300
100
80
250
MF
60
200
% RTG
150
40
100
20
50
0
0
0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
mM
1
* = MF exceeds GEF (126 x 10-6)
Treatment/recovery (hrs)
CHO
MLA
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)
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
% cells with CA, excl gaps
%Relative PD
90
120
*
80
100
70
60
80
50
60
40
30
% cells with
CA
Rel PD
40
20
*
20
10
0
0
0
1
2
mM
3
10
* = statistically significant, p<0.001
MF x 10-6
%RTG.
1600
120
*
1400
100
MF 3 hr -S9
1200
80
1000
*
800
*
*
*
600
*
400
40
*
*
200
*
*
60
20
*
0
0
0
1
2
3
4
mM
5
6
7
8
9
* = Induced MF exceeds GEF
MF 3 hr +S9
% RTG 3 hr S9
% RTG 3 hr
+S9
MF x 10-6
%RTG.
700
*
600
120
100
500
80
MF
400
60
300
% RTG
40
#
200
20
100
0
0
0
1
mM
2
* = 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 high
levels 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?
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Leave the top concentration at 10 mM
◦ Lack of harmony with ICH
◦ High potential for misleading positives
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Reduce top concentration to 1 mM
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Reduce top concentration to 2 mM
◦ Harmonise with ICH
◦ Reduce frequency of misleading positives
◦ Risk a small number of false negatives (<<0.5%,
methylolacrylamide, 2-mercaptobenzothiazole,
others?)
◦ Current data indicate no false negatives
◦ Reduce frequency of misleading positives
◦ Lack of harmony with ICH
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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
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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?