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- Questionable Probable genotoxic Mode of carcinogenic
mutagenic) carcinogens, carcinogens carcinogens action unknown, in vivo
tumour promoters or negative genotoxicity unknown or
for genotoxicity in vivo unclear
Chlorendic acid Toluene Caffeic acid Allyl isovalerate
Clofibrate Furosemide 3-(p-Chlorophenyl)-1-1- Benzofuran
dimethylurea (AKA Monuron)
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- Questionable Probable genotoxic Mode of carcinogenic
mutagenic) carcinogens, carcinogens carcinogens action unknown, in vivo
tumour promoters or negative genotoxicity unknown or
for genotoxicity in vivo unclear
Chlorendic acid Toluene Caffeic acid Allyl isovalerate
Clofibrate Furosemide 3-(p-Chlorophenyl)-1-1- Benzofuran
dimethylurea (AKA Monuron)
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 +ve at 4 mM +ve at 1 mM*
(49% RPD) (45% RPD) (54% RPD)

MLA 3 hr –S9 3 hr +S9 24 hr –S9

+ve at 4 mM -ve up to 6 mM +ve 0.4 mM
(7% RTG) (6% RTG) (28% RTG)

* 14.5% cells with CA

MF x 10-6 %RTG
400 120

350 *
* 100
300 *
80
250
MF RTG
200 60

150
40
100
20
50

0 0
0 0.1 0.2 0.3 0.4 0.5 0.6
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 -ve up to 10 mM -ve up to 10 mM
(Non-toxic) (Non-toxic) (84% RPD)

MLA 3 hr –S9 3 hr +S9 24 hr –S9

-ve up to 10 mM -ve up to 10 mM -ve up to 10 mM
(62% RTG) (Non-toxic) (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 3+17 –S9 3+17 +S9 20+0 –S9
-ve up to 10 mM +ve at 4 mM* -ve up to 10 mM
(87% RPD) (55% RPD) (93% RPD)

MLA 3 hr –S9 3 hr +S9 24 hr –S9

-ve up to 10 mM +ve at 0.8 mM -ve up to 10 mM
(72% RTG) (16% RTG) (45% RTG)

* 6% cells with CA
% cells with CA, excl gaps %Relative PD
35 120
*
30 100
25 % cells with
80 CA
20
60
Rel PD
15
40
10
* *
5 20

0 0
0 1 4 5 10
mM

* = statistically significant, p<0.001

MF x 10-6 %RTG.
450 120
400
* * 100
350
*
300 80 MF

250
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 1
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 +/- at 3 mM +ve at 2 mM*
(72% RPD) (49% RPD) (67% RPD)

MLA 3 hr –S9 3 hr +S9 24 hr –S9

+ve at 3 mM +ve at 4 mM +ve at 2 mM**
(74% RTG) (59% RTG) (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
% cells with
60 80 CA
50
60
40 Rel PD

30 40
20 * 20
10
0 0
0 1 2 3 10
mM

* = statistically significant, p<0.001

MF x 10-6 %RTG.
1600 120

1400 *
100
1200 MF 3 hr -S9
80 MF 3 hr +S9
1000

800 * * 60 % RTG 3 hr -
* * S9
% RTG 3 hr
600
* 40 +S9
* *
400 *
* 20
200 *
0 0
0 1 2 3 4 5 6 7 8 9
mM
* = Induced MF exceeds GEF
MF x 10-6 %RTG.
700 120
*
600 100
500
80 MF
400
60
300 % RTG
40
200 #

100 20

0 0
0 1 2
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 -ve up to 2.8 mM -ve up to 3.2 mM
(10% RTG) (7% RTG) (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; -ve up to 10 mM (CA & MLA)

11.25 mM in 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?