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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the various test chemicals
Cross-referenceopen allclose all
Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats
Test concentrations with justification for top dose:
6 different concentrations were used; 10 mg/plate was the maximum concentration
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).
Statistics:
No data
Species / strain:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Gene mutation toxicity study was performed to evaluate the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA100, TA1535, TA1537, TA97, TA98
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers
Test concentrations with justification for top dose:
0, 10, 33, 100, 333, 1000, 1666, 3333, 6666 or 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (TA98 and TA1538; Without S9); 2-aminoanthracene (With S9; all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA100, TA1535, TA1537, TA97, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential

Table: Results for the test chemical

Dose (µg/plate)

TA100

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

97

6.4

120

4.4

179

4.6

109

14.4

168

4.4

10

94

7.0

 

 

 

 

 

 

 

 

33

96

9.9

 

 

 

 

 

 

 

 

100

98

7.2

93

4.8

170

1.9

115

3.9

178

3.5

300

91

0.9

116

11.1

185

0.9

112

3.7

191

4.4

1000

85

4.4

121

4.4

172

9.0

110

6.2

189

6.6

1666

 

 

 

 

 

 

 

 

 

 

3333

 

 

95

4.8

168

9.4

100

4.8

153

5.3

6666

 

 

86

3.8

 

 

 

 

 

 

10000

 

 

 

 

122

7.9

95

14.0

135

6.4

Positive control

383

4.7

416

43.7

593

21.6

407

21.8

614

15.8

 

Dose (µg/plate)

TA1535

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

27

4.3

9

2.2

10

1.5

7

1.8

13

0.6

10

16

2.4

 

 

 

 

 

 

 

 

33

20

3.7

 

 

 

 

10

0.9

 

 

100

17

2.2

8

0.9

12

1.5

8

1.9

14

1.3

300

10

2.4

6

0.9

11

0.0

9

2.3

11

1.3

1000

12

2.5

6

0.6

12

2.0

11

0.3

10

1.2

1666

 

 

 

 

 

 

6

1.0

 

 

3333

 

 

6

2.4

9

1.3

 

 

11s

1.2

6666

 

 

7

1.5

 

 

 

 

 

 

10000

 

 

 

 

0s

0.0

 

 

0s

0.0

Positive control

331

11.2

141

17.2

205

21.1

167

4.5

60

3.0

 

Dose (µg/plate)

TA1537

-S9

30% HLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

0

13

1.0

8

0.9

12

2.5

10

 

 

 

 

 

 

33

10

1.9

 

 

 

 

100

11

3.8

7

0.7

7

2.0

300

8

0.9

8

1.0

10

2.2

1000

5

1.3

9

0.3

10

1.2

1666

1s

1.0

 

 

 

 

3333

 

 

7

0.9

10s

3.1

6666

 

 

 

 

 

 

10000

 

 

1s

1.0

2s

1.7

Positive control

241

17.4

56

3.8

42

3.2

 

Dose (µg/plate)

TA97

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

172

10.4

171

8.6

232

5.5

197

6.4

205

7.4

10

170

3.0

 

 

 

 

 

 

 

 

33

174

7.3

 

 

 

 

188

10.8

 

 

100

167

7.1

169

5.5

206

7.9

208

3.8

219

2.9

300

160

6.5

201

10.4

201

7.8

200

3.3

220

9.6

1000

140s

18.4

182

6.2

201

9.1

186

8.2

215

12.7

1666

 

 

 

 

 

 

184

12.2

 

 

3333

 

 

166

14.3

183

8.7

 

 

142s

18.3

6666

 

 

161

10.5

 

 

 

 

 

 

10000

 

 

 

 

136s

9.1

 

 

0s

0.0

Positive control

617

90.0

406

20.2

395

1.3

465

33.2

351

5.8

 

Dose (µg/plate)

TA98

-S9

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

19

1.2

30

1.2

33

2.6

33

2.3

48

1.8

10

13

0.9

 

 

 

 

 

 

 

 

33

16

2.3

 

 

 

 

26

4.3

 

 

100

14

2.8

30

3.2

31

1.5

26

3.3

40

1.5

300

12

2.0

28

6.4

29

2.3

27

1.8

36

2.6

1000

17

3.8

28

2.2

28

3.9

19

0.7

21

3.0

1666

 

 

 

 

 

 

31

5.2

 

 

3333

 

 

23

3.9

24

3.7

 

 

14s

0.7

s: slight clearing of background lawn

Conclusions:
The test chemical did not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 10, 33, 100, 333, 1000, 1666, 3333, 6666 or 10000 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The test chemical did not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Data source

Reference
Reference Type:
review article or handbook
Title:
WoE of gene mutation in vitro toxicity study for CAS no 97-89-2
Author:
Sustainability Support Services (Europe) AB
Year:
2018
Bibliographic source:
WoE report, Sustainability Support Services (Europe) AB, 2018

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
WoE derived based on the experimental data from various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Isoamyl isovalerate
- IUPAC name: Butanoic acid, 3-methyl-, 3-methylbutyl ester
- Molecular formula: C10H20O2
- Molecular weight: 172.266 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: 96.8%
- Impurities (identity and concentrations): 4.2%

Method

Target gene:
Histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98
Remarks:
1
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537
Remarks:
2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The liver microsome fraction (S-9) was prepared from the liver of Fischer rats
Test concentrations with justification for top dose:
1. 6 different concentrations were used; 10 mg/plate was the maximum concentration
2. 0, 10, 33, 100, 333, 1000, 1666, 3333, 6666 or 10000 µg/plate
Vehicle / solvent:
1./2. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
1
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine (TA98 and TA1538; -S9) and 2-aminoanthracene (all strains; +S9)
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.
Rationale for test conditions:
No data
Evaluation criteria:
1. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

2. The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.
Statistics:
1. No data
2. The initial test of a chemical was without activation and with 10% S-9. If a positive result was obtained, the positive trial(s) was repeated. If the trials were negative the chemical was retested without S-9 and with 30% S-9. If all trials were negative, no further testing was performed.

Initial testing was in strains TA98 and TA100 without activation and with 30% rat and hamster S-9. If a positive response was obtained in one or both strains, only the positive test condition(s) was repeated. If an equivocal or weak positive response was obtained in one or both of these
strains, the corresponding plasmid-free parent strain( s), TA1535 and/or TA1538 was used to determine whether a more definitive response could be obtained. If the results with TA98 and TA100 were negative, strains TA97 and TA1535 were used without activation and with 30% S-9. If a positive response was obtained, a confirmation test was run. If a negative response was obtained in these two strains, the test was repeated using all four strains without activation and with 10% S-9.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98
Remarks:
1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium, other: TA97, TA98, TA100, TA1535, TA1537
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
1. ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.
Executive summary:

Data available for the test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Gene mutation toxicity study was also performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 10, 33, 100, 333, 1000, 1666, 3333, 6666 or 10000 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. The test chemical did not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.