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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
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

Data source

Reference
Reference Type:
publication
Title:
Salmonella Mutagenicity Tests: V. Results from the Testing of 311 Chemicals.
Author:
Errol Zeiger, Beth Anderson, Steve Haworth, Timothy Lawlor, and Kristien Mortelmans
Year:
1992
Bibliographic source:
Environmental and Molecular Mutagenesis Volume 19, Supplement 21: 2-141 (1992)

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Similar to OECD 471
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 gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4,6-trichloroaniline
EC Number:
211-219-8
EC Name:
2,4,6-trichloroaniline
Cas Number:
634-93-5
Molecular formula:
C6H4Cl3N
IUPAC Name:
2,4,6-trichloroaniline
Details on test material:
- Name of test material: 2,4,6 trichloroaniline
- Molecular formula: C6H4Cl3N
- Molecular weight: 196.464 g/mol
- Substance type: organic

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 100, TA 1535, TA97 and TA98
Remarks:
Lab 1
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:
S9 metabolic activation 10% and 30% hamster liver S9 and 10% and 30% rat liver S9
Test concentrations with justification for top dose:
Lab 1: 0, 1.0, 3.3, 10, 33, 67, 100, 200, 333, 667 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: sodium azide
Remarks:
For strain TA1535 and TA100
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
Remarks:
For strain TA97
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Remarks:
For strain TA98
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene.
Remarks:
For metabolic activation with all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 2 days
- Expression time (cells in growth medium): 2 days

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:
Mean, SEM

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 100, TA 1535, TA97 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:
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

Any other information on results incl. tables

Table: Results for the test chemical

Dose (µg/plate)

TA100

NA

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

147

7.5

102

3.5

150

8.3

121

4.5

176

6.6

1

107

5.2

 

 

 

 

 

 

 

 

3.3

120

7.8

101

6.1

 

 

115

1.5

 

 

10

115

3.4

110

3.2

147

13.7

108

9.6

178

4.9

33

115

1.5

115

3.5

141

9.4

110

3.2

162

7.4

67

 

 

119

6.3

148

15.4

115

3.7

166

5.0

100

 

 

63s

23.2

 

 

26s

22.9

 

 

200

 

 

 

 

129

5.9

 

 

6s

4.7

333

 

 

 

 

T

 

 

 

T

 

667

 

 

 

 

 

 

 

 

 

 

Positive control

444

19.3

279

10.1

658

122.3

1197

32.6

1262

30.1

 

Dose (µg/plate)

TA1535

NA

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

41

8.4

23

0.7

15

3.9

25

3.5

17

0.6

6.6

1

42

4.4

 

 

 

 

 

 

 

 

3.3

41

1.9

18

1.2

12

0.99

25

2.0

10

0.6

10

42

1.5

22

1.5

14

1.2

25

1.9

14

0.7

33

36

2.1

25

0.7

17

0.9

24

2.9

14

1.5

67

23s

5.0

 

 

 

 

 

 

 

 

100

 

 

17

3.3

23

2.1

17

2.1

18

0.9

200

 

 

6s

1.0

 

 

7s

3.5

 

 

333

 

 

 

 

17s

1.7

 

 

4s

2.5

667

 

 

 

 

 

 

 

 

 

 

Positive control

277

14.0

65

6.9

249

1.8

271

7.7

266

7.8

 

Dose (µg/plate)

TA97

NA

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

118

4.5

135

6.9

176

4.9

117

5.2

190

3.0

1

115

4.4

 

 

 

 

 

 

 

 

3.3

119

4.3

103

4.0

189

5.8

140

7.0

172

10.5

10

117

9.3

120

12.3

187

7.8

135

7.0

155

9.5

33

111

2.8

124

1.8

214

4.7

145

7.3

192

5.0

67

96s

5.5

 

 

 

 

 

 

 

 

100

 

 

153s

10.0

218

10.6

159s

5.6

199

18.4

200

 

 

91s

25.0

 

 

86s

1.0

 

 

333

 

 

 

 

146s

15.4

 

 

T

 

667

 

 

 

 

 

 

 

 

 

 

Positive control

387

72.7

1061

4.3

1341

17.0

1993

3.9

1232

71.6

 

Dose (µg/plate)

TA98

NA

10% HLI

30% HLI

10% RLI

30% RLI

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

Mean

SEM

0

24

2.0

39

0.3

28

4.3

37

5.5

29

3.3

1

20

1.5

 

 

 

 

 

 

 

 

3.3

25

1.7

39

4.8

 

 

35

2.6

 

 

10

25

2.6

41

3.8

33

4.1

37

4.6

32

3.5

33

19

1.9

37

3.8

28

2.9

36

0.9

27

0.6

67

14s

1.8

 

 

 

 

 

 

 

 

100

 

 

37

3.7

32

1.5

26

2.8

34

3.8

200

 

 

18s

3.2

 

 

14s

10.5

6s

4.7

333

 

 

 

 

19s

9.5

 

 

6s

2.8

667

 

 

 

 

T

 

 

 

T

 

Positive control

309

19.1

230

7.5

138

12.3

201

11.2

335

9.3

 

T: Toxic

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutation in Salmonella typhimurium TA98, TA100, TA1538 in the presence and absence of S9 metabolic activation system and hence 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 TA98, TA97, TA100 and TA1535 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 1.0, 3.3, 10, 33, 67, 100, 200, 333, 667 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 gene mutation in Salmonella typhimurium TA98, TA100, TA1538 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.