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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

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.

Chromosome aberration study:

In vitro mammalian chromosome aberration test for the test chemical. The study was performed usingChinese hamster lung (CHL) cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of0.04-0.16 mg/mL (24 hrs; -S9), 0.04-0.24 mg/mL (48 hrs; -S9), 0.025-0.1 mg/mL (6 hrs, -S9) and 0.025-0.2 mg/mL (6 hrs, +S9). 6 hrs treatment was followed by 18 hrs recovery period. The test chemical did not induce chromosomal aberration in the Chinese hamster lung (CHL) cells and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
Histidine
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
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
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

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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 authoritative database
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro mammalian chromosome aberration test for the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
mammalian cell line, other: CHL cells
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Rat, Liver, S-9, Sodium Phenobarbital And 5,6-Benzoflavone
Test concentrations with justification for top dose:
Continuous treatment (24 hrs; -S9): 0.04-0.16 mg/mL
Continuous treatment (48 hrs; -S9): 0.04-0.24 mg/mL
Short term treatment (6 hrs, -S9): 0.025-0.1 mg/mL
Short term treatment (6 hrs, +S9): 0.025-0.2 mg/mL
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:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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: No data
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for chromosomal aberrations
Statistics:
No data
Species / strain:
mammalian cell line, other: CHL cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce chromosomal aberration in the Chinese hamster lung (CHL) cells and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

In vitro mammalian chromosome aberration test for the test chemical. The study was performed usingChinese hamster lung (CHL) cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of0.04-0.16 mg/mL (24 hrs; -S9), 0.04-0.24 mg/mL (48 hrs; -S9), 0.025-0.1 mg/mL (6 hrs, -S9) and 0.025-0.2 mg/mL (6 hrs, +S9). 6 hrs treatment was followed by 18 hrs recovery period. The test chemical did not induce chromosomal aberration in the Chinese hamster lung (CHL) cells and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo:

Gene mutation in vivo was performed for the test chemical using Drosophila melanogaster. 72-h-old larvae of Drosophila melanogaster, trans-heterozygous for the mutations multiple wing hair (mwh, 3-0.0) and flare (fir, 3-38.8), were fed the test compound (5mM (9823 mg/L)) for 48 h. Test compound was dissolved in an aqueous solution containing 3% absolute ethanol and 1% Tween 80 and used to prepare Drosophila Instant Medium (Formula 4- 24, Carolina Biological Supply Company, Burlington, NC, U.S.A.) at dose of 5mM. In order to determine the spontaneous mutation frequency, additional larvae were simultaneously exposed to the solvent only. The larvae were treated, and the wings prepared and scored for induced spots. Statistical analysis of the data was carried out as described by Frei and Würgler (1988). The test chemical is considered to be positive for inducing somatic and recombinant mutation using Drosophila melanogaster.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: gene mutation
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
Qualifier:
according to guideline
Guideline:
other: Refer below principle
Principles of method if other than guideline:
The test chemical was investigated for genotoxicity in Drosophila.
GLP compliance:
not specified
Type of assay:
somatic mutation and recombination test in Drosophila
Species:
Drosophila melanogaster
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Age at study initiation: 72 hr old larvae
- Diet (e.g. ad libitum): Drosophila Instant Medium
Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: absolute ethanol and Tween 80
- Concentration of test material in vehicle: 5mM (9823 mg/L)
- Amount of vehicle (if gavage or dermal): No data

Details on exposure:
Details on exposure
For oral route
PREPARATION OF DOSING SOLUTIONS: Test compound were dissolved in an aqueous solution containing 3% absolute ethanol and 1% Tween 80 and used to prepare Drosophila Instant Medium (Formula 4- 24, Carolina Biological Supply Company, Burlington, NC, U.S.A.).

Duration of treatment / exposure:
48 hrs
Frequency of treatment:
No data available
Post exposure period:
No data available
Remarks:
Doses / Concentrations:
5mM (9823 mg/L)
Basis:
nominal in diet
No. of animals per sex per dose:
No data
Control animals:
yes, concurrent vehicle
Positive control(s):
No data
Tissues and cell types examined:
Multiple wing hair and flare
Details of tissue and slide preparation:
No data
Evaluation criteria:
Induced spots on wings were examined acc. to Graf et al.
Statistics:
Statistical analysis of the data was carried out as described by Frei and Würgler (1988).
Sex:
not specified
Genotoxicity:
positive
Toxicity:
not specified
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
No data

Compound and concentration

Number of wings

Spots per wing and statistical diagnoses

Small single spots

m= 2.0

Large single spots

m= 5.0

Twin spots m= 5.0

Total spots m= 2.0

2,4,6-TCA

5 mM

80

0.45 +

0.08 -

0.01 i

0.54 +

Note: += positive; -= negative; i= inconclusive

Conclusions:
The test chemical is considered to be positive for inducing somatic and recombinant mutation using Drosophila melanogaster.
Executive summary:

Gene mutation in vivo was performed for the test chemical using Drosophila melanogaster. 72-h-old larvae of Drosophila melanogaster, trans-heterozygous for the mutations multiple wing hair (mwh, 3-0.0) and flare (fir, 3-38.8), were fed the test compound (5mM (9823 mg/L)) for 48 h. Test compound was dissolved in an aqueous solution containing 3% absolute ethanol and 1% Tween 80 and used to prepare Drosophila Instant Medium (Formula 4- 24, Carolina Biological Supply Company, Burlington, NC, U.S.A.) at dose of 5mM. In order to determine the spontaneous mutation frequency, additional larvae were simultaneously exposed to the solvent only. The larvae were treated, and the wings prepared and scored for induced spots. Statistical analysis of the data was carried out as described by Frei and Würgler (1988). The test chemical is considered to be positive for inducing somatic and recombinant mutation using Drosophila melanogaster.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Gene mutation in vitro:

Data available for the test chemical was reviewed to determine the mutagenic nature of 2,4,6 -trichloroaniline, 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 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.

In vitro mammalian chromosome aberration test for the test chemical. The study was performed usingChinese hamster lung (CHL) cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of0.04-0.16 mg/mL (24 hrs; -S9), 0.04-0.24 mg/mL (48 hrs; -S9), 0.025-0.1 mg/mL (6 hrs, -S9) and 0.025-0.2 mg/mL (6 hrs, +S9). 6 hrs treatment was followed by 18 hrs recovery period. The test chemical did not induce chromosomal aberration in the Chinese hamster lung (CHL) cells and hence it is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, 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, 3.0, 10, 33, 66, 100, 166, 333, 666, 1000, 3333 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 (30% RLI), TA1538 in the presence and absence of S9 metabolic activation system. It however induced gene mutation in Salmonella typhimurum strain TA100 in the presence of 30% HLI. Based on the observations made, the test chemical is not likely to classify as a gene mutant in vitro.

Salmonella/microsome test was performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537 and TA100 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO. To 4 ml of top agar at 45°C, 0.2 ml of S. typhimurium (about 4 × 108bacteria) 0.1 ml of drug in DMSO, and 1.0 ml of S9 mix were added. The resulting mixture was then dispersed rapidly with a 5-ml disposable pipette and 2 ml was plated on top of 20 ml agar in each of 2 plates. Top agar was spread by tilting the plates, allowed to harden and incubated for 40 h at 37°.The revertant count for each point was the average of duplicate plates scored on a modified Artek Model 870 Colony Counter.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1537 and TA100 in the presence and absence of S9 metabolic activation systemand hence it is not likely to classify as a gene mutant in vitro.

Suspension assay was also performed to determine the mutagenic nature of test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1537 and TA100 in the presence and absence of S9 metabolic activation system. To 0.2 ml of cells, 0.02 ml of drug in DMSO, and 1.0 ml of S9 mix were added in a 13 × 100 mm tube and incubated for 30 min at 37°C. Top agar was then added and the mixture was plated for 40 h at 37°.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1537 and TA100 in the presence and absence of S9 metabolic activation system in the suspension assay and hence it is not likely to classify as a gene mutant in vitro.

In yet another study, the hepatocyte/DNA repair test which measures unscheduled DNA synthesis (UDS) was performed to determine the genotoxicity of the test chemical using male ACI rat hepatocytes. The test was performed basically in accordance with the method of Williams et al. The test material was dissolved in DMSO, used at dose level of 10-3, 10-4, 10-5, 10-6M and the positive control used was N-2-fluorenylacetamide. The isolated hepatocytes were allowed to attach for 2 h on plastic coverslips in primary culture using Williams' Medium E. The cultures were then washed and exposed to the test chemical and [Me- 3H]thymidine (10 µCi/ml; 49 Ci/mmole) for 20 h. At the end of incubation, the cultures were washed, and the coverslips were mounted on glass slides. The slides were dipped in Sakura NR-M2 photographic emulsion and exposed for 14 days. Autoradiographic grains were counted on a television screen (Olympus, type S) with a microscopic attachment. The data were expressed as the average net counts/nucleus for 3 coverslips + the standard deviation (50 cells/coverslip). The test chemical was considered positive when the mean net nuclear grain count was more than 5 grains above background and statistically greater than that of controls. The test chemical did not induce unscheduled DNA synthesis in the rat hepatocyte/DNA repair test using male ACI rat hepatocytes and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

Gene mutation in vivo was performed for the test chemical using Drosophila melanogaster. 72-h-old larvae of Drosophila melanogaster, trans-heterozygous for the mutations multiple wing hair (mwh, 3-0.0) and flare (fir, 3-38.8), were fed the test compound (5mM (9823 mg/L)) for 48 h. Test compound was dissolved in an aqueous solution containing 3% absolute ethanol and 1% Tween 80 and used to prepare Drosophila Instant Medium (Formula 4- 24, Carolina Biological Supply Company, Burlington, NC, U.S.A.) at dose of 5mM. In order to determine the spontaneous mutation frequency, additional larvae were simultaneously exposed to the solvent only. The larvae were treated, and the wings prepared and scored for induced spots. Statistical analysis of the data was carried out as described by Frei and Würgler (1988). The test chemical is considered to be positive for inducing somatic and recombinant mutation using Drosophila melanogaster.

In vivo mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using rats. The test chemical was dissolved in DMSO and used at dose level of 0, 0.4, 4 or 40 mg/Kg/day. Rats treated orally with 2,4,6-trichloroaniline at 40 mg/kg-day (but not 0.4 or 4 mg/kg-day) for 6 months showed a small but statistically significant increase (p < 0.05) in the number of bone marrow cells containing chromosomal aberrations when compared with controls (1.6% vs. 0.4%, respectively). Based on the observations made, the given test material is positive for inducing chromosome aberrations in the bone marrow cells of rat.

However, the studies mentoined above does not give a complete description to prove the mutagenic nature of the test chemical in vivo.

Based on the observations made, the test chemical does not exibit gene mutation in vitro and in vivo. Hence it is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the observations made, the test chemical 2,4,6 -trichloroaniline (CAS no 634 -93 -5) does not exibit gene mutation in vitro and in vivo. Hence it is not likely to classify as a gene mutant in vitro and in vivo as per the criteria mentioned in CLP regulation.