Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-12-2018 - 21-12-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
-Substance Name: triclocarban
-Molecular Formula: C13H9Cl3N2O
-Molecular weight: 315.58 g/mol
-SMILES: Clc2ccc(NC(=O)Nc1ccc(Cl)cc1)cc2Cl
- Substance type: organic
- Physical state: solid

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
Aroclor 1254 induced S9 metabolic activation system
Test concentrations with justification for top dose:
0.0 (NC), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- 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: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data

Results and discussion

Test results
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True 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: Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions and evident to the unaided eye. Test item dissolved in DMSO at 50 mg/mL concentration was checked for precipitation. Different amounts of formulated test item preparation (50 mg/mL) were added to overlay agar (top agar) in test tubes to give various test item concentration of (maximum 5 mg/plate) and plated on minimal glucose agar (MGA) plates. Precipitation was noticed at 5 mg/plate, 3.75 mg/plate and 2.5 mg/plate concentration which were assumed to interfere with the scoring. At treatment concentration 1.25 mg/plate slight precipitation was observed which was assumed to be non-interfering with the scoring. Therefore 1.25 mg/plate was selected as highest concentration for pre-experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.000 – 1.25 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentration 1.25 (T8) mg/plate and no reduction in colony count as well as in background lawn in treated concentrations 0.396 (T7) mg/plate – 0.0003 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.0(VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The concentrations used in the experiment (pre-experiment, Trial-I, Trial-II) were placed with (√10) half log interval.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Without metabolic activation (-S9)

With metabolic activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

106

20

113

20

R2

110

19

116

18

R3

109

18

121

21

VC

(0.00)

R1

116

24

125

24

R2

120

23

121

27

R3

117

25

123

26

T1

(0.0003)

R1

102

20

110

20

R2

101

20

105

21

R3

108

21

116

18

T2

(0.001)

R1

110

18

119

16

R2

104

22

120

22

R3

102

17

117

20

T3

(0.004)

R1

106

21

119

19

R2

112

23

115

22

R3

110

17

118

21

T4

(0.013)

R1

112

16

119

19

R2

116

24

125

23

R3

116

25

118

25

T5

(0.040)

R1

104

21

122

23

R2

117

22

120

25

R3

110

24

117

21

T6

(0.125)

R1

110

20

120

25

R2

115

18

123

23

R3

114

22

122

22

T7

(0.396)

R1

116

24

117

20

R2

106

23

122

24

R3

115

18

124

21

T8

(1.25)

R1

119

21

123

27

R2

118

18

120

26

R3

115

17

123

24

PC

R1

1608

904

1600

1280

R2

1592

912

1528

1248

R3

1616

1008

1544

1328

NC          =    Negative control

VC          =  Vehicle Control

PC          =    Positive control

R             =    Replicate

T             =    Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

11

20

106

256

R2

4

9

19

110

224

R3

5

10

18

109

220

VC

(0.00)

R1

8

15

24

116

256

R2

5

16

23

120

260

R3

8

14

25

117

272

T1

(0.004)

R1

6

12

21

106

232

R2

5

11

23

112

236

R3

5

9

17

110

246

T2

(0.013)

R1

7

10

16

112

240

R2

5

13

24

116

234

R3

5

11

25

116

238

T3

(0.040)

R1

6

13

21

104

242

R2

7

11

22

117

236

R3

5

12

24

110

250

T4

(0.125)

R1

7

14

20

110

256

R2

6

15

18

115

238

R3

6

13

22

114

252

T5

(0.396)

R1

8

14

24

116

240

R2

6

13

23

106

250

R3

6

12

18

115

262

PC

R1

180

1188

904

1608

1808

R2

188

1240

912

1592

1760

R3

156

1232

1008

1616

1860

 

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

6

11

20

113

250

R2

5

11

18

116

232

R3

4

10

21

121

220

VC

(0.00)

R1

6

15

24

125

276

R2

7

14

27

121

260

R3

8

17

26

123

258

T1

(0.004)

R1

5

12

19

119

242

R2

6

11

22

115

240

R3

6

13

21

118

238

T2

(0.013)

R1

7

13

19

119

222

R2

6

12

23

125

244

R3

5

10

25

118

246

T3

(0.040)

R1

6

11

23

122

250

R2

5

15

25

120

246

R3

6

14

21

117

252

T4

(0.125)

R1

7

15

25

120

244

R2

6

14

23

123

238

R3

6

15

22

122

246

T5

(0.396)

R1

6

16

20

117

262

R2

7

14

24

122

256

R3

7

12

21

124

252

PC

R1

172

448

1280

1600

1344

R2

180

420

1248

1528

1352

R3

 190

388

1328

1544

1312

 

NC= Negative Control,VC= Vehicle Control,T =Test concentrati388on (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                                2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                               Sodium azide [10μg/plate]: TA 1535, TA 100                                                                                                                                

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose

(mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

4

10

20

110

232

R2

5

12

17

112

224

R3

5

13

20

113

240

VC

(0.00)

R1

6

12

25

125

276

R2

7

14

26

128

268

R3

8

13

23

125

250

T1

(0.004)

R1

5

12

21

110

236

R2

5

14

20

116

222

R3

6

10

22

118

240

T2

(0.013)

R1

5

12

23

119

242

R2

4

14

20

122

248

R3

6

12

22

120

230

T3

(0.040)

R1

7

13

20

119

236

R2

5

12

24

116

238

R3

5

15

25

116

234

T4

(0.125)

R1

7

14

21

117

268

R2

5

13

22

118

242

R3

6

11

21

120

244

T5

(0.396)

R1

6

12

24

121

260

R2

6

13

25

122

256

R3

7

12

23

123

258

PC

R1

188

1120

956

1088

1680

R2

200

1232

940

1272

1632

R3

192

1256

912

1128

1568

 

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

5

12

22

115

242

R2

5

11

17

107

230

R3

5

13

20

109

244

VC

(0.00)

R1

6

16

25

123

270

R2

7

13

23

136

264

R3

7

14

25

125

264

T1

(0.004)

R1

4

12

22

107

246

R2

5

12

21

105

242

R3

7

13

20

123

242

T2

(0.013)

R1

6

12

22

124

236

R2

5

13

20

120

250

R3

7

15

24

119

248

T3

(0.040)

R1

4

16

23

121

254

R2

6

13

24

124

252

R3

7

12

21

126

248

T4

(0.125)

R1

4

14

20

117

258

R2

5

13

26

117

262

R3

6

15

25

123

258

T5

(0.396)

R1

7

16

24

129

250

R2

6

11

21

124

248

R3

6

12

20

120

262

PC

R1

200

380

1280

1520

1440

R2

186

432

1344

1472

1448

R3

212

448

1360

1488

1520

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate

PC= Positive control                                                                                 2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                                 Sodium azide [10μg/plate]: TA 1535, TA 100,                                                                                                                                     

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]     Methyl methanesulfonate [4μl/plate]: TA 102

TABLE 4 -   MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.33

1.53

10.00

1.00

19.00

1.00

108.33

2.08

233.33

19.73

VC

(0.00)

7.00

1.73

15.00

1.00

24.00

1.00

117.67

2.08

262.67

8.33

T1

(0.004)

5.33

0.58

10.67

1.53

20.33

3.06

109.33

3.06

238.00

7.21

T2

(0.013)

5.67

1.15

11.33

1.53

21.67

4.93

114.67

2.31

237.33

3.06

T3

(0.040)

6.00

1.00

12.00

1.00

22.33

1.53

110.33

6.51

242.67

7.02

T4

(0.125)

6.33

0.58

14.00

1.00

20.00

2.00

113.00

2.65

248.67

9.45

T5

(0.396)

6.67

1.15

13.00

1.00

21.67

3.21

112.33

5.51

250.67

11.02

PC

174.67

16.65

1220.00

28.00

941.33

57.87

1605.33

12.22

1809.33

50.01

 

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.00

1.00

10.67

0.58

19.67

1.53

116.67

4.04

234.00

15.10

VC

(0.00)

7.00

1.00

15.33

1.53

25.67

1.53

123.00

2.00

264.67

9.87

T1

(0.004)

5.67

0.58

12.00

1.00

20.67

1.53

117.33

2.08

240.00

2.00

T2

(0.013)

6.00

1.00

11.67

1.53

22.33

3.06

120.67

3.79

237.33

13.32

T3

(0.040)

5.67

0.58

13.33

2.08

23.00

2.00

119.67

2.52

249.33

3.06

T4

(0.125)

6.33

0.58

14.67

0.58

23.33

1.53

121.67

1.53

242.67

4.16

T5

(0.396)

6.67

0.58

14.00

2.00

21.67

2.08

121.00

3.61

256.67

5.03

PC

180.67

9.02

418.67

30.02

1285.33

40.27

1557.33

37.81

1336.00

21.17

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

2-Aminoanthracene [10μg/plate]:TA 102                                            

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD
(TRIAL II)

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.67

0.58

11.67

1.53

19.00

1.73

111.67

1.53

232.00

8.00

VC

(0.00)

7.00

1.00

13.00

1.00

24.67

1.53

126.00

1.73

264.67

13.32

T1

(0.004)

5.33

0.58

12.00

2.00

21.00

1.00

114.67

4.16

232.67

9.45

T2

(0.013)

5.00

1.00

12.67

1.15

21.67

1.53

120.33

1.53

240.00

9.17

T3

(0.040)

5.67

1.15

13.33

1.53

23.00

2.65

117.00

1.73

236.00

2.00

T4

(0.125)

6.00

1.00

12.67

1.53

21.33

0.58

118.33

1.53

251.33

14.47

T5

(0.396)

6.33

0.58

12.33

0.58

24.00

1.00

122.00

1.00

258.00

2.00

PC

193.33

6.11

1202.67

72.59

936.00

22.27

1162.67

96.77

1626.67

56.19

 

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.00

0.00

12.00

1.00

19.67

2.52

110.33

4.16

238.67

7.57

VC

(0.00)

6.67

0.58

14.33

1.53

24.33

1.15

128.00

7.00

266.00

3.46

T1

(0.004)

5.33

1.53

12.33

0.58

21.00

1.00

111.67

9.87

243.33

2.31

T2

(0.013)

6.00

1.00

13.33

1.53

22.00

2.00

121.00

2.65

244.67

7.57

T3

(0.040)

5.67

1.53

13.67

2.08

22.67

1.53

123.67

2.52

251.33

3.06

T4

(0.125)

5.00

1.00

14.00

1.00

23.67

3.21

119.00

3.46

259.33

2.31

T5

(0.396)

6.33

0.58

13.00

2.65

21.67

2.08

124.33

4.51

253.33

7.57

PC

199.33

13.01

420.00

35.55

1328.00

42.33

1493.33

24.44

1469.33

44.06

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, vehicle and positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methodsi.e.Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test item with the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.