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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:
key study
Study period:
22-03-2018 - 20-04-2018
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:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of test chemical 1-phenylethan-1-ol to induce gene muta¬tions in comparison to negative 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
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-phenylethanol
EC Number:
202-707-1
EC Name:
1-phenylethanol
Cas Number:
98-85-1
Molecular formula:
C8H10O
IUPAC Name:
1-phenylethanol
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report):Methyl benzyl alcohol
- Molecular formula (if other than submission substance):C8H10O
- Molecular weight (if other than submission substance):122.166 g/mol
- Substance type:Organic- Physical state:Liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report):1-phenylethan-1-ol
- Molecular formula (if other than submission substance):C8H10O
- Molecular weight (if other than submission substance):122.166 g/mol
- Substance type:Organic- Physical state:Liquid
-Purity ; 99.8%

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:
other:
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), 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate .
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
RO water
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); positive controls are used as per OECD 471 point number 25 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9);
Remarks:
S9 is also charecterised with mutagen that requires metabolic activation by microsomal enzyme such as DMSO,Aflatoxin B1,2-Aminoanthracene ,Benzo (a) Pyrene,2 amino3,4 dimethylimindazo (4,4 Aquinoline)
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 negative 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 such an increase is not considered biologically relevant.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, 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
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 precipitation was noted at a dose upto 5 mg/plate in the 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), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 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 a clearing of the bacterial background lawn. (Concentration of the substance is as per OECD guideline 471 point number 20.)

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentration 5 mg/plate (T8) and no reduction in colony count as well as in background lawn in treated concentrations 1.582 (T7) mg/plate – 0.002 (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: 0.0158, 0.050, 0.158, 0.501, 1.582 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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

119

22

125

24

R2

115

20

123

22

R3

123

23

120

20

T1

(0.002)

R1

103

15

111

20

R2

119

17

106

18

R3

107

17

103

18

T2

(0.005)

R1

107

16

113

19

R2

105

16

109

20

R3

104

17

113

19

T3

(0.0158)

R1

107

20

117

18

R2

111

18

109

20

R3

115

21

112

21

T4

(0.050)

R1

112

20

117

19

R2

115

21

114

20

R3

109

19

109

22

T5

(0.158)

R1

114

21

117

21

R2

109

21

113

20

R3

117

20

120

22

T6

(0.501)

R1

110

21

115

21

R2

116

21

119

21

R3

112

21

120

22

T7

(1.582)

R1

115

21

118

21

R2

117

22

121

23

R3

115

21

120

21

T8

(5)

R1

109

23

119

23

R2

117

19

123

22

R3

120

20

119

23

PC

R1

1152

1020

1464

1230

R2

1140

1002

1424

1152

R3

1122

978

1480

1194

NC           =     Negative 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 Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

8

15

24

125

266

R2

7

16

22

123

284

R3

8

14

20

120

276

T1

(0.0158)

R1

4

11

18

117

224

R2

4

10

20

109

228

R3

5

10

21

112

218

T2

(0.050)

R1

5

11

19

117

224

R2

5

12

20

114

234

R3

5

13

22

109

226

T3

(0.158)

R1

6

12

21

117

240

R2

5

12

20

113

236

R3

5

13

22

120

250

T4

((0.501)

R1

6

14

21

115

246

R2

7

14

21

119

254

R3

6

13

22

120

262

T5

(1.582)

R1

7

14

21

118

276

R2

8

13

23

121

264

R3

6

15

21

120

278

PC

R1

174

446

1230

1464

1242

R2

185

414

1152

1424

1374

R3

167

346

1194

1480

1248

 

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

16

22

119

271

R2

9

14

20

115

265

R3

8

13

23

123

289

T1

(0.0158)

R1

4

10

20

107

215

R2

4

11

18

111

220

R3

4

10

21

115

226

T2

(0.050)

R1

4

11

20

112

217

R2

4

12

21

115

229

R3

5

10

19

109

231

T3

(0.158)

R1

5

12

21

114

233

R2

5

10

21

109

241

R3

6

13

20

117

244

T4

((0.501)

R1

5

12

21

110

251

R2

6

13

21

116

253

R3

6

13

21

112

261

T5

(1.582)

R1

6

14

21

115

248

R2

7

15

22

117

268

R3

8

13

21

115

274

PC

R1

176

1160

1020

1152

1872

R2

182

1236

1002

1140

1640

R3

165

1232

978

1122

1688

NC= Negative Control,T=Test concentration (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 Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

16

27

119

251

R2

7

16

28

120

263

R3

8

15

26

118

270

T1

(0.0158)

R1

4

11

20

101

234

R2

4

12

20

104

238

R3

4

11

19

108

242

T2

(0.050)

R1

5

12

22

106

236

R2

4

13

23

107

240

R3

5

12

21

110

242

T3

(0.158)

R1

5

13

22

109

250

R2

5

12

24

113

241

R3

6

14

23

107

248

T4

((0.501)

R1

6

13

24

116

245

R2

5

15

25

114

252

R3

6

15

26

117

256

T5

(1.582)

R1

7

16

26

117

262

R2

8

15

27

120

266

R3

6

15

25

119

258

PC

R1

162

340

1328

1448

1434

R2

181

430

1360

1518

1432

R3

183

376

1384

1480

1512

 

 

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

6

17

27

121

258

R2

7

16

28

119

262

R3

8

15

27

117

274

T1

(0.0158)

R1

4

10

21

104

230

R2

5

11

21

102

227

R3

4

11

20

105

244

T2

(0.050)

R1

5

12

19

111

242

R2

5

10

23

107

246

R3

4

12

21

109

262

T3

(0.158)

R1

5

13

23

112

256

R2

6

16

22

115

260

R3

4

14

22

117

248

T4

(0.501)

R1

6

15

25

114

264

R2

5

14

23

113

259

R3

6

13

24

116

264

T5

(1.582)

R1

7

15

25

119

265

R2

7

16

26

120

256

R3

6

14

27

115

268

PC

R1

182

1160

894

1128

1568

R2

176

1224

928

1086

1608

R3

180

1272

952

1164

1656

NC= Negative 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 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)

7.67

0.58

15.00

1.00

22.00

2.00

122.67

2.52

275.33

9.02

T1

(0.0158)

4.33

0.58

10.33

0.58

19.67

1.53

112.67

4.04

223.33

5.03

T2

(0.050)

5.00

0.00

12.00

1.00

20.33

1.53

113.33

4.04

228.00

5.29

T3

(0.158)

5.33

0.58

12.33

0.58

21.00

1.00

116.67

3.51

242.00

7.21

T4

(0.501)

6.33

0.58

13.67

0.58

21.33

0.58

118.00

2.65

254.00

8.00

T5

(1.582)

7.00

1.00

14.00

1.00

21.67

1.15

119.67

1.53

272.67

7.57

PC

175.33

9.07

402.00

51.07

1192.00

39.04

1456.00

28.84

1288.00

74.54

 

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)

8.00

1.00

14.33

1.53

21.67

1.53

119.00

4.00

275.00

12.49

T1

(0.0158)

4.00

0.00

10.33

0.58

19.67

1.53

111.00

4.00

220.33

5.51

T2

(0.050)

4.33

0.58

11.00

1.00

20.00

1.00

112.00

3.00

225.67

7.57

T3

(0.158)

5.33

0.58

11.67

1.53

20.67

0.58

113.33

4.04

239.33

5.69

T4

(0.501)

5.67

0.58

12.67

0.58

21.00

0.00

112.67

3.06

255.00

5.29

T5

(1.582)

7.00

1.00

14.00

1.00

21.33

0.58

115.67

1.15

263.33

13.61

PC

174.33

8.62

1209.33

42.77

1000.00

21.07

1138.00

15.10

1733.33

122.46

NC= Negative 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 Methyl methanesulfonate [4μl/plate]: TA 102

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]

 

 


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

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)

7.33

0.58

15.67

0.58

27.00

1.00

119.00

1.00

261.33

9.61

T1

(0.0158)

4.00

0.00

11.33

0.58

19.67

0.58

104.33

3.51

238.00

4.00

T2

(0.050)

4.67

0.58

12.33

0.58

22.00

1.00

107.67

2.08

239.33

3.06

T3

(0.158)

5.33

0.58

13.00

1.00

23.00

1.00

109.67

3.06

246.33

4.73

T4

(0.501)

5.67

0.58

14.33

1.15

25.00

1.00

115.67

1.53

251.00

5.57

T5

(1.582)

7.00

1.00

15.33

0.58

26.00

1.00

118.67

1.53

262.00

4.00

PC

175.33

11.59

382.00

45.30

1357.33

28.10

1482.00

35.04

1459.33

45.62

 

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)

7.00

1.00

16.00

1.00

27.33

0.58

119.00

2.00

264.67

8.33

T1

(0.0158)

4.33

0.58

10.67

0.58

20.67

0.58

103.67

1.53

233.67

9.07

T2

(0.050)

4.67

0.58

11.33

1.15

21.00

2.00

109.00

2.00

250.00

10.58

T3

(0.158)

5.00

1.00

14.33

1.53

22.33

0.58

114.67

2.52

254.67

6.11

T4

(0.501)

5.67

0.58

14.00

1.00

24.00

1.00

114.33

1.53

262.33

2.89

T5

(1.582)

6.67

0.58

15.00

1.00

26.00

1.00

118.00

2.65

263.00

6.24

PC

179.33

3.06

1218.67

56.19

924.67

29.14

1126.00

39.04

1610.67

44.06

NC= Negative 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 1-phenylethan-1-ol 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 test was performed to investigate the potential of 1-phenylethan-1-ol – (CAS No. 98-85-1) to induce gene muta­tions in comparison to negative 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 both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.0158, 0.050, 0.158, 0.501, 1.582 and 5 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), 0.0158, 0.050, 0.158, 0.501, 1.582 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 1-phenylethan-1-ol – (CAS No. 98-85-1) 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, 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 methods i.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 1-phenylethan-1-ol – (CAS No. 98-85-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.