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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study contains experimental data of the registered substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted: 21st July 1997, Corrected: 26th June 2020
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-xylenol
EC Number:
208-395-3
EC Name:
2,3-xylenol
Cas Number:
526-75-0
Molecular formula:
C8H10O
IUPAC Name:
2,3-xylenol
Test material form:
other: Colorless crystalline solid or brown chunky solid
Specific details on test material used for the study:
Purity: 98.06%

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Cofactors complemented S9 homogenate obtained from phenobarbitone and β-naphthoflavone-induced rat liver
Test concentrations with justification for top dose:
0 mg/plate (negative control)
0mg/plate (vehicle control)
0.0390625 mg/plate
0.078125 mg/plate
0.15625 mg/plate
0.3125 mg/plate
0.625 mg/plate

Justification for the top dose: A preliminary cytotoxicity test was conducted with test concentrations of 0 (NC), 0 (VC, DMSO), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate and with and without S9 metabolic activation using S. typhimurium TA 98 and TA 100. No revertant colony and complete inhibition of the background lawn were observed at 5 and 2.5 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation, compared to the vehicle and negative control data. A substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 1.25 mg/plate. A moderate decrease in the revertant count and no inhibition in the background lawn was noted at 0.625 mg/plate both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. No reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.3125 to 0.039625 mg/plate, neither in the presence (10% v/v S9 mix) nor the absence of metabolic activation when compared to the vehicle control data. Based on the results of the pre-test, 0.625 mg/plate was selected as the top concentration for the main test.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test chemical is soluble in DMSO
Controls
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Nitrofluoren: TA98 (-S9) Sodium azide: TA100, TA1535 (-S9) 9-Aminoacridine: TA1537 (-S9) Mitomycin C: TA102 (-S9) Benzo[a]pyrene: TA98, TA100, TA1535, TA1537, TA102 (+S9)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: Two
Trial I: plate incorporation assay
Trial II: pre-incubation assay

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added in medium; in agar (plate incorporation); pre-incubation; in suspension; as impregnation on paper disk: In medium (plate incorporation, in suspension (pre-incubation assay)

TREATMENT AND HARVEST SCHEDULE:
- Pre-incubation period, if applicable: 20 min at 37 ± 2°C
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times):48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g., background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: cytotoxicity was determined by the reduction in revertant colony count and/or inhibition of the background lawn
- Any supplementary information relevant to cytotoxicity: NA
Rationale for test conditions:
Test Item Solubility and Precipitation:

The test item was insoluble in distilled water (50 mg/ml) and was soluble in dimethyl sulfoxide (DMSO). The precipitation test was performed by adding 100 µl of the highest test item concentration (50 mg/ml) to 2 ml of top agar and plated on to minimal glucose agar plate.
Test Item Dilution Preparation for Cytotoxicity Test
250.4 mg of the test item was dissolved in 5.008 ml of DMSO to get a stock concentration of 50 mg/ml. The stock solution was subsequently diluted using a twofold spacing factor to achieve the concentrations of 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5 and 25 mg/ml. All stock solution preparations and subsequent serial dilutions were performed inside the laminar airflow unit freshly before treatment.

Test Item Dilution Preparation of Trial I and Trial II:

In Trial I, 31.4 mg of the test item was dissolved in 5.024 ml of DMSO to get a stock concentration of 6.25 mg/ml. The stock solution was subsequently diluted with DMSO using a twofold spacing factor to achieve the concentrations of 0.390625.0.78125, 1.5625 and 3.125 mg/ml.
In Trial II, 62.5 mg of the test item was dissolved in 10 ml of DMSO to get a stock concentration of 6.25 mg/ml. The stock solution was subsequently diluted with DMSO using a twofold spacing factor to achieve the concentrations of 0.390625.0.78125, 1.5625 and 3.125 mg/ml.
All the stock solution preparations and subsequent serial dilutions of test item concentrations were performed inside the laminar airflow unit before treatment.

Sterility Check:
The sterility of the top agar, minimal glucose agar plates, S9 cofactor mix, phosphate buffer, Test Item and the vehicle were confirmed in all the study phases.
Evaluation criteria:
A test item was considered a mutagen if a biologically relevant increase in the mean number of revertants exceeded the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control was observed.
A dose-dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
A dose-dependent increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent experiment.
A dose-dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent experiment.
Statistics:
No statistics was used.

Results and discussion

Test results
Key result
Species / strain:
other:
Remarks:
TA98, TA100, TA1535, TA1537, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 0.625 mg/plate, moderate reductions in revertant colony counts were noted in TA98, TA100, TA1535, TA1537 and TA102 with no inhibition of the background lawn.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Cytotoxicity test:
A preliminary cytotoxicity test was conducted with test concentrations of 0 (NC), 0 (VC, DMSO), 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5 and 5 mg/plate in triplicates along with the negative, vehicle and positive controls using S. typhimurium TA 98 and TA 100 tester strains. The test was performed both in the presence and absence of S9 metabolic activation (liver S9 microsomal fraction was obtained from phenobarbitone and β-naphthoflavone-induced rats). No revertant colony and complete inhibition of the background lawn were observed at 5 and 2.5 mg/plate, both in the presence (10% v/v S9 mix) and absence of metabolic activation, compared to the vehicle and negative control data. A substantial reduction in the revertant count and moderate inhibition of the background lawn was observed at 1.25 mg/plate. A moderate decrease in the revertant count and no inhibition in the background lawn was noted at 0.625 mg/plate both in the presence (10% v/v S9 mix) and absence of metabolic activation compared to vehicle control data. No reduction in the revertant count and/or inhibition in the background lawn was noted at concentrations from 0.3125 to 0.039625 mg/plate, neither in the presence (10% v/v S9 mix) nor the absence of metabolic activation when compared to the vehicle control data. Based on the results of the pre-test, 0.625 mg/plate was selected as the top concentration for the main test.
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Mean Revertant Colony Count – Preliminary Cytotoxicity Assay

Test Item

Concentration

(mg/plate)

TA 98

TA 100

- S9

+ S9

- S9

+ S9

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC

(Distilled water)

21.67

(NI)

1.53

22.33

(NI)

1.53

102.67

(NI)

1.15

103.67

(NI)

5.86

VC

(Dimethyl sulfoxide)

21.67

(NI)

2.52

22.00

(NI)

1.00

105.33

(NI)

3.06

100.33

(NI)

2.08

T1

(0.0390625)

23.00

(NI)

2.00

20.67

(NI)

3.06

98.33

(NI)

4.04

98.33

(NI)

4.04

T2

(0.078125)

21.33

(NI)

1.53

21.00

(NI)

2.00

96.00

(NI)

6.24

100.33

(NI)

4.16

T3

(0.15625)

22.67

(NI)

1.53

21.67

(NI)

1.53

97.33

(NI)

2.89

97.67

(NI)

4.04

T4

(0.3125)

22.33

(NI)

1.15

19.33

(NI)

1.15

98.33

(NI)

3.21

96.67

(NI)

2.52

T5

(0.625)

17.33

(NI)

1.53

17.33

(NI)

1.15

87.33

(NI)

6.03

81.67

(NI)

2.52

T6

(1.25)

9.00

(MI)

1.73

9.67

(MI)

1.53

21.00

(MI)

4.36

22.00

(MI)

3.61

T7

(2.5)

0.00

(CI)

0.00

0.00

(CI)

0.00

0.00

(CI)

0.00

0.00

(CI)

0.00

T8

(5.0)

0.00

(CI)

0.00

0.00

(CI)

0.00

0.00

(CI)

0.00

0.00

(CI)

0.00

PC

343.00

9.85

333.00

10.15

715.67

17.62

714.00

7.94

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T8 = Test Item concentration from lower to higher.

Mean Revertant Colony Count in Trial I (Plate Incorporation Method)

Absence of
metabolic activation (-S9)

Presence of
metabolic activation (+S9 10 % v/v S9 Mix)

Test Item Concentration

(mg/plate)

TA 1535

TA 1537

TA 102

TA 1535

TA 1537

TA 102

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (Distilled water)

16.00

1.00

7.00

1.00

238.67

5.03

15.67

1.15

6.67

2.31

236.67

4.04

VC (Dimethyl sulfoxide)

17.33

1.15

6.67

1.15

236.67

7.23

15.33

0.58

7.00

1.73

237.33

6.66

T1 (0.0390625)

17.00

1.73

6.00

1.73

239.00

3.61

14.67

2.08

6.33

1.15

237.00

2.65

T2 (0.078125)

15.33

1.15

5.33

1.53

233.00

6.00

14.67

2.52

6.67

1.53

233.67

3.06

T3 (0.15625)

14.67

1.15

6.33

0.58

237.00

5.00

14.33

1.15

6.00

0.00

236.00

6.00

T4 (0.3125)

13.67

1.15

5.33

0.58

238.67

2.52

13.33

1.15

6.00

1.73

233.33

2.08

T5 (0.625)

10.67

0.58

3.33

1.15

216.00

7.21

11.33

1.53

2.67

0.58

212.33

5.86

PC

332.67

11.59

200.33

12.06

1667.33

8.62

327.67

8.02

193.67

4.51

1717.00

27.62

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation, T1-T5 = Test Item concentration from lower to higher.

Mean Revertant Colony Count in Trial II (Preincubation Method)

Absence of metabolic activation

Presence of metabolic activation (+S9 10% v/v S9 Mix)

Test Item

Concentration

(mg/plate)

TA 1535

TA 1537

TA 102

TA 98

TA 100

TA 1535

TA 1537

TA 102

TA 98

TA 100

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

NC (Distilled water)

16.33

1.53

6.33

1.15

238.00

6.56

20.7

1.5

102.67

3.21

15.67

1.53

6.00

1.73

238.33

4.04

23.00

1.73

100.33

2.08

VC (Dimethyl sulfoxide)

16.33

1.15

7.00

1.73

236.00

4.36

21.0

1.0

100.67

3.06

16.33

0.58

5.67

0.58

228.33

2.52

21.00

1.00

100.33

1.53

T1(0.0390625)

15.67

0.58

7.00

1.73

227.00

2.00

20.7

1.5

99.00

2.00

15.00

1.00

5.67

2.31

236.33

0.58

22.33

1.53

97.00

2.08

T2 (0.078125)

17.00

1.00

5.67

1.53

235.00

4.36

19.3

2.3

100.00

5.57

14.33

0.58

6.00

1.73

231.33

6.11

20.33

0.58

96.33

1.53

T3 (0.15625)

16.67

1.15

6.00

1.73

232.67

5.13

21.3

3.1

99.00

3.61

14.00

1.73

5.67

0.58

233.67

8.02

22.00

2.65

95.00

2.00

T4 (0.3125)

16.33

1.53

6.67

1.53

229.33

6.66

20.0

1.7

99.33

2.31

14.67

0.58

5.00

1.73

235.00

7.21

21.00

1.00

94.33

1.15

T5 (0.625)

12.33

1.53

3.67

1.66

213.00

4.00

16.7

0.6

83.67

3.79

10.33

0.58

3.67

0.58

212.00

2.65

16.00

1.73

83.00

1.73

PC

339.33

9.07

224.00

4.58

1714.33

33.17

346.7

7.8

732.00

7.55

332.67

8.96

217.67

8.02

1691.00

22.87

342.00

4.58

733.67

2.08

Key:     NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, SD = Standard Deviation,T1-T5 = Test Item concentration from lower to higher.

Fold increase

Trial I - Plate Incorporation Method

Trial II – Preincubation Method

Test Item

Concentration

(mg/plate)

TA 98

TA 100

TA 1535

TA 1537

TA 102

TA 98

TA 100

TA 1535

TA 1537

TA 102

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

NC (Distilled water)

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

VC (Dimethyl sulfoxide)

1.00

0.99

1.03

0.97

1.08

0.98

0.95

1.05

0.99

1.00

1.02

0.91

0.98

1.00

1.00

1.04

1.11

0.94

0.99

0.96

T1

(0.0390625)

1.06

0.94

0.93

0.98

0.98

0.96

0.90

0.90

1.01

1.00

0.98

1.06

0.98

0.97

0.96

0.92

1.00

1.00

0.96

1.04

T2

(0.078125)

0.98

0.95

0.91

1.00

0.88

0.96

0.80

0.95

0.98

0.98

0.92

0.97

0.99

0.96

1.04

0.88

0.81

1.06

1.00

1.01

T3

 (0.15625)

1.05

0.98

0.92

0.97

0.85

0.93

0.95

0.86

1.00

0.99

1.02

1.05

0.98

0.95

1.02

0.86

0.86

1.00

0.99

1.02

T4

(0.3125)

1.03

0.88

0.93

0.96

0.79

0.87

0.80

0.86

1.01

0.98

0.95

1.00

0.99

0.94

1.00

0.90

0.95

0.88

0.97

1.03

T5

(0.625)

0.80

0.79

0.83

0.81

0.62

0.74

0.50

0.38

0.91

0.89

0.79

0.76

0.83

0.83

0.76

0.63

0.52

0.65

0.90

0.93

PC

15.83

15.14

6.79

7.12

19.19

21.37

30.05

27.67

7.05

7.23

16.51

16.29

7.27

7.31

20.78

20.37

32.00

38.41

7.26

7.41

Key:   NC = Negative Control, VC = Vehicle control, PC = Positive Control, -S9 = Absence of metabolic activation, +S9 = Presence of metabolic activation, T1-T5 = Test Item concentration from lower to higher.

Applicant's summary and conclusion

Conclusions:
The registered chemical 3-dimethylphenol (CAS 526-75-0) did not induce gene mutations by base pair changes or frameshifts in the His gene of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102, neither the presence nor in the absence of S9 metabolic activation system in a study performed according to OECD TG 471.
Executive summary:

A bacterial reverse mutation assay (OECD TG 471) was performed to investigate the potential of the registered chemical i. e. 3-dimethylphenol (CAS 526-75-0), to induce gene mutations in comparison to vehicle control following the plate incorporation (Trial I) and the pre-incubation methods (Trial II) using 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 the S9 metabolic activation system (S9 liver microsomal fraction obtained fromphenobarbitone and β-naphthoflavone-induced rats).The test concentrations were selected based on the results of the preliminary cytotoxicity tests: 0.625 mg/plate was selected as the highest test concentration that produced moderate decreases in revertant colony counts in TA98 and TA100. The mutagenicity test was performed with the following test substance concentrations: 0 (NC), 0 (VC), 0.0390625, 0.078125, 0.15625,0.3125 and 0.625 mg/plate in both trials and the presence (+S9) and absence of metabolic activation (-S9). Results: No significant increase in the number of revertant colonies in the presence and absence of metabolic activation was observed at any concentrations tested compared to the vehicle control. Furthermore, no trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle controls were within the historical range. Each strain-specific positive control in both trials showed a significant increase in the number of revertant colonies. Conclusion: The registered chemical, i.e. 3-dimethylphenol (CAS 526-75-0), did not induce gene mutations by base pair changes or frameshifts in the His gene of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 neither in the presence nor in the absence of S9 metabolic activation in a study performed according to OECD TG 471.

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