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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial gene mutation: Key study: OECD Guideline 471 and GLP. The test substance did not show any mutagenic activity.

In vitro cytogenicity in mammalian cells: Key study: OECD Guideline 473 and GLP. The test substance did not show any clastogenic activity.

In vitro gene mutation in mammalian cells: Key study: OECD Guideline 476 and GLP. Based on read-across approach from the analogue acetone oxime, Wasox-MMAC2 was determined not to be mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 11 - November 5, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD Guideline 471, with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine-requiring gene
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
other: S. typhimurium TA 97a
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
5000, 1667, 556, 185, and 62 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance hydrolyses rapidly in water
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA97a: 4-nitro-o-phenylene-diamine- 10 µg
Positive controls:
yes
Remarks:
(with activation)
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
TA97a:7,12-dimethylbenz(a)anthracene-10 µg
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
2-nitrofluorene
Remarks:
TA98: 2-nitrofluorene - 2 µg
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
sodium azide
Remarks:
TA100: sodium azide-2 µg and TA1535: sodium azide- 1 µg
Positive controls:
yes
Remarks:
(with activation)
Positive control substance:
other: 2- aminoanthracene
Remarks:
TA98:2- aminoanthracene- 1 µg , TA1535: aminoanthracene- 2 µg and TA100: aminoanthracene- 2 µg
Positive controls:
yes
Remarks:
(with activation)
Positive control substance:
other: 1,8-Dihydroxy-anthraquinone
Remarks:
TA102: 1,8-Dihydroxy-anthraquinone - 50 µg
Positive controls:
yes
Remarks:
(without activation)
Positive control substance:
other: t-Butyl-hydroperoxide
Remarks:
TA102: t-Butyl-hydroperoxide-50 µg
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation). Bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. For each sample the following solutions were combined:
- 0.1 mL of the overnight culture of the bacteria,
- 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
- 0.1 mL of the appropriate test- or reference substance solution and
- 2 mL of top agar.

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates (an independent repetition of the experiment was performed)

DETERMINATION OF CYTOTOXICITY
Method: the citotoxicity was rated as follows:
A reduced bacterial background lawn (mottled instead of homogeneous).
Microcolonies of bacteria instead of a homogeneous background lawn.
No background lawn.
Clearly reduced numbers of revertant colonies.

Evaluation criteria:
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
- For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2.5 fold of the amount of the spontaneous revertants.
- For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of the Ames test.
Key result
Species / strain:
other: TA97a, TA98, TA100, TA102, and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was seen in any on the concentrations groups

RANGE-FINDING/SCREENING STUDIES:
Preliminary experiment was performed: different concentrations of test substance solutions were mixed with phosphate buffer, bacteria (TA100) and top-agar, as described in 3.5 and spread over a plate with minimal agar. The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.

COMPARISON WITH HISTORICAL CONTROL DATA:
The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

Individual numbers of revertants per plates

 

StrainTA97a: experiment no.1

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

34

53

40

62

85

81

1667

64

61

79

128

82

88

556

72

70

59

90

111

123

185

82

70

104

139

96

116

62

61

58

58

125

88

111

solvent

41

94

66

98

82

78

solvent

70

94

70

133

102

110

positive

219

1

200

517

482

460

StrainTA97a: experiment no.2

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

166

133

136

148

128

145

1667

111

119

111

149

111

128

556

140

140

136

149

111

131

185

123

137

148

151

154

128

62

123

136

123

165

192

183

solvent

123

142

131

148

151

168

solvent

133

175

123

125

119

145

positive

465

675

569

645

526

601

solvent:     DMSO

positive:    without metabolisation: 4-Nitro-o-phenylene-diamine, 10 µg / plate

                  with metabolisation: 7,12-Dimethylbenz[a]anthracene, 10 µg / plate


StrainTA98: experiment no.1

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

9

6

9

13

9

11

1667

11

11

9

5

9

9

556

8

12

7

4

9

8

185

7

10

10

10

11

13

62

15

10

9

3

11

13

solvent

11

10

10

9

12

12

solvent

6

8

9

14

6

12

positive

155

131

10

216

227

209

StrainTA98: experiment no.2

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

8

6

8

10

4

9

1667

10

8

12

13

10

19

556

12

9

6

5

15

14

185

8

9

10

9

7

10

62

8

14

15

10

10

15

solvent

13

10

8

10

13

14

solvent

11

5

4

12

11

16

positive

343

450

306

338

273

329

solvent:     DMSO

positive:    without metabolisation: 2-Nitrofluorene, 2 µg / plate

                  with metabolisation: 2-Amino-anthracene, 1 µg / plate

 

StrainTA100:  experiment no.1

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

44

44

38

78

76

110

1667

76

67

84

114

111

98

556

76

79

93

87

85

85

185

88

84

105

84

84

84

62

93

90

101

81

93

116

solvent

73

59

88

102

101

122

solvent

66

82

102

113

108

73

positive

410

399

375

1096

1321

1081

StrainTA100: experiment no.2

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

64

47

38

56

62

59

1667

72

69

81

55

66

76

556

94

94

73

81

69

87

185

66

87

75

105

69

67

62

79

59

76

93

117

82

solvent

87

59

69

84

91

81

solvent

87

84

67

94

84

76

positive

181

149

160

469

393

375

solvent:     DMSO

positive:    without metabolisation: Sodium azide, 2 µg / plate

                  with metabolisation: 2 -Amino-anthracene, 2 µg / plate

 

StrainTA102: experiment no.1

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

139

128

146

175

261

253

1667

174

172

136

229

245

203

556

131

137

177

259

236

236

185

155

159

210

230

245

221

62

159

163

134

279

259

282

solvent

169

162

157

251

250

265

solvent

169

206

187

248

239

241

positive

629

742

634

629

588

576

StrainTA102: experiment no.2

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

216

198

213

253

236

244

1667

172

209

174

251

273

235

556

197

203

197

265

261

235

185

192

219

195

251

241

256

62

198

323

204

241

261

255

solvent

183

206

183

242

267

224

solvent

157

183

148

238

247

248

positive

975

902

666

477

442

533

solvent:     DMSO

positive:    without metabolisation: t-Butyl-hydroperoxide, 50 µg / plate

                  with metabolisation: 1,8-Dihydroxy-anthraquinone, 50 µg / plate

 

StrainTA1535: experiment no.1

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

10

11

7

9

12

6

1667

11

6

15

10

7

10

556

12

7

12

8

13

12

185

10

10

13

10

5

9

62

6

14

8

11

17

10

solvent

6

10

6

12

5

8

solvent

8

4

8

10

15

10

positive

256

229

207

181

127

175

StrainTA1535: experiment no.2

 

Concentration
µg/plate

Revertants
without metabolisation

Revertants
with metabolisation

5000

5

14

12

16

18

12

1667

11

10

15

12

11

14

556

18

12

12

8

13

7

185

16

9

7

6

6

12

62

12

15

11

16

14

11

solvent

14

18

12

18

12

11

solvent

14

18

11

10

12

13

positive

253

244

274

154

154

184

solvent:     DMSO

positive:    without metabolisation: Sodium azide, 1 µg / plate

                  with metabolisation: 2-Amino-anthracene, 2 µg / plate

Conclusions:
According to the results obtained in this study, the test substance is non-mutagenic in the Ames test with the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 5000 µg/plate.
Executive summary:

The Bacterial Reverse mutation Assay (Ames test) for the test substance Wasox-MMAC2 was performed according to OECD Guideline 471 with five histidine dependents strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535. Plate Incorporation Method was carried out at the concentrations of 5000, 1667, 556, 185, and 62 µg/plate in the presence and absence of metabolic activation. No precipitation of the test substance and no signs of cytotoxicity were noted at any dose level. In none of the concentrations tested and with none of the strains used an significant increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535 and 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results. According to the obtained results, the test substance is non-mutagenic in the Ames test with and without an external metabolizing system up to 5000 µg/plate.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 24, 2004 - March 24, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD guideline 473, with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5.000, 1.670, 0.560, and 0.185 µL/mL
Untreated negative controls:
yes
Remarks:
The culture medium
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation system
Untreated negative controls:
yes
Remarks:
The culture medium
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation system
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium. 10 mL of RPMI medium (3 hours treatment) or of complete culture medium (for 20 hours treatment) containing the test substance at the appropriate concentrations. Each culture with the use of a metabolic activation system contained 5% S9-Mix (v/v)
Evaluation criteria:
A statistically significant increase in the number of metaphases with aberrations or a concentration-relate increase in this number is considered as a positive result. However, a result can also be regarded as positive when other than merely statistical considerations, for example, the kind of aberration are taken into account.
Statistics:
The Chi2-Test (two-tailed, p<0.05) was used for the comparison between the negative control and the substance cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.
Key result
Species / strain:
lymphocytes: (3 h treatment)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 5.00 µL/mL)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: (3 h treatment)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: (20 h treatment)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(at 5.00 and 1.67 µL/mL)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
All figures were within the range of historical negative controls.

Concurrent Positive Controls

The mitotic indices were between 61.1 % and 103.8 % of the negative controls. The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system. Additional statistically significant differences to the negative controls like the number of gaps, the number of chromatid-type aberrations.

 

Mitotic index

In the test substance cultures without a metabolic activation system the mitotic indices were markedly reduced to 54.6 % of the corresponding negative controls after 3 hours of treatment with 5.00 µL/mL. After 20 hours of treatment with 5.00 µL/mL they were reduced to 6.5 % of the negative controls and to 44.9 % at 1.67 µL/mL. At all other concentrations the mitotic indices were between 70.5 and 100.0 % of those of the corresponding negative controls. In the experiments with a metabolic activation system the mitotic indices were in the test substance treated cultures between 89.7 % and 103.6 % of those of the corresponding negative controls.

 

Structural aberrations

No statistically significant increases in the number of metaphases with structural aberrations were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not. All figures were within or very close to the range of historical negative controls.

 

Gaps

No statistically significant increases in the number of gaps were noted at any concentration analysed compared to the concurrent negative controls, neither without nor with the use of a metabolic activation system. All figures were within or very close to the range of historical negative controls.

 

Numerical aberrations

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Conclusions:
Under study conditions, there was no relevant evidence that the test substance Wasox-MMAC2 did induce structural chromosomal aberrations in cultured human Iymphocytes, neither without nor with the use of a metabolic activation system.
Executive summary:

An in vitro Mammalian Chromosome Aberration Test in human lymphocytes was performed according to OECD Guideline 473 to determine possible mutagenic properties of the test substance. A total of four experiments were performed: two of them without and two with the use of a metabolic activation system. A concentration range between 5.000 and 0.185 µL of test substance per mL of medium was tested. The test substance caused marked and severe cytotoxic effects at the concentrations of 5.00 (3 and 20 h of treatment) and 1.67 (20 h of treatment) µL/mL medium when no metabolic activation system was used. There was, under the conditions of this study, no relevant evidence that "WASQX-MMAC2" did induce structural chromosomal aberrations in cultured human lymphocytes, neither without nor with the use of a metabolic activation system. The conclusion is based on a lack of a statistically significant increase of metaphases with structural aberrations in any experiment and on the finding that all figures were within the range of historical negative controls.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 10, 2012 - August 23, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to EU Method B.17 and OECD Guideline 476. GLP study.
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Rat Liver Homogenate S9 Fraction)
Test concentrations with justification for top dose:
Assay 1, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL
Assay 1, 3-hour treatment without metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL
Assay 2, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL
Assay 2, 24-hour treatment without metabolic activation: 5000; 4000; 3000; 2000; 1000; 500; 250 and 125 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the result of the preliminary solubility test the test item was insoluble in Distilled water at 500 mg/mL concentration, but it was soluble in Dimethyl sulfoxide (DMSO) at the same concentrations.
Untreated negative controls:
yes
Remarks:
Untreated control
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO 1% v/v)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.15 μg/mL (3h treatment) and 0.1 μg/mL (24h treatment) in DMSO
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
4 μg/mL in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (RPMI-5 medium)

DURATION
- Exposure duration: Assay 1: 3h (with and without metabolic activation); Assay 2: 3h (with metabolic activation), 24h (without metabolic activation)
- Expression time (cells in growth medium, to allow expresion of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival

OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies were calculated.
Evaluation criteria:
The test item was considered to be mutagenic if all the following criteria were met:
1. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency observed in treated cultures compared to the corresponding negative (solvent) control values at one or more concentrations.
3. Increases in mutation frequency reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. Significant concentration-relationship indicated by the linear trend analysis (p < 0.05).
5. Mutation frequency at the test concentration showing the largest increase at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (solvent) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: No insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment.

RANGE-FINDING/SCREENING STUDIES: No insolubility, but cytotoxicity was observed in the preliminary experiment in one case (24-hour treatment without metabolic activation). The recommended maximum concentration of 5000 μg/mL was selected as highest examined concentration in the main experiments in each case. The lower test concentrations were generally separated by factor of two. More closely spaced concentrations were used in the expected cytotoxic concentration range to cover the concentration range from the maximum cytotoxicity (resulting approximately 10-20% relative survival) to little or no cytotoxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the main test up to 5000 µg/mL.

In Assay 1 and 2, both with and without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all the six examined concentrations. No biologically relevant or statistically significant increase was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

The experiments were performed using appropriate untreated, negative (solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (solvent) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the solvent controls at the end of the expression period were accepted in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, all the validity criteria were fulfilled and the study was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.

Conclusions:
No mutagenic effect of Acetonoxim was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the analogue substance acetone oxime to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Dimethyl sulfoxide was used as the solvent of the test item in this study. The test item was examined up to 5000 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays: Assay 1, 3-hour treatment with and without metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL; Assay 2, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL and 24-hour treatment without metabolic activation: 5000; 4000; 3000; 2000; 1000; 500; 250 and 125 μg/mL In Assays 1 and 2, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment. In both assays no cytotoxicity of the test item was observed. Therefore, an evaluation was made using data of all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. All the validity criteria were fulfilled and the overall study was considered to be valid. In conclusion, no mutagenic effect of Acetonoxim was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Wasox-MMAC2 undergoes rapid hydrolysis in aqueous to acetone oxime and the corresponding silanols. These silanols undergo continuous condensation reactions to produce higher molecular weight siloxanes which are considered biologically unavailable. Therefore, the observed toxicity is likely due to the acetone oxime and their values are comparable.
See attached the reporting format and read-across rationale.
Reason / purpose for cross-reference:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Based on read-across approach, Wasox-MMAC2 was determined to be not mutagenic under test conditions.
Conclusions:
No mutagenic effect of Acetonoxim was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Based on read-across approach, Wasox-MMAC2 was determined to be not mutagenic under test conditions.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the analogue substance acetone oxime to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Dimethyl sulfoxide was used as the solvent of the test item in this study. The test item was examined up to 5000 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays: Assay 1, 3-hour treatment with and without metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL; Assay 2, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL and 24-hour treatment without metabolic activation: 5000; 4000; 3000; 2000; 1000; 500; 250 and 125 μg/mL In Assays 1 and 2, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment. In both assays no cytotoxicity of the test item was observed. Therefore, an evaluation was made using data of all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. All the validity criteria were fulfilled and the overall study was considered to be valid. In conclusion, no mutagenic effect of Acetonoxim was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. Based on these results, the read-across approach was applied and the target substance Wasox-MMAC2 was determined to be not mutagenic under test conditions.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro bacterial gene mutation: Key study: The Bacterial Reverse mutation Assay (Ames test) for the test substance Wasox-MMAC2 was performed according to OECD Guideline 471 and GLP. According to the results obtained in this study, the test substance is non-mutagenic in the Ames test to the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 with and without metabolic activation up to 5000 µg/plate.

In vitro cytogenicity in mammalian cells: Key study: In vitro Mammalian Chromosome Aberration Test in human lymphocytes was performed according to OECD Guideline 473. Under study conditions, there was no relevant evidence that the test substance Wasox-MMAC2 did induce structural chromosomal aberrations in cultured human Iymphocytes, neither without nor with the use of a metabolic activation system.

In vitro gene mutation in mammalian cells: Read-across approach from experimental results on an analogue substance: Key study: An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of acetone oxime to cause gene mutation and/or chromosome damage. The test item was determined to be non mutagenic either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Justification for classification or non-classification

Based on the available information on genetic toxicity in vitro, the substance is considered to be negative, and therefore, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.