A mixture of: propan-2-one-O,O'-(methoxymethylsilandiyl)dioxime; propan-2-one-O-(dimethoxymethylsilyl)oxime; propan-2-one-O,O',O''-(methylsilantriyl)trioxime

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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.

Reference 3

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.

Reference 4

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.