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

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

460-110-3

EC Name:

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

Cas Number:

797751-43-0

Molecular formula:

not applicable, multiconstituent substance

IUPAC Name:

2,5,8-trimethyl-5-{[(propan-2-ylidene)amino]oxy}-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene; 3-methoxy-3,6-dimethyl-2,4-dioxa-5-aza-3-silahept-5-ene; 5-methoxy-2,5,8-trimethyl-4,6-dioxa-3,7-diaza-5-silanona-2,7-diene

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Wasox-MMAC2
- Physical state: Clear liquid.
- Lot/batch No.: 1000024854.
- Expiration date of the lot/batch: December 2005.
- Storage condition of test material: Storage under a nitrogen atmosphere at ambient temperature
- Other: Handling precautions: Exothermic reaction with water or humidity from the air.

Method

Target gene:

histidine-requiring gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

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

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Controls 2

Positive controls:

yes

Remarks:

(without activation)

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

TA97a: 4-nitro-o-phenylene-diamine- 10 µg

Controls 3

Positive controls:

yes

Remarks:

(with activation)

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

TA97a:7,12-dimethylbenz(a)anthracene-10 µg

Controls 4

Positive controls:

yes

Remarks:

(without activation)

Positive control substance:

2-nitrofluorene

Remarks:

TA98: 2-nitrofluorene - 2 µg

Controls 5

Positive controls:

yes

Remarks:

(without activation)

Positive control substance:

sodium azide

Remarks:

TA100: sodium azide-2 µg and TA1535: sodium azide- 1 µg

Controls 6

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

Controls 7

Positive controls:

yes

Remarks:

(with activation)

Positive control substance:

other: 1,8-Dihydroxy-anthraquinone

Remarks:

TA102: 1,8-Dihydroxy-anthraquinone - 50 µg

Controls 8

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.