3-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]- 2-hydroxypropyl methacrylate and 1-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-3-hydroxypropan-2-yl methacrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 18 June 2008 and 08 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

10-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-11-yl 2-methylprop-2-enoate; 11-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-10-yl 2-methylprop-2-enoate

EC Number:

700-334-3

Molecular formula:

C17H38O6Si3

IUPAC Name:

10-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-11-yl 2-methylprop-2-enoate; 11-hydroxy-2,2,4-trimethyl-4-[(trimethylsilyl)oxy]-3,8-dioxa-2,4-disilaundecan-10-yl 2-methylprop-2-enoate

Test material form:

other: liquid

Details on test material:

identification: SiMAA2
Description : Clear colourless liquid
Lot number : 003068
Date received : 25 March 2008
Storage conditions: Approximately 4°C in the dark

Method

Target gene:

Histidine (for Salmonell), tryptophan (for E.coli).

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta-naphthoflavone induced rat liver (S9 mix)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation Test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

ENNG, 9AA, 4NQO used without S9 mix. BP and 2AA used with S9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 48 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours


NUMBER OF REPLICATIONS: Triplicate plates


NUMBER OF CELLS EVALUATED: 10-9 cells per ml


DETERMINATION OF CYTOTOXICITY
- Method: other: Evalaution of background lawn, frequency of revertant colonies.

Evaluation criteria:

There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation.  Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity.  Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

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

Test results 2

Species / strain:

E. coli WP2 uvr A

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:

- Precipitation: An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA^-).  The test material formulation and S9-mix used in this experiment were both shown to be sterile. The number of revertant colonies for the toxicity assay were;

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	79	72	81	80	99	77	89	77	80	112	72P
+	TA100	70	81	67	73	73	76	82	89	61	73	80P
-	WP2uvrA^-	29	20	28	29	27	21	17	22	23	20	36P
+	WP2uvrA^-	28	21	23	22	25	32	28	35	34	31	42P

Mutation test:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).  The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable.  These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1:Spontaneous Mutation Rates (Concurrent Negative Controls)

Range finding test: Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
67		15		30		13		7
79	(79)	12	(14)	23	(24)	19	(17)	7	(9)
90		15		19		18		13

Main test: Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
117		20		29		18		7
129	(130)	22	(20)	34	(29)	15	(17)	6	(6)
143		19		25		17		5

Table 2: Range finding test-without metabolic activation.

Test Period		From: 28 June 2008					To: 01 July 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	86 70 70	(75) 9.2#	18 21 18	(19) 1.7	30 34 25		(30) 4.5	18 19 11	(16) 4.4	14 15 11		(13) 2.1
-	50	71 75 70	(72) 2.6	23 16 16	(18) 4.0	19 41 24		(28) 11.5	24 20 12	(19) 6.1	13 12 14		(13) 1.0
-	150	70 71 71	(71) 0.6	16 23 16	(18) 4.0	23 20 24		(22) 2.1	20 16 22	(19) 3.1	15 12 13		(13) 1.5
-	500	71 79 79	(76) 4.6	18 19 21	(19) 1.5	24 29 20		(24) 4.5	15 19 13	(16) 3.1	12 11 13		(12) 1.0
-	1500	87 70 71	(76) 9.5	19 13 25	(19) 6.0	19 31 30		(27) 6.7	15 20 16	(17) 2.6	14 13 12		(13) 1.0
-	5000	79 P 75 P 78 P	(77) 2.1	20 P 24 P 19 P	(21) 2.6	31 P 24 P 20 P		(25) 5.6	13 P 15 P 18 P	(15) 2.5	11 P 14 P 12 P		(12) 1.5
Positive controls S9-Mix -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		514 556 511	(527) 25.2	255 151 150	(185) 60.3	716 668 647		(677) 35.4	222 203 218	(214) 10.0	2113 2210 1974	(2099) 118.6

Table 3: Range finding test-with metabolic activation.

Test Period		From: 28 June 2008					To: 01 July 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	71 74 74	(73) 1.7#	11 14 14	(13) 1.7	30 38 30		(33) 4.6	20 19 18	(19) 1.0	16 15 11	(14) 2.6
+	50	71 70 71	(71) 0.6	13 10 10	(11) 1.7	33 31 27		(30) 3.1	19 20 18	(19) 1.0	15 11 12	(13) 2.1
+	150	71 70 76	(72) 3.2	13 13 10	(12) 1.7	23 33 22		(26) 6.1	14 16 16	(15) 1.2	14 11 13	(13) 1.5
+	500	78 73 76	(76) 2.5	12 11 12	(12) 0.6	30 31 35		(32) 2.6	16 20 16	(17) 2.3	11 14 12	(12) 1.5
+	1500	73 77 81	(77) 4.0	11 12 12	(12) 0.6	29 31 35		(32) 3.1	19 16 18	(18) 1.5	13 10 13	(12) 1.7
+	5000	74 P 73 P 79 P	(75) 3.2	13 P 13 P 12 P	(13) 0.6	32 P 26 P 36 P		(31) 5.0	20 P 20 P 15 P	(18) 2.9	13 P 16 P 14 P	(14) 1.5
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1306 1178 1426	(1303) 124.0	277 376 345	(333) 50.6	299 352 293		(315) 32.5	511 387 483	(460) 65.0	260 398 389	(349) 77.2

Table 4: Maint test- without metabolic activation:

Test Period			From: 05 July 2008						To: 08 July 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
-	0	95 99 98	(97) 2.1#	20 14 20	(18) 3.5	22 14 25		(20) 5.7	26 22 22	(23) 2.3	5 11 3		(6) 4.2
-	50	84 93 125	(101) 21.5	13 24 18	(18) 5.5	31 30 16		(26) 8.4	21 16 34	(24) 9.3	12 4 7		(8) 4.0
-	150	112 108 124	(115) 8.3	8 21 9	(13) 7.2	30 24 16		(23) 7.0	19 25 24	(23) 3.2	10 4 5		(6) 3.2
-	500	99 104 93	(99) 5.5	15 11 7	(11) 4.0	29 31 23		(28) 4.2	18 26 21	(22) 4.0	9 4 10		(8) 3.2
-	1500	119 100 103	(107) 10.2	19 16 9	(15) 5.1	36 20 30		(29) 8.1	20 18 18	(19) 1.2	3 7 7		(6) 2.3
-	5000	99 P 97 P 133 P	(110) 20.2	13 P 10 P 21 P	(15) 5.7	27 P 21 P 20 P		(23) 3.8	24 P 15 P 18 P	(19) 4.6	2 P 7 P 4 P		(4) 2.5
Positive controls S9-Mix -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		539 547 509	(532) 20.0	264 196 214	(225) 35.2	709 818 823		(783) 64.4	178 136 183	(166) 25.8	997 970 1262	(1076) 161.4

Table 5: Main test-with metabolic activation:

Test Period		From: 05 July 2008					To: 08 July 2008
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA‑			TA98		TA1537
+	0	91 108 114	(104) 11.9#	9 13 13	(12) 2.3	23 35 25		(28) 6.4	14 18 21	(18) 3.5	9 10 8	(9) 1.0
+	50	123 92 97	(104) 16.6	21 22 10	(18) 6.7	32 29 42		(34) 6.8	18 18 19	(18) 0.6	10 4 10	(8) 3.5
+	150	84 88 119	(97) 19.2	13 13 19	(15) 3.5	29 23 40		(31) 8.6	13 22 19	(18) 4.6	10 7 8	(8) 1.5
+	500	107 95 103	(102) 6.1	13 15 7	(12) 4.2	29 29 29		(29) 0.0	31 19 21	(24) 6.4	13 8 12	(11) 2.6
+	1500	101 99 101	(100) 1.2	20 9 14	(14) 5.5	30 25 25		(27) 2.9	25 31 16	(24) 7.5	5 9 9	(8) 2.3
+	5000	80 P 106 P 103 P	(96) 14.2	15 P 9 P 8 P	(11) 3.8	33 P 31 P 29 P		(31) 2.0	22 P 11 P 18 P	(17) 5.6	5 P 7 P 11 P	(8) 3.1
Positive controls S9-Mix +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		723 882 811	(805) 79.7	272 274 227	(258) 26.6	465 475 459		(466) 8.1	344 326 317	(329) 13.7	431 322 368	(374) 54.7

Results of range finding test without metabolic activation.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.