Amino functional alkoxysilanes

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:

2012-04-26 to 2012-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Method

Target gene:

His

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: compatible with bacterial survival and S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

A. dest., BSL Lot No. 120417, 120504

Negative solvent / vehicle controls:

yes

Remarks:

acetone, Merck Lot No. K4258814

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Positive control without metabolic activation: sodium azide for tester strains TA100, TA1535; 4-nitro-o-phenylene-diamine for TA98, TA1537; methylmethanesulfonate for TA102. Positive control with metabolic activation: 2-aminoanthracene for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min; 100 µl of the test item preparation was pre-incubated with the tester strains (100 µl) and sterile buffer or the metabolic activation system (500 µl) for 60 min at 37 °C prior to adding the overlay agar (2000 µl) and pouring onto the surface of a minimal agar plate.
- Exposure duration: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used. A pre-experiment for toxicity was carried out. The experiment was repeated: the first main experiment used the plate incorporation method, the second experiment included pre-incubation.


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

METABOLIC ACTIVATION: Phenobarbital and beta-naphthoflavone induced rat liver S9: S9 mix included glucose-6-phosphate and NADP as co-factors. S9 mix contained 5% S9, and 0.5 ml S9 mix was added to a total of 2.7 ml top agar, giving a final concentration of approximately 1% S9. The activity of the S9 had been checked by the supplier for promutagen activity using 2-aminoanthracene and benzo[a]pyrene.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I, 5000 µg/plate (without metabolic activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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 4

Key result

Species / strain:

S. typhimurium 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 5

Key result

Species / strain:

S. typhimurium TA 102

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:

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiments I and II with the exception of a toxic effect observed in tester strain TA 1537 at a concentration of 5000 µg/plate in experiment I.

Table 1 Pre-experiment for toxicity: mutation factor

Concentration μg/plate	TA 98		TA 100
	-MA	+MA	-MA	+MA
0*	1.0	1.0	1.0	1.0
3.16	0.7	1.4	1.0	1.2
10.0	0.7	1.1	1.0	1.2
31.6	0.9	1.5	0.8	1.3
100	1.0	1.1	0.9	1.1
316	1.1	1.0	1.0	1.2
1000	0.5	1.1	1.0	1.0
2500	0.9	1.2	0.9	1.1
5000	0.9	1.7	1.0	1.1
Positive control	19.9	58.9	7.9	6.4

* Solvent control with acetone

Table 2 Experiment 1 plate incorporation: revertants per plate (mean of 3 plates)

Concentration μg/plate	TA 98		TA 100		TA 1535		TA 1537		TA 102
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0**	21	22	111	114	12	9	8	13	242	311
0*	20	17	128	107	12	8	10	12	279	256
31.6	17	25	108	137	10	10	9	11	272	298
100	20	19	112	114	7	9	9	9	275	294
316	21	17	126	129	11	9	8	10	273	262
1000	10	19	122	104	12	7	8	13	265	274
2500	17	20	121	122	10	8	7	9	248	313
5000	18	29	125	115	8	7	1.0	15	256	287
Positive control	391	1001	1007	683	1447	27	103	1435	517	517

* Solvent control with acetone

**Distilled water

Table 3 Experiment 2 pre-incubation: revertants per plate (mean of 3 plates)

Concentration μg/plate	TA 98		TA 100		TA 1535		TA1537		TA 102
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
0**	23	29	112	99	12	8	12	9	249	285
0*	23	31	104	91	8	12	7	8	229	258
31.6	19	26	100	109	10	9	9	7	213	298
100	19	28	112	108	12	4	5	8	235	312
316	27	23	118	99	8	9	10	7	262	304
1000	28	25	105	107	11	11	7	7	245	319
2500	26	25	116	116	10	12	11	6	244	347
5000	26	29	106	91	10	8	5	9	222	294
Positive control	659	1816	947	2229	1051	84	115	109	2029	596

* Solvent control with acetone

**Distilled water

Applicant's summary and conclusion

Conclusions:

Trimethoxy(methyl)silane and its reaction products with 3-aminopropyltriethoxysilane and [3-(2,3-epoxypropoxy)propyl]trimethoxysilane have been tested in a study conducted according to OECD Test Guideline 471 and in compliance with GLP (BSL BIOSERVICE, 2012). No test-substance related increase in the number of revertants was observed with or without metabolic activation in either the initial plate incorporation assay or the repeat experiment using the pre-incubation method in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 up to limit concentrations. Toxicity was observed in one strain in the absence of metabolic activation at the highest concentration tested. Appropriate positive and solvent controls were included and gave expected results, apart from the solvent and positive controls for TA 1535 with metabolic activation in experiment 1, where the mean values for both controls were just below the range of historical controls. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.