Dimethylbis(octadecyloxy)silane

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:

2016-05-26 to 2016-06-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Reaction mass of octadecan-1-ol and trimethyl(octadecyloxy)silane

Molecular formula:

C21H46OSi

IUPAC Name:

Reaction mass of octadecan-1-ol and trimethyl(octadecyloxy)silane

Test material form:

other: Off-white waxy solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0008287484
- Expiration date of the lot/batch: 2017-03-03
- Purity test date: 2015-11-18

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored sealed away from air exposure
- Stability under test conditions: no data available
- Solubility and stability of the test substance in the solvent/vehicle: the test item was soluble and stable in anhydrous tetrahydrofuran (THF)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no correction was made for the purity/composition of the test item
- Final dilution of a dissolved solid, stock liquid or gel: the test item was dissolved in anhydrous tetrahydrofuran (THF)

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: Each strain contained the following mutations: rfa: defective lipopolysaccharide cellcoat; gal: mutation in the galactose metabolism; chl: mutation in nitrate reductase; bio: defective biotin synthesis; uvrB: loss of the excision repair system

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Dose-range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate (TA100 and WP2uvrA)
Experiment 1: 52, 164, 512, 1600, 5000 µg/plate (TA1535, TA1537, TA98)
Experiment 2: 275, 492, 878, 1568, 2800, 5000 µg/plate (all test strains)
Experiment 3: 86, 154, 275, 492, 878 µg/plate (all test strains)
Limited solubility was exhibited at 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: anhydrous tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: the test item was soluble and stable in the vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 10⁹ cells/ml

ACTIVATION SYSTEM: 10 mL of S9 mix contained: 30 mg NADP, 15.2 mg glucose-6-phosphate in 5.5 ml Milli-Q water or 5.0 ml Milli-Q water, 2 ml 0.5 M sodium phosphate buffer pH 7.4, 1 ml 0.08 M MgCl, 1 ml 0.33 M KCl. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 ml of S9-mix components 1.0 ml S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

DURATION
- Exposure duration: 48 h +/- 4 h
- Mutation assay details:
Dose-range finding study: eight different doses of the test item were tested with and without metabolic activation in TA100 and WP2uvrA
Experiment 1: five different doses of the test item were tested with and without metabolic activation in TA1535, TA1537 and TA98
Experiment 2: five different doses of the test item were tested with and without metabolic activation in all test strains
Experiment 3: five different doses of the test item were tested with and without metabolic activation in all test strains

SELECTION AGENT (mutation assays): Selective agar plates were used: biotin and histidine-containing agar plates for Salmonella typhimurium; tryptophan-containing plates for Escherichia coli

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants, reduction of bacterial background lawn

Rationale for test conditions:

Three different experiments were performed with different dose selection until no precipitate of the test item was observed.

Evaluation criteria:

The test item is considered negative when:
- the total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 and TA98 is not greater than three times the concurrent vehicle control;
- the negative control should be reproducible in at least one follow-up experiment;

Statistics:

Not used

Results and discussion

Test results

Test results 1

Species / strain:

bacteria, other: TA1535, TA1537 and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 512 µg/plate TA98, with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

bacteria, other: TA1535, TA1537, TA98, TA100 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 512 in TA98 with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Species / strain:

bacteria, other: TA1535, TA1537, TA100, TA98 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: precipitation was observed at the start and the end of the incubation period at concentration of 512 µg/plate
Experiment 2: precipitation was observed at the start of the incubation period at the concentration of 492 µg/plate and higher, and at all dose levels at the end of the incubation period except in strains TA98 (+/- S9 mix), TA100 (+/- S9 mix) and TA1537 (+ S9 mix) at 275 µg/plate.
Experiment 3: precipitation was observed at the start of the incubation period at 275 µg/plate and higher, and at 878 µg/plate at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES:
Precipitation of the test item was observed at the start of the incubation period at 164 µg/plate and higher and at the end of the incubation period at 1600 and 5000 µg/plate.

HISTORICAL CONTROL DATA
- Positive historical control data: positive control results were within the ranges of historical control data
- Negative (solvent/vehicle) historical control data: negative control results were within the ranges of historical control data

Remarks on result:

other:

Remarks:

Experiment 1

Any other information on results incl. tables

Table 1: Mean number of revertant colonies in dose-range finding test

Concentration µL/plate	TA 100		WP2uvrA
	+MA	-MA	+MA	-MA
Positive control	1285	1098	501	1337
Negative control	112	128	42	36
1.7	110	111	41	35
5.4	125	124	42	34
17	121	105	45	39
52	122	113	45	40
164	140	118	48	42
512	126np	124np	58np	40np
1600	126sp	135sp	44sp	30sp
5000	111mp	121mp	47mp	36mp

Table 2: Mean number of revertant colonies in experiment 1

Concentration µL/plate	TA 98		TA 1535		TA 1537
	+MA	-MA	+MA	-MA	+MA	-MA
Positive control	1254	1217	193	974	379	638
Negative control	19	12	6	7	7	6
52	15	14	8	4	8	9
164	19np	22np	7np	9 np	5np	9np
512	7sp	11sp	10sp	11sp	7sp	5sp
1600	hp	hp	hp	hp	hp	hp
5000	hp	hp	hp	hp	hp	hp

Table 3: Mean number of revertant colonies in experiment 2

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Positive control	148	698	300	808	459	1595	803	960	315	1135
Solvent control	14	14	9	5	20	21	96	123	60	42
275	8sp	10sp	5np	4sp	13np	12np	95np	125np	43sp	25sp
492	13sp	12sp	8sp	9sp	16np	16sp	92sp	115sp	39mp	19mp
878	11sp	9mp	8sp	6np	14mp	13mp	89sp	127sp	27mp	24mp
1568	8mp	13mp	4mp	3np	8mp	8mp	75mp	99mp	28mp	20mp
2800	9mp	hp	6mp	hp	hp	hp	88mp	hp	31mp	hp
5000	10mp	hp	3mp	hp	hp	hp	81mp	hp	24mp	hp

Table 3: Mean number of revertant colonies in experiment 3

Concentration µg/plate	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA
Positive control	158	846	244	1132	603	1290	956	951	288	1285
Solvent control	6	11	7	5	27	13	105	135	38	32
86	11	10	7	8	25	16	101	112	42	30
154	8	10	9	6	25	23	112	113	56	44
275	11	14	4	9	25	20	107	111	51	41
492	19np	16np	8np	10np	23np	22np	110np	131np	52np	42np
878	17sp	21sp	8sp	12sp	26sp	19sp	109sp	124sp	52sp	42sp

hp	Heavy Precipitate,the number of revertant colonies could not be determined.
np	No precipitate
sp	Slight Precipitate

Applicant's summary and conclusion

Conclusions:

Trimethyl(octadecyloxy)silane in stearyl alcohol was tested in a valid bacterial reverse mutation assay, conducted according to OECD TG 471 and in compliance with GLP using Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and Escherichia coli (WP2uvrA). No increase in the number of revertant was observed in any test strains , with or without metabolic activation, when tested up to precipitating concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.