Methyl [3-(trimethoxysilyl)propyl]carbamate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-13 to 2014-09-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Results of dose range finding test are in contrast to the results observed in the main study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Methyl [3-(trimethoxysilyl)propyl]carbamate

EC Number:

245-659-7

EC Name:

Methyl [3-(trimethoxysilyl)propyl]carbamate

Cas Number:

23432-62-4

Molecular formula:

C8H19NO5Si

IUPAC Name:

methyl N-[3-(trimethoxysilyl)propyl]carbamate

Details on test material:

- Name of test material (as cited in study report): Methyl [3-(trimethoxysilyl)propyl]carbamate
- Physical state: liquid
- Stability under test conditions: stable
- Storage condition of test material: at RT, protect from light, avoid humidity

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamine
- Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium)
- Exposure medium: for 3 hour exposure: cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium); for 24 hour exposure:
cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/ml trifluorothymidine (TFT)
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats treated with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).

Test concentrations with justification for top dose:

Experiment 1:
- 1.7, 5.4, 17, 52, 164, 512, 1600, 2373 µg/ml with and without metabolic activation (3 h)

Experiment 2:
- 1.7, 5.4, 17, 52, 164, 512, 1600, 2373 µg/ml without metabolic activation (24 h)

Vehicle / solvent:

- Vehicle solvent used: DMSO; 1% (v/v)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - Methyl methanesulfonate: 15 µg/ml (3 h) and 5 µg/ml (24 h) in DMSO without S9-mix - Cyclophosphamide: 7.5 µg/ml in HBSS (3, 24 h) with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in suspension

DURATION:
1st experiment: 3 h exposure with and without S9 mix
2nd experiment: 24 h exposure without S9 mix
- Expression time (cells in growth medium): 2 days after the end of the treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtiter plates containing TFT selective medium. The microtitre plates were incubated for 11 or 12 days.
- Selection time: 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days

SELECTION AGENT (mutation assays):
- 5 µg/ml trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- two

DETERMINATION OF CYTOTOXICITY:
- Method: cloning efficiency and relative total growth

OTHER:
- Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at > 52 µg/ml with metabolic activation (3 h)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was only observed in the presence of S9-mix (at > 512 µg/ml), resulting in a relative total growth of 40%.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 52 to 2373 µg/ml in the absence and presence of S9-mix with a 3 hour treatment period and at 10 to 475 µg/ml in the absence of S9-mix with a 24 hour treatment period. No toxicity in the suspension growth was observed up to and including the highest test substance concentration of 2373 μg/ml compared to the suspension growth of the solvent control both in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Any other information on results incl. tables

Experiment 1: Cytotoxic and mutagenic response of methyl-N- [3-(trimethoxysilyl)propyl]carbamate in the mouse lymphoma L5178Y test system

Dose (µg/ml)	RSG (%)	CEday2 (%)	RSday2 (%)	RTG (%)	Mutation frequency per 10^6survivors
					total	small	large
without metabolic activation 3 hours treatment
SC1	100	102	100	100	81	63	16
SC2		84			106	84	19
1.7	88	86	93	82	87	66	18
5.4	95	81	88	83	76	60	14
17	88	93	100	86	102	80	19
52	93	63	68	63	125	106	17
164	91	72	78	71	79	55	22
512	81	90	97	79	111	71	35
1600	84	79	85	72	107	83	21
2373	85	81	88	74	109	78	27
MMS	60	55	59	36	1084	792	184
with metabolic activation 3 hours treatment
SC1	100	81	100	100	62	27	33
SC2		69			88	32	53
1.7	88	83	110	97	75	34	39
5.4	83	97	128	107	89	47	38
17	102	105	140	143	75	31	41
52	135	99	132	178	82	37	41
164	90	89	118	105	130	69	54
512	74	77	102	76	379	182	143
1600	61	61	81	50	604	258	237
2373	52	58	76	40	714	366	212
CP	47	19	26	12	2839	1426	1033

Experiment 2: Cytotoxic and mutagenic response of methyl-N-[3-(trimethoxysilyl)propyl]carbamate in the mouse lymphoma L5178Y test system

Dose (µg/ml)	RSG (%)	CEday2 (%)	RSday2 (%)	RTG (%)	Mutation frequency per 10^6survivors
					total	small	large
without metabolic activation 24 hours treatment
SC1	100	89	100	100	62	24	36
SC2		90			77	32	42
1.7	87	78	87	76	57	15	41
5.4	91	88	98	89	68	18	48
17	95	85	95	90	80	21	57
52	103	79	89	91	83	19	62
164	99	93	104	102	69	21	47
512	94	105	118	110	53	17	35
1600	90	80	90	81	103	49	50
2373	89	81	91	81	80	33	45
MMS	88	77	86	76	687	234	367

Note: All calculations were made without rounding off

RSG= Relative Suspension Growth; CE= Cloning Efficiency: RS= Relative Survival; RTG= Relative Total Growth; SC= Solvent control= dried dimethyl sulfoxide; MMS= Methylmethanesulfonate; CP= Cyclophosphamide

Historical control data of the spontaneous mutation frequencies of the solvent controls for the mouse lymphoma assay

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment
Range	[50 – 119]	[50 – 150]	[50 – 167]
Mean	73	70	81
SD	16	19	25
n	152	132	283

Historical control data of the mutation frequencies of the positive controls for the mouse lymphoma assay

		Mutation frequency per 10^6survivors
		- S9-mix		+ S9-mix
		3 hour treatment	24 hour treatment
Range		[501 – 3165]	[504 – 1445]	[593 – 4356]
Mean		902	750	1377
SD		367	198	593
n		78	67	141

SD = Standard deviation

n = Number of observations

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative without metabolic activation
positive with metabolic activation after 3 h

The in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells performed according to OECD guideline 476 and in compliance with GLP, employing doses of up to 2373 µg/ml, revealed that the test substance does not show mutagenic potential without metabolic activation. In contrast, the test material is mutagenic under the given experimental conditions with metabolic activation. In summary, the mutagenic activity is confined only to incubations with metabolic activation.