Carbamic acid, N-[(dimethoxymethylsilyl)methyl]-, methyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation:
Bacterial reverse mutation assay (Ames test / OECD 471): negative
In vitro mammalian chromosome aberration test (CA / OECD 473): ambiguous
In vitro mammalian cell micronucleus test (MNT / OECD 487): negative
In vitro mammalian cell gene mutation assay (MLA / OECD 490): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Nov 2003 - 07 Jan 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

yes

Remarks:

2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, München, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/beta-Naphtoflavone.

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Remarks:

Without S9: sodium azide (10 µg/plate for TA 100 and TA 1535); 4-NOPD (10 and 40 µg/plate for TA 98 and TA 1537); MMS (1 µL/plate for TA 102); With S9: 2-AA (2.5 µg/plate for TA 98, TA 100, TA 1535, TA 1537; 10 µg/plate for TA 102)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 plates per concentration in two independent experiments (plate incorporation and preincubation)

DETERMINATION OF CYTOTOXICITY
- Method: reduced background lawn, reduction in the number of revertants, degree of survival of treated cultures

METABOLIC ACTIVATION
The S9 fraction was stored in liquid nitrogen. The S9 mix was freshly prepared on the day of the test according to AMES (1933): containing 15% S9 and the following components (final concentrations):
- 8 mM MgCl2
- 33 mM KCI-salt solution
- 5 mM glucose-6-phosphate
- 5 mM NADP
in 100 mM phosphate buffer, pH 7.4

Evaluation criteria:

A test item is considered as mutagenic if:
- a dose-related increase in the number of revertants occurs and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation
A biologically relevant increase is described as follows:
- if in strains TA 100 and TA 102 the number of reversions is at least twice as high
- if in strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher
as compared to the spontaneous reversion rate.

Statistics:

Mean values and standard deviation were calculated

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and 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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and 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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and 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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and 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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and 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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was reported

RANGE-FINDING/SCREENING STUDIES:
In a preliminary toxicity test TA 98 and TA 100 were treated with the test item up to 5000 µg/plate. No toxicity was observed up to 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA: no data given

Table 1: Test results of main test 1 (Plate incorporation test).

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate in the plate incorporation test
	(μg/plate)	(average of 3 plates±standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA 102	TA 1535	TA1537	TA98
–	A. dest.	124±8	327±6	27±4	12±7	31±3
–	Solvent control	99±11	273±16	28±7	11±6	33±2
–	31.6	106±19	286±16	24±5	14±6	36±2
–	100	109±4	279±11	22±6	15±5	27±8
–	316	104±9	301±7	23±5	15±2	33±6
–	1000	115±4	294±8	21±4	17±3	27±2
–	2500	103±14	319±26	19±4	12±3	28±8
–	5000	116±5	308±19	24±9	12±6	30±8
Positive controls, –S9	Name	sodium azide	MMS	sodium azide	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10	1 µL/plate	10	40	10
	Average of 3 plates±sd	402±28	1724±129	1032±46	179±11	774±59
+	A. dest.	124±22	355±56	12±2	15±4	46±9
+	Solvent control	116±13	289±31	11±3	13±1	36±7
+	31.6	116±7	330±28	15±7	17±2	38±2
+	100	108±16	369±40	21±1	14±1	41±4
+	316	116±14	330±23	16±7	13±9	38±12
+	1000	113±17	360±19	16±4	19±3	35±1
+	2500	128±4	352±5	11±3	13±4	34±4
+	5000	130±9	375±23	15±6	13±3	38±6
Positive controls,+S9	Name	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene
	Concentrations (μg/plate)	2.5	10	2.5	2.5	2.5
	Average of 3 plates±sd	1991±36	804±20	226±29	282±9	1207±142

 

Table 2: Test results of main test 2 (Pre-incubation test).

With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate in the pre-incubation test
	(μg/plate)	(average of 3 plates±standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA 102	TA 1535	TA1537	TA98
–	A. dest.	113±11	192±19	21±4	17±6	35±5
–	Solvent control	99±5	141±8	17±4	11±	27±4
–	31.6	128±3	166±12	16±4	17±7	25±4
–	100	115±18	159±11	20±1	17±4	26±3
–	316	117±21	151±1	24±2	15±4	25±3
–	1000	114±1	154±20	17±4	17±1	25±3
–	2500	123±5	131±9	24±1	17±2	24±5
–	5000	123±10	166±6	23±3	17±1	31±4
Positive controls, –S9	Name	sodium azide	MMS	sodium azide	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10	1 µL/plate	10	40	10
	Average of 3 plates±sd	540±49	2246±80	1168±84	152±15	848±29
+	A. dest.	143±12	304±32	16±6	24±8	45±5
+	Solvent control	128±9	211±39	11±2	11±0	39±11
+	31.6	132±10	258±15	18±2	13±3	38±10
+	100	127±12	246±1	13±6	18±8	43±12
+	316	120±8	223±17	15±3	14±2	33±4
+	1000	128±3	221±25	19±4	11±4	42±2
+	2500	122±25	209±12	12±6	15±4	38±8
+	5000	122±22	233±31	21±3	16±2	35±9
Positive controls,+S9	Name	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene	2-aminoanthracene
	Concentrations (μg/plate)	2.5	10	2.5	2.5	2.5
	Average of 3 plates±sd	2373±144	839±197	178±31	204±44	2033±238

MMS - Methylmethanesulfonate

4 -NOPD - 4 -nitro-o-phenylene-diamine

Conclusions:

Interpretation of results::
negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Sep 2003 - 05 Jan 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, München, Germany

Type of assay:

other: in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Complete culture medium: MEM medium supplemented with 10% (v/v) fetal calf serum (FCS)
Treatment medium: MEM medium supplemented with 10% (v/v) fetal calf serum (FCS)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction of phenobarbital and β-naphtoflavone induced rats

Test concentrations with justification for top dose:

- Pre-experiment:
- 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500, 5000 μg/mL (with and without metabolic activation)
Experiment I:
- 125, 250, 500, 1000, 2500, 5000 μg/mL (with and without metabolic activation)
Experiment II:
- 125, 250, 500, 1000, 2500, 5000 μg/mL (without metabolic activation)
- 500, 1000, 2000, 3000, 4000, 5000 μg/mL (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate: 600 μg/mL in nutrient medium, -S9; Cyclophosphamide: 0.83 μg/mL in nutrient medium, +S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: 4 and 20 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 24 h; 20 h treatment: 20 h

SPINDLE INHIBITOR:
- colcemide 0.2 μg/mL medium

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells

OTHER EXAMINATIONS:
Determination of polyploidy: yes

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells).
According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of the results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data both, biological and if evaluated, statistical significance should be considered together. A test item is considered to be negative if there is no biologically relevant increase in the percentage of aberrant cells above concurrent control levels, at any dose group.

Statistics:

Mean values and standard deviation have been calculated.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

positiv at 2500 and 5000 µg/mL in experiment I, negativ in experiment II

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥4000 µg/mL (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

negative in experiment I and positive at 2500 µg/mL in experiment II

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥250 µg/mL (experiment I) and ≥500 µg/mL (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TESTSPECIFIC CONFOUNDING FACTORS
Precipitation: No precipitation of the test item was noted in all dose groups evaluated and no influence on the pH values for the cell culture medium was detected.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I without metabolic activation the test item did not increase the frequency of the cells with aberrations as compared to the concurrent solvent control. In the cultures treated 1.5 – 5.0% aberrant cells were found which was slightly above the range of the historical negative control values. The results with metabolic activation showed a distinct increase in the aberrant cells at the highest dose groups evaluated. In the cultures treated 5.0 – 8.5% aberrant cells were found. Compared to the solvent controls the test item increased the frequency of aberrant cells at doses of 2500 µg/mL and 5000 µg/mL. The solvent controls showed values of 3.0% aberrant cells with and 2.0% aberrant cells without metabolic activation.
In experiment II without metabolic activation the number of aberrant cells observed at a dose of 2500 µg/mL were above the range of the concurrent solvent control value (4.0%) and even the historical negative control data. In the treated cultures 3.0 – 7.0% aberrant cells were found. The highest evaluable concentration (2500 µg/mL) showed a distinct but not statistically significant increase of aberrant cells. In experiment II with metabolic activation closer concentration steps were chosen to verify the results of experiment I. The increase in aberrant cells in experiment I could not be verified. The number of aberrant cells was 4.5 – 5.5%. The values were slight above the concurrent solvent control but not statistically different from the solvent control. The concurrent negative controls showed values of 1.0 and 3.0% aberrant cells (experiment I) and 2.0 and 1.8% aberrant cells (experiment II) without and with metabolic activation, respectively.
The positive controls EMS and CPA showed a distinct and biologically relevant increase of cells with structural chromosome aberrations above the historical positive control level (experiment II without metabolic activation). As experiment I (positive with metabolic activation) and experiment II (positive without metabolic activation) gave contradictory results there is no conclusive evidence as to whether the test substance is clastogenic or not under the applied test conditions. Therefore the results are considered ambiguous.

RANGEFINDING/SCREENING STUDIES:
According to the used OECD guideline the highest recommended concentration tested was 5000 μg/mL. Cytotoxicity was observed at concentrations ≥500 µg/mL (without metabolic activation) and 5000 µg/mL (with metabolic activation), respectively.

POLYPLOIDY:
No increase in the frequencies of polyploidy cells was found after treatment with the test item as compared to the controls in both experiments.

Table 1: Chromosome aberrations in Chinese hamster V79 cells in the absence/presence of metabolic activation

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 4 h, fixation time 20 h, without S9-mix
Control	0	112	3.5	1.0
DMSO	0	100	3.0	2.0
EMS	600	138	12.0	9.5
Test substance	125	91	8.0	5.0
	250	65	6.0	4.0
	1000	63	2.0	2.0
	5000	48	3.0	1.5
Exposure period 4 h, fixation time 20 h, with S9-mix
Control	0	119	5.0	3.0
DMSO	0	100	6.0	3.0
CPA	0.83	69	17.5	13.0
Test substance	1000	120	8.0	5.0
	2500	98	10.0	8.5
	5000	104	8.0	7.5
Exposure period 20 h, fixation time 20 h, without S9-mix
Control	0	72	4.0	2.0
DMSO	0	100	5.0	4.0
EMS	600	51	28.5	24.5
Test substance	500	42	8.5	3.0
	1000	36	7.0	4.0
	2500	47	7.5	7.0
Exposure period 4 h, fixation time 20 h, with S9-mix
Control	0	69	2.5	1.8
DMSO	0	100	6.0	4.0
CPA	0.83	54	16.4	12.7
Test substance	3000	78	7.0	5.5
	4000	66	8.3	4.5
	5000	66	7.3	5.3

EMS: Ethylmethanesulfonate

CPA: Cyclophosphamide

 

Table 2: Test for cytotoxicity

Concentration [µg/ml]	Mitotic index metabolic activation
	without S9-mix relative [%]		with S9-mix relative [%]
Control	122	144	70	90
DMSO	85	100	78	100
15.6	75	88	79	101
31.3	80	94	81	104
62.5	74	87	55	71
125	65	76	81	104
250	79	93	69	88
500	52	61	63	81
1000	45	53	56	72
2500	49	58	71	91
5000	63	74	53	68

 

Conclusions:

Interpretation of results: ambiguous

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item SLM 449008 led to inconclusive results in the V79 chinese hamster cell line. Therefore, it was not possible to classify the test item as clastogenic or non-clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-09 to 2016-03-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit

Type of assay:

other: gene mutation assay in mammalian cells

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time or proliferation index: 10-12 h
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 complete
medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphtholflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Preliminary experiment:
Without S9 mix: 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10 mM (4 h)
With S9 mix: 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10 mM (4 h)

Experiment:
Without S9 mix: 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10 mM (4 h)
With S9 mix: 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10 mM (4 h)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: THF was selected based on the solubility test of the test item; the solvent was compatible with the survival of the cells and S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in RPMI medium with 5% horse serum
- Cell density at seeding: adjusted daily to 3 x 10E5

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: only negative and solvent controls were plated in duplicate; mutant frequency was counted in 4 plates for all treatment groups.

NUMBER OF CELLS EVALUATED: four 96-well plates at a density of approximately 2000 cells/well

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth (SG; the number of times the cell number increased from the starting
cell density); relative total growth (RTG; the product of the relative suspension growth [RTG] and the
relative cloning efficiency [RCE])

Rationale for test conditions:

The assay was considered acceptable if the following criteria were met:

1. At least 3 out of 4 of the 96-well plates were scorable;
2. The cloning efficiency of the negative and/or solvent controls was between 65-120%;
3. The spontaneous mutant frequency in the negative and/or solvent controls was between 50 to 170 mutant per 10E6 cells;
4. The cell number of negative and/or solvent controls increased between 8 to 32 fold during the 2-day growth period; and
5. Positive controls responded appropriately and produced either at least 300 mutants per 10E6 cells with at least 40% of the colonies being small OR indcued a small colony mutant frequency of at least 150 mutants per 10E6 cells. The RTG for positive controls also must be greater than 10%.

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.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

Statistical significance at the 5% level (p < 0.05) was evaluated for mutant frequency by means of the non-parametric Mann-Whitney test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Results showed that there was a statistically significant increase in mutant frequency in mouse lymphoma cells at 7.5 and 10 mM with metabolic activation when compared to solvent controls; however, these increases did not exceed 126 mutants per 10E6 cells. Therefore, these increases were not considered to be a positive response.

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [mM]	Relative cloning efficiency [%]	Relative Total Growth [%]	Mutants per 10E+06 surviving cells	Mutation frequency
Negative 1	105.0	109.2	115.2	---
Negative 2	78.9	78.9	119.8	---
Solvent (THF) 1	100.0	100.0	122.4	---
Solvent (THF) 2	100.0	100.0	95.1	---
0.10	106.9	99.6	112.4	3.7
0.25	90.5	97.4	124.8	16.1
0.5	86.4	87.1	111.2	2.4
1.0	96.5	97.1	93.3	-15.4
2.5	96.5	87.2	126.9	18.2
5.0	103.2	98.9	135.0	26.2
7.5	80.1	72.7	221.1	112.3 *
10	96.5	92.5	203.4	94.6 *
B[a]P, 3.5 µg/mL	66.4	48.7	750.5	641.7

B[a]P  Benzo[a]pyrene

*        p < 0.05

 

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [mM]	Relative cloning efficiency [%]	Relative Total Growth [%]	Mutants per 10E+06 surviving cells	Mutation frequency
Negative 1	103.4	105.4	103.5	---
Negative 2	107.0	114.2	95.7	---
Solvent (THF) 1	100.0	100.0	109.7	---
Solvent (THF) 2	100.0	100.0	84.2	---
0.10	100.0	96.2	121.7	24.8
0.25	95.2	96.2	137.2	40.2
0.5	108.9	102.1	108.8	11.8
1.0	86.6	75.3	116.3	19.3
2.5	98.3	91.8	120.0	23.0
5.0	112.9	97.8	88.9	-8.1
7.5	100.0	87.2	93.4	-3.6
10	96.7	86.2	112.3	15.3
EMS, 300 µg/mL	85.3	76.5	910.9	813.9
MMS, 10 µg/mL	69.5	57.1	615.9	518.9

EMS    Ethylmethanesulfonate

MMS   Methylmethanesulfonate

 

Conclusions:

In an OECD 490 study, the test material is not considered to be mutagenic in an in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells both with and without metabolic activation.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Mar - 27 Jun 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487: In vitro mammalian cell micronucleus test (2004)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, München, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Type and identity of media:
Complete culture medium: MEM medium supplemented with
- 10%(v/v) fetal calf serum (FCS)
- 220 µg/ml penicillin/streptomycin
Treatment medium: MEM medium supplemented with
- 220 µg/ml penicillin/streptomycin
- 1.5 µg/ml cytochalasine B
- 220 µg/ml sodium pyruvate

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction of phenobarbital and β-naphtoflavone induced rats

Test concentrations with justification for top dose:

Pre-experiment:
- 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500, 5000 µg/mL
Main experiment I:
- 125, 250, 500, 1000, 2500, 5000 µg/mL (with and without metabolic activation)
Main experiment II:
- 31.3, 62.5, 125, 250, 500, 1000, 2500, 5000 µg/mL (without metabolic activation)
- 500, 1000, 2000, 3000, 4000, 5000 µg/mL (with metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulfonate, 600 and 900 µg/mL in MEM, -S9; Cyclophosphamide, 2.5 µg/mL in MEM, +S9 (clastogenic control substance); Colcemide 0.08 and 0.8 µg/mL (aneugenic control substance)

Details on test system and experimental conditions:

Example:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 24 h; 24 h treatment: 24 h;

STAIN (for cytogenetic assays): Acridine Orange solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation index (CBPI)

Evaluation criteria:

There are several criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells containing micronuclei
- a biologically relevant increase in the number of cells containing micronuclei for at least one of the dose groups, which is higher than the laboratory negative control range.
Statistical methods may be used as an aid in evaluating the test results. Statistical significance should not be the only determination of a positive response. A test item is considered to be negative if there is no biologically relevant increase in the percentages of cells with micronuclei above concurrent control levels, at any dose group.

Statistics:

χ² test, one-sided, p < 0.05

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/mL (main experiment I), ≥ 1000 µg/mL (main experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 2500 µg/mL (main experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was noted in all dose groups evaluated.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I and II (with and without metabolic activation) the micronucleated cells frequencies of the negative control and the solvent control were within the historical control data of the negative control. The number of micronucleated cells found after treatment with the test item was within the historical control data range of the negative control.

RANGE-FINDING/SCREENING STUDIES:
According to the used draft guideline the highest recommended concentration tested was 5000 µg/mL.

Table 1: CBPI and micronucleus induction in V79 cells

Test item	Concentration	CBPI	Number of cells with MN [%]
	in µg/mL	in %
Exposure period 4 h, fixation time 24 h, without S9-mix
Negative control	0	95	0.95
DMSO	0	100	0.75
EMS	900	74	7.15
Colcemid	0.8	81	3.05
Test substance	1000	120	1.05
	2500	110	1.05
	5000	103	1.05
Exposure period 4 h, fixation time 24 h, with S9-mix
Negative control	0	92	1.0
DMSO	0	100	0.9
CPA	2.5	55	10.7
Test substance	1000	64	1.9
	2500	70	1.8
	5000	78	1.65
Exposure period 4 h, fixation time 24 h, with S9-mix
Negative control	0	111	1.9
DMSO	0	100	1.2
CPA	2.5	68	8.6
Test substance	500	95	1.55
	1000	67	1.6
	2000	50	2.2
	4000	59	2.1
Exposure period 24 h, fixation time 24 h, without S9-mix
Negative control	0	101	1.75
DMSO	0	100	2.1
EMS	600	41	7.45
Colcemid	0.08	72	15.7
Test substance	1000	93	1.6
	2500	57	1.35
	5000	54	1.75

EMS: Ethylmethanesulfonate (positive control)

CPA: Cyclophosphamide (positive control)

CBPI: Cytokinesis-Block Proliferation Index

Conclusions:

Interpretation of results: negative

Based on the results of the study the test material is considered not to be clastogenic in this mammalian micronucleus test in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity (mutagenicity) in bacteria in vitro

A key genetic toxicity study addressing mutagenicity in bacteria in vitro with methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate (CAS 23432-65-7) is available and was performed according to OECD TG 471 (Ames test) and in compliance with GLP (BSL, 2004).

The strains Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were tested according to the plate incorporation (experiment I) and pre-incubation (experiment II) procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). Two independent experiments were conducted in three repetitions at each concentration from 31.6 to 5000 µg/plate. No significant increase in the number of revertants was observed in any of the tester strains with and without metabolic activation. No cytotoxicity and no precipitation of the test material were observed at any tested concentration. Appropriate negative, solvent (DMSO) and positive controls were included into the test and gave the expected results. Based on the results of the study the test material was considered to be not mutagenic in bacteria under the conditions of the test.

 

Genetic toxicity (clastogenicity) in mammalian cells in vitro

Two studies assessing clastogenicity in mammalian cells in vitro with methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate (CAS 23432-65-7) are available.

In a chromosome aberration study performed according to OECD TG 473 and in compliance with GLP Chinese hamster lung fibroblasts (V79) were treated with methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate or vehicle (DMSO) in two independent experiments in the absence or presence of a metabolic activation system (Aroclor-induced rat liver S9-mix) at concentrations of 125, 250, 500, 1000, 2500, 5000 μg/mL (experiment I with and without S9 -mix, experiment II without S9-mix) and 500, 1000, 2000, 3000, 4000, 5000 μg/mL (experiment II with S9-mix) for 4 or 20 hours (without S9-mix) and 4 hours (with S9-mix) (BSL, 2004). Fixation of the cells was performed 20 hours after start of exposure with the test material. Appropriate solvent, negative and positive controls were included in the test and gave the expected results. Clastogenicity was recorded at concentrations of 2500 and 5000 µg/mL (experiment I with S9-mix) and 2500 µg/mL (experiment II without S9-mix). Cytotoxicity was observed at concentrations greater than 4000 µg/mL (experiment II with S9-mix) and ≥250 µg/mL (experiment I) and ≥500 µg/ml (experiment II) without S9-mix. No precipitation of the test material was noted at any tested concentration. The results of the study are inconclusive as no clear dose response relation could be observed and clastogenicity mainly occurred at cytotoxic concentration. Therefore, an additional in vitro micronucleus test was performed.

In this in vitro micronucleus test performed according to OECD TG 487 and in compliance with GLP Chinese hamster lung fibroblasts (V79) were treated with methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate or vehicle (DMSO) in two independent experiments in the absence or presence of a metabolic activation system (Aroclor-induced rat liver S9-mix) at concentrations of 125, 250, 500, 1000, 2500, 5000 μg/mL (experiment I with and without S9-mix), 31.3, 62.5, 125, 250, 500, 1000, 2500, and 5000 μg/mL (experiment II without S9-mix) and 500, 1000, 2000, 3000, 4000 and 5000 µg/mL (experiment II with S9-mix) for 4 or 24 hours (without S9-mix) and 4 hours (with S9-mix) (BSL, 2006). Fixation of the cells was performed 24 hours after start of exposure with the test material. Appropriate solvent, negative and positive controls were included in the test and gave the expected results. The number of micronucleated cells found after treatment with the test item was within the historical control data range of the negative control. Cytotoxicity was observed at concentrations of 1000 µg/mL (experiment I) and ≥1000 µg/mL (experiment II) with metabolic activation and ≥2500 µg/mL (experiment II) without metabolic activation. No precipitation of the test material was noted at any tested concentration. Based on the results of the study the test material is considered not to be clastogenic in this mammalian micronucleus test in vitro.

Based on the results of these two studies taken together, it is assumed that the substance has no clastogenic potential.

 

Genetic toxicity (mutagenicity) in mammalian cells in vitro

An in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells both with and without metabolic activation was performed using methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate (Eurofins, 2016). The study was conducted in accordance with OECD 490. For the study, mouse lymphoma L5178Y cells were treated with the test material or vehicle (tetrahydrofuran) at concentrations of 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10 mM for 4 hours when in the presence of a metabolic activation system (phenobarbital/β-naphtholflavone induced rat liver S9-mix). The test material was also evaluated without a metabolic activation system at concentrations of 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5, or 10 mM for 4 hours. Appropriate solvent and positive controls were included in the test and gave the expected result. No cytotoxicity was observed either with or without metabolic activation. Mutant frequency was comparable to negative controls in the assay conducted without metabolic activation. Results showed that there was a statistically significant increase in mutant frequency in mouse lymphoma cells at 7.5 and 10 mM with metabolic activation when compared to solvent controls; however, these increases did not meet the criteria of a positive response (i.e., exceeding 126 mutants per 10E6 cells). Therefore, these increases were not considered to be a positive response. Based on these results, the test material is not considered to be mutagenic in an in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells both with and without metabolic activation.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance methyl-N-{[dimethoxy(methyl)silyl]methyl}carbamate (CAS 23432-65-7) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.