Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test) (OECD TG 471): negative with and without activation in all strains tested

Cytogenicity in mammalian cells (OECD TG 473): negative with and without metabolic activation in chinese hamster V79 cells

Mutagenicity in mammalian cells (OECD TG 490): negative in L5178Y mouse lymphoma cell

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
July 29, 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Target gene:
TK 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:
rat liver S9-mix
Test concentrations with justification for top dose:
Experiment I:
- 0.225, 0.250, 0.275, 0.300, 0.325, 0.350, 0.375, 0.400 μL/mL (without metabolic activation)
- 0.300, 0.325, 0.350, 0.375, 0.400, 0.425, 0.450, 0.475, 0.500 μL/mL (with metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran
- 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
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.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG was 10.1% for 0.400 mg/mL, -S9, and 9.6% at 0.500 mg/mL, +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutant frequencies without metabolic activation were not statistically significantly increased over the solvent controls. With metabolic activation, there was a statically significant increase in mutant frequencies over the solvent controls at 0.350, 0.375, and 0.475 mg/mL; however, these increases were not considered treatment related because there was no evidence of a dose-relationship.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH dectected was within the normal physiological range.
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: test item is water-reactive
- Precipitation: No precipitation was observed in the main experiment; precipitation was observed in the pre-experiment at a concentration of 2 mg/mL.

RANGE-FINDING/SCREENING STUDIES: A pre-experiment was conducted at concentrations up to 2 mg/mL.

HISTORICAL CONTROL DATA
- Positive historical control data: EMS (300 μL/mL): 726.5±203.5; MMS (10 μL/mL): 763.4±421.6; B[a]P (1.5 μL/mL): 535.5±152.5
- Negative (solvent/vehicle) historical control data: Negative control, -S9: 87.9±25.5; Negative control, +S9: 85.1±24.3; THF, -S9: 105.9±30.0; THG, +S: 10.6±23.5

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative total growth (RTG)
- Other observations when applicable: Growth inhibition was observed both with and without metabolic activation. Without metabolic activation, the relative total growth (RTG) was 10.1% for the highest concentration (0.400 mg/mL); with metabolic activation, the RTG was 9.6% at 0.500 mg/L.

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 factor

Negative 1

96.5

87.7

90.4

/

Negative 2

77.0

71.7

113.2

/

Solvent (THF) 1

100.0

100.0

70.1

/

Solvent (THF) 2

65.6

/

0.300

101.3

95.9

82.9

15.1

0.325

103.0

98.5

96.6

28.8

0.350

93.6

83.9

114.8*

47.0

0.375

90.7

82.2

115.7*

47.9

0.400

101.3

72.2

60.4

-7.4

0.425

103.0

18.9

65.0

-2.9

0.450

104.7

33.3

66.8

-1.0

0.475

88.0

25.1

102.0*

34.2

0.500

96.5

9.6

54.1

-13.7

B[a]P, 3.5 µg/mL

90.7

61.3

537.1*

469.2

B[a]P  Benzo[a]pyrene

*         Statistically significant

 

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 factor

Negative 1

93.0

91.2

91.8

/

Negative 2

99.2

106.1

72.6

/

Solvent (THF) 1

100.0

100.0

114.7

/

Solvent (THF) 2

82.8

/

0.225

93.0

78.0

86.5

-12.2

0.250

104.3

92.4

52.6*a

-46.2

0.275

87.4

68.1

90.5

-8.2

0.300

113.9

93.6

85.9

-12.9

0.325

94.5

67.4

88.9

-9.9

0.350

81.1

40.9

84.8

-14.0

0.375

84.8

34.6

109.4

10.7

0.400

73.2

10.1

99.0

0.3

EMS, 300 µg/mL

90.2

71.3

595.3*

496.5

MMS, 10 µg/mL

70.1

55.5

690.4*

591.7

EMS    Ethylmethanesulfonate

MMS   Methylmethanesulfonate

*         Statistically significant

a         Significantly decreased compared to the solvent control, therefore not relevant for interpretationof results

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.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July, 1997
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
bacterial reverse mutation assay
Target gene:
His operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: rfa, uvrB and pKM101 (TA98, TA100 and TA102)
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphthoflavone
Test concentrations with justification for top dose:
Experiment I: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0 and 2.5 µL/plate
Experiment II: 0.0020, 0.0063, 0.020, 0.063, 0.20, 0.63 and 2.0 µL/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest. and tetrahydrofuran
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine for TA98 and TA1537, -S9; 2-aminoanthracene for TA98, TA100, TA1535, TA1537 and TA102, +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was detected by a 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.
Evaluation criteria:
A test was considered acceptable if for each strain:
- the bacteria demonstrated their typical responses to ampicillin (TA98, TA100, TA102)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges:

- S9 + S9
min max min max
TA 98 11 58 15 59
TA 100 49 155 62 160
TA 1535 4 41 3 38
TA 1537 3 35 3 36
TA 102 141 472 157 586

- corresponding background growth on negative control, solvent control and test plates was observed
- the positive controls showed a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain were analysable.

A test item was 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 was described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions was at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions was at least three times higher than the reversion rate of the solvent control

Statistics:
A statistical evaluation of the results is not regarded as necessary
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at concentrations of 1.0 µL/plate and higher (with and without metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at concentrations of 1.0 µL/plate and higher (without metabolic activation) and at a concentration of 2.5 µL/plate (with metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at a concentration of 2.5 µL/plate (with and without metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at a concentration of 2.5 µL/plate (with and without metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at a concentration of 2.5 µL/plate (with and without metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 102
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at a concentration of 2.0 µL/plate (with and without metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at a concentration of 0.63 µL/plate and higher (with and without metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
toxic effects at concentrations of 0.63 µL/plate and higher (without metabolic activation) and at a concentration of 2.0 µL/plate (with metabolic activation)
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
During the described mutagenicity test and under the experimental conditions reported, trichloro(4-methyl)silane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, trichloro(4-methyl)silane is considered to be non-mutagenic in this bacterial reverse mutation assay.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July, 2016
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 30, 2008
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit
Type of assay:
other: in vitro mammalian chromosome aberration test (migrated information)
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
MEDIA USED
Complete culture medium, as well as long-term treatment and post incubation: MEM medium supplemented with 10% fetal bovine serum (FBS); 100 U/100 µg/mL penicillin/streptomycin solution; 2 mM L-glutamine; 2.5 µg/mL amphotericin; and 25 mM HEPES

Treatment medium (short-term exposure): Complete culture medium without FBS

Metabolic activation:
with and without
Metabolic activation system:
cofactor 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 body weight)
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 325, 350, 375 and 400 µg/mL
with metabolic activation: 500, 550 and 600 µg/mL

Experiment II:
without metabolic activation: 300, 350 and 400 µg/mL
without metabolic activation (repetition): 250, 300 and 325 µg/mL

A pre-experiment was conducted under identical conditions as the main experiment. The concentrations tested in the main experiment based on the results obtained in the pre-experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water free tetrahydrofurane (0.5%, v/v)
- Justification for choice of solvent/vehicle: based on the results of the solubility test
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: approximately 1 x 10E4 cells/mL

DURATION
- Preincubation period:
- Exposure duration: 4 h for Experiment I, with and without metabolic activation; 21 h Experiment II, without metabolic activation
- Expression time (cells in growth medium): 17 h for Experiment I
- Selection time (if incubation with a selection agent): 17.5 h
- Fixation time (start of exposure up to fixation or harvest of cells): 21 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colcemid (0.2 µg/mL culture medium) was added to the cultures around 17.5 h after the start of the treatment. About 2.5 h later preparation was started. At first cells were trypsinated and resuspended in about 9 mL complete culture medium. An aliquot of each culture was removed to determine the cell count by a cell counter (AL-Systems). Then cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for 15-20 min. After hypotonic treatment the cells were fixed at least two times with 3 + 1 methanol + glacial acetic acid and spread onto the slides. After the fixation steps the slides were dried and stained with Giemsa. The slides were coverslipped using 2-3 drops of Eukitt(R). Afterwards they were air dried.

NUMBER OF CELLS EVALUATED: 300 well spread metaphases

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 300 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: not reported
Evaluation criteria:
The chromosomal aberration assay was considered acceptable if it met the following criteria:

- the number of aberration found in the negative and/or solvent controls fell within the range of historical laboratory control data / was considered acceptable for addition to the laboratory historical negative control database.
- concurrent positive controls induced responses that are compatible with those generated in the historical positive control data base and produced a statistically significant increase compared with the concurrent negative control
- the proliferation criteria in the solvent control was similar to the corresponding negative control (where applicable)
- all three experimental conditions were tested unless one resulted in positive results
- an adequate number of cells and concentrations were analysable
- the criteria for the selection of top concentration were consistent with those described earlier

The test substance was considered to be clearly positive if, in any of the experimental conditions examined: a) at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control; b) the increase was dose-related when evaluated with an appropriate trend test; and c) any of the results were outside the distribution of the historical negative control data

The test substance was considered clearly negative if, in all experimental conditions examined: a) none of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control; b) there was no concentration-related increase when evaluated with an appropriate trend test; c) all results were inside the distribution of the historical negative control data.
Statistics:
Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment I
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects at concentrations of 550 µg/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment I
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects at concentrations of 375 µg/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster lung fibroblasts (V79)
Remarks:
Experiment II
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxic effects at concentrations of 300 (repetition experiment) or 350 (main experiment) µg/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In experiment, I, precipitation of the test item was noted without metabolic activation at concentrations of 400 µg/mL and higher with the unaided eye. No precipitation of the test item was noted in experiment I with metabolic activation and in experiments II without metabolic activation in all concentrations evaluated.

RANGE-FINDING/SCREENING STUDIES: For the pre-experiment, precipitation of the test item was noted at concentrations of 500 µg/mL and higher. The highest dose group evaluated in the pre-experiment was 2000 µg/mL. The relative increase in cell count (RICC) was used as parameter for toxicity.

HISTORICAL CONTROL DATA
- Positive historical control data: yes, see Table 1
- Negative (solvent/vehicle) historical control data:
4 h, -S9: -0.28% - 3.70% aberrant cells excl. gaps
4 h, +S9: -0.23% - 3.95% aberrant cells excl. gaps
21 h, -S9, -0.20% - 2.71% aberrant cells excl. gaps

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative increase in cell count (RICC)
Remarks on result:
other:
Remarks:
no precipitation noted

Table 1. Positive control data

PC general

Number of aberrant cells

metabolic activation

with (CPA)

without (EMS)

 

 +gaps

 -gaps

 +gaps

 -gaps

mean [%]

12.5

10.1

12.9

10.5

SD

3.01

2.84

4.22

4.00

RSD [%]

24.1

28.0

32.8

38.0

min [%]

7.3

4.5

6.0

5.0

max [%]

23.0

21.5

34.4

30.4

n

193

193

215

215

 

 

LCL

6.49

4.45

4.44

2.53

UCL

18.54

15.80

21.33

18.54

 

PC:        Positive Control (EMS without metabolic activation, CPA with metabolic activation)

mean:  mean number of aberrant cells

SD:        Standard Deviation

RSD:      relative Standard Deviation

min.:     minimum number of aberrant cells

max.:     maximum number of aberrant cells

n:                     Number of assays

LCL:                Lower control limit (95%, mean-2SD)

UCL:               Upper control limit (95%, mean+2SD)

Table 2:  Summary: Experiment I, without and with metabolic activation

 

Dose Group

Concentration [µg/mL]

RICC [%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

without

4 h treatment,

21 h preparation interval

C

0

122

1.3

0.7

-0.28% - 3.70% aberrant cells excl. gaps

-

-

S

0

100

3.0

2.0

-

/

5

325

83

2.3

2.3

-

-

6

350

89

3.0

2.3

-

-

7

375

55

2.0

0.7

-

-

8

400

38

1.7

0.7

+

-

EMS

600

117

6.7

5.3

-

+

  

with

4 h treatment, 21 h preparation interval

C

0

105

4.0

2.3

-0.23% - 3.95% aberrant cells excl. gaps

-

-

S

0

100

5.0

3.3

-

/

4

500

92

3.3

2.3

-

-

5

550

63

3.0

2.3

-

-

6

600

43

4.7

3.7

-

-

CPA

0.83

100

10.0

9.0

-

+

C:       Negative Control (Culture Medium)

S:        Solvent Control (THF)

EMS: Ethylmethanesulfonate

CPA:  Cyclophosphamide

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the solvent control groups. The cell count was determined by a cell counter per culture for each test group.

a:        - without precipitation, + with precipitation

b:        statistical significant increase compared to solvent controls (Fisher’s exact test, p< 0.05),
+: significant; -not significant

Table 3:  Summary: Experiment II, without metabolic activation

 

Dose Group

Concentration [µg/mL]

RICC
[%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

Experiment II

21 h treatment,

21 h preparation interval

C

0

126

3.7

1.7

-0.20% - 2.71% aberrant cells excl. gaps

-

-

S

0

100

3.3

1.7

-

/

5

300

81

1.0

0.0

-

-

6

350

69

2.0

1.3

-

-

7

400

39

0.0

0.0

-

-

EMS

400

90

9.3

7.3

-

+

C:       Negative Control (Culture Medium)

S:        Solvent Control (THF)

EMS: Ethylmethanesulfonate

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the              solvent control groups. The cell count was determined by a cell counter per culture for each test group.

a:        - without precipitation, + with precipitation

b:        statistical significant increase compared to solvent controls (Fishers exact test, p< 0.05),
+: significant; -not significant

 

Table 4:  Summary: Experiment II (Repetition), without metabolic activation

 

Dose Group

Concentration [µg/mL]

RICC
[%]

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

Experiment II

21 h treatment,

21 h preparation interval

C

0

119

1.3

0.7

-0.20% - 2.71% aberrant cells excl. gaps

-

-

S

0

100

3.0

2.3

-

/

2

250

79

2.3

1.3

-

-

3

300

56

2.2

0.9

-

-

4

325

44

2.3

1.6

-

-

EMS

400

90

10.3

8.7

-

+

 

C:       Negative Control (Culture Medium)

S:        Solvent Control (THF)

EMS: Ethylmethanesulfonate

RICC: Relative Increase in Cell Count, calculated by the increase in cell number of the test groups compared to the solvent control groups. The cell count was determined by a cell counter per culture for each test group.

a:        - without precipitation, + with precipitation

b:        statistical significant increase compared to solvent controls (Fishers exact test, p< 0.05),
+: significant; -not significant

Conclusions:
In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item trichloro(4-methyl)silane did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item trichloro(4-methyl)silane is considered to be non-clastogenic in this chromosome aberration test.
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 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 trichloro(4 -methylphenyl)silane (Eurofins, 2017). The study was conducted in accordance with OECD 490 and GLP. In the study, mouse lymphoma L5178Y cells were treated with the test material or vehicle (tetrahydrofuran) at concentrations of 0.300, 0.325, 0.350, 0.375, 0.400, 0.425, 0.450, 0.475, 0.500 μL/mL 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 0.225, 0.250, 0.275, 0.300, 0.325, 0.350, 0.375, 0.400 μL/mL for 4 hours. Appropriate solvent and positive controls were included in the test and gave the expected result. Growth inhibition was observed both with and without metabolic activation. Without metabolic activation, the relative total growth (RTG) was 10.1% for the highest concentration (0.400 mg/mL); with metabolic activation, the RTG was 9.6% at 0.500 mg/L. Mutant frequencies without metabolic activation were not statistically significantly increased over the solvent controls. With metabolic activation, there was a statically significant increase in mutant frequencies over the solvent controls at 0.350, 0.375, and 0.475 mg/mL; however, these increases were not considered treatment related because there was no evidence of a dose-relationship. Additionally, historical data for mutant frequencies were within the historical data. Therefore, these increases were not considered to be a positive response. Based on these results, trichloro(4 -methylphenyl)silane 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.

 

Genetic toxicity (mutagenicity) in bacterial cells in vitro

An in vitro bacterial cell gene mutation assay with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537, and TA 102 both with and without metabolic activation was performed using trichloro(4 -methylphenyl)silane (Eurofins, 2018). The study was conducted in accordance with OECD 471 and GLP. In two independent experiments several concentrations of the test substance were used (0.0020 to 2.5 µL/plate). Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. No precipitation of the test substance was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test substance were noted in all tester strains used in experiment I and II: In experiment I, toxic effects of the test substance were observed at concentrations of 1.0 µL/plate and higher (with and without metabolic activation), depending on the particular tester strain. In experiment II, toxic effects of the test item were noted at concentrations of 0.63 µL/plate and higher (with and without metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with trichloro(4 -methyl)silane at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, trichloro(4 -methyl)silane did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, trichloro(4 -methyl)silane is considered to be non-mutagenic in this bacterial reverse mutation assay.

 

Cytogenicity in vitro

An in vitro chromosome aberration assay in Chinese hamster V79 cells both with and without metabolic activation was performed using trichloro(4 -methylphenyl)silane (Eurofins, 2018). The metaphases were prepared 21 h after start of treatment with the test substance. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 21 h without metabolic activation. Duplicate cultures were treated at each concentration. 150 metaphases per culture were scored for structural chromosomal aberrations. Concentrations between 250 and 600 µg/mL were evaluated. In experiment I, precipitation of the test substance was noted without metabolic activation at concentrations of 400 µg/mL and higher with the unaided eye. No precipitation of the test substance was noted in experiment I with metabolic activation and in experiments II without metabolic activation in all concentrations evaluated. In experiment I without metabolic activation, cytotoxic effects of the test substance were noted at concentrations of 375 µg/mL and higher. With metabolic activation cytotoxic effects of the test substance were noted at concentrations of 550 µg/mL and higher. In experiment II without metabolic activation, cytotoxic effects of the test substance were observed at concentrations of 350 µg/mL and higher. In the repetition experiment II without metabolic activation, cytotoxic effects of the test substance were observed at concentrations of 300 µg/mL and higher. In all experiments, no biologically relevant increase of aberration rates was noted after treatment with the test substance without and with metabolic activation. The aberration rates of all dose groups treated with the test substance were within the historical control data of the negative control. Moreover, no statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the dose groups of the test substance determined with the Fisher´s exact test. The chi² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations, but no statistically significant increase was observed in all experimental conditions. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the solvent controls.

EMS (400 and 600 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases of chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test substance trichloro(4 -methyl)silane did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test substance trichloro(4 -methyl)silane is considered to be non-clastogenic in this chromosome aberration test.

 

In conclusion, based on the available data with the registered substance it was concluded that the registered substance does not have genotoxic potential.

Justification for classification or non-classification

The available in vitro genotoxicity data are reliable and suitable for classification. Based on this data, classification for mutagenicity according to Regulation 67/584/EEC and (EC)1272/2008 is not warranted.