(3-chloropropyl)triethoxysilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without activation in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 (OECD Test Guideline 471) (Laboratory of Pharmacology and Toxicology KG 2002).
Cytogenicity in mammalian cells: negative with and without metabolic activation in cultured human lymphocytes (OECD Test Guideline 473) (Harlan Laboratories Ltd 2010a).
Mutagenicity in mammalian cells: negative with and without metabolic activation in L5178Y mouse lymphoma cells (OECD Test Guideline 476) (Harlan Laboratories 2010b).

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:

2002-04-03 to 2002-05-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

100-5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: none given in report

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100 and TA 1535 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

TA 102 without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthracene amide

Remarks:

TA 98, TA 102, TA 1537 with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

TA 100, TA 1535 with metabloic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation;
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

NUMBER OF REPLICATIONS: 3 plates per concentration and 2 independent experiments

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn

Evaluation criteria:

A response is considered positive if there is a statistically significant dose-related increase in the number of revertants relative to the solvent control, by at least 2-fold (TA 98, 100 and 102) or 3-fold (TA 1535 or 1537), and that this is reproducible and revertants are confirmed by streaking to histidine-free agar plates.

Statistics:

Mann and Whitney U-test and Spearman's rank correlation coefficient.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preincubation method: 316 µg/plate -S9; 3160 µg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preincubation method: 5000 µg/plate -S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preincubation method: 1000 µg/plate - S9 and +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Plate incorporation method: 5000 µg/plate -S9; 316 µg/plate + S9 Preincubation method: 316 µg/plate -S9; 3160 µg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Pre-incubation method: 316 µg/plate -S9; 1000 µg/plate +S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Table 1 Plate incorporation test average number of revertants per plate (mean of 3 plates)

 

Concentration (µg/plate)	TA 98		TA 100		TA 102		TA 1535		TA 1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
5000	35.3	34.3	179.7	157.3	150.7	151.7	0.0*	0.0*	4.0*	3.7*
3160	31.7	26.3	134.0	137.3	172.3	120	39.7*	0.0*	2.7*	5.7*
1000	40.3	19.0	122.7	128.0	161.7	155.7	22.7	0.0*	5.0	5.3*
316	32.0	27.3	121.3	166.0	172.3	202	28.0	1.3	2.7	5.3
100	46.7	28.3	109.3	159.7	197.3	240.7	22.7	37.7	4.7	4.3
Negative control 100 µL/plate	32.0	36	119.0	153.0	220.7	292.0	16.3	27.7	5.3	3.7
Positive control	817.0	333	447.3	491.7	1231.0	905.7	957.3	841.3	290.3	290.7

 * = scarce background lawn

Table 2 Preincubation test average number of revertants per plate (mean of 3 plates)

Concentration (µg/plate)	TA 98		TA 100		TA 102		TA 1535		TA 1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
5000	0.0*	0.0*	31.0*	72.7*	1.3*	0.0*	0.0*	0.0*	0.0*	0.0*
3160	10.3*	0.0*	65.3*	80.3*	0.0*	0.0*	0.0*	7.0*	0.0*	2.3*
1000	10.7*	50.0*	93.0*	116.3*	74.3*	0.0*	0.0*	18.7*	0.0*	0.0*
316	12.3	25.7	119.7	135	280	340.7	3.3	20.7	3	13.3
100	19.7	29.7	102.3	132.3	275.3	370.7	11	17.3	3.7	12.3
Negative control 100 µl/plate	32.3	35	119.7	126	246.7	364.7	12	18.7	6.3	14.7
Positive control	303.3	313	464	477	1131	1153.7	891	984.3	286.3	288.7

* = scarce background lawn

Conclusions:

(3-chloropropyl)triethoxysilane has been tested in a valid and reliable study according to OECD Test Guideline 471 and in compliance with GLP. No increase in the number of revertants was observed in any strain with or without activation in either the initial plate incorporation assay or the subsequent preincubation assay. The solvent and positive controls gave the expected results. It is concluded that the substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-21 to 2009-11-30

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

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

primary culture, other: peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone and beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

18.75 - 250 µg/ml (4 h exposure, +/-S9; 20 h exposure -S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: recommended by sponsor and formed clear solution suitable fo rdosing

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

ACTIVATION: S9 mix contained NADP as cofactor and S9 was present at a final concentration of 2%

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hours (with and without activation) 24 hours (without activation)
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 µg/ml) two hours before harvest
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Duplicate cultures.

NUMBER OF CELLS EVALUATED: 2000 (mitotic index); 100/culture (chromosome damage)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A result was considered positive if the percentage of cells with aberrations, excluding gaps markedly exceeded that seen in the concurrent control,
either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

Only applied in event of a positive result.

Species / strain:

primary culture, other: peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

150 µg/ml (4 h exp -S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: controls were within the range of historical data

Table 1Results of Chromosome Aberration Test 4 hours incubation, without metabolic activation (S9)

 

Concentration µg/ml	No. of cells examined	Mitotic index % mean	Cells with aberrations % mean including gaps	Cells with aberrations % mean excluding gaps
Vehicle control	200	100	0.5	0.5
18.75	200	82	0.5	0
37.5	200	111	1	0.5
75	200	107	0	0
150	200	33	2	1.5
MMC 0.4	100a	55	59	56

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

 

Table 2 Results of Chromosome Aberration Test 4 hours incubation, with metabolic activation (S9)

Concentration µg/ml	No. of cells examined	Mitotic index % mean	Cells with aberrations % mean including gaps	Cells with aberrations % mean excluding gaps
Vehicle control	200	100	0.5	0
75	200	95	0	0
150	200	90	0	0
200	200	45	0	0
CP 5	150	31	26.7	26.7

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

Table 3 Results of Chromosome Aberration Test 24 hours incubation, without metabolic activation (S9)

Concentration µg/ml	No. of cells examined	Mitotic index % mean *	Cells with aberrations % mean including gaps	Cells with aberrations % mean excluding gaps
Vehicle control	200	100	0.5	0.5
37.5	200	124	1	1
75	200	132	1.5	1
150	200	86	0.5	0
200	200	25	b	b
MMC 0.2	100a	41	33	33

a = Slide evaluation terminated at 50 cells because at least 30% cells with aberrations had been observed

b = Too toxic for metaphase analysis

Conclusions:

(3-chloropropyl)triethoxysilane has been tested in a valid and reliable study according to OECD Test Guideline 473 and in compliance with GLP. No increase in the number of cells with aberrations was observed with or without activation. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in peripheral human lymphocytes under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-12-15 to 2010-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997 (prior to guideline 490)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone, beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

4.69 to 150 µg/ml (4 and 24 h exposure without activation); 9.38 to 300 µg/ml (4 hour exposure with activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

ACTIVATION: S9 mix contained NADP as cofactor and S9 was present at a final concentration of 2%
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 4 hour (with and without activation); 24 hour (without activation)
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10-14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): 4 µg/ml 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate treatments

NUMBER OF CELLS EVALUATED: 2000 cells plated in selective medium for mutant frequency; 2 cells per well to evaluate viability

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

OTHER EXAMINATIONS:
- Other: Large and small colony evaluation

OTHER:

Evaluation criteria:

To be considered positive, a substance must induce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. The mutation frequency must also exceed the vehicle value by the Global Evaluation Factor (GEF) of 126 x 10-06 (International Workshop on Genotoxicity Test Procedures, Moore et al 2003, 2006)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

150 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Table 1 Summary of Results, 4 hour treatment

Treatment (µg/ml)	4-hours -MA			Treatment (µg/ml)	4-hours +MA
	%RSG	RTG	MF		%RSG	RTG	MF
0	100	1	122.75	0	100	1	153.76
4.69	99	NE	NE	9.38	90	1.23	125.84
9.38	103	NE	NE	18.75	90	1.16	151.25
18.75	100	0.92	129.75	37.5	92	1.17	135.38
37.5	111	0.94	131.85	75	85	1.05	169.31
75	96	0.91	148.54	150	60	0.68	148.29
100	76	0.7	140.85	200	3	0.02	333.47
125	35	0.33	118.37	250	1	NE	NE
150	5	0.04	178.71	300	1	NE	NE
EMS 400	79	0.7	650.26	CP 2	57	0.2	1243.63

NE not evaluated

RSG Relative suspension growth

RTG Relative total growth

MF mutant frequency

Table 2 Summary of Results, 24 hour treatment

Treatment (µg/ml)	24-hours -MA
	%RSG	RTG	MF
0	100	1	102.37
4.69	96	NE	NE
9.38	92	0.94	97.25
18.75	94	0.98	108.58
37.5	89	0.99	93.55
75	72	0.88	73.5
100	49	0.57	129.81
125	30	0.41	81.87
150	3	NE	NE
EMS 150	61	0.49	911.5

NE not evaluated

RSG Relative suspension growth

RTG Relative total growth

MF mutant frequency

Conclusions:

(3-chloropropyl)triethoxysilane has been tested in a valid and reliable study according to OECD Test Guideline 476 and in compliance with GLP. No increase in the number of revertants was observed with or without metabolic activation. The vehicle and positive controls gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Information is available from reliable studies for all the required in vitro endpoints. The results of most of the studies were in agreement. A positive result was obtained in one study in a single strain of bacteria in the presence of activation, but only when ethanol was used as the solvent. This result was not confirmed in a more recent study also using ethanol as solvent, nor in the other available bacterial mutagenicity assays, so it is concluded that the positive response was not biologically relevant, and the most recent study was chosen as the key study.

No test substance related increase in the number of revertants relative to the solvent control was observed when (3-chloropropyl)triethoxysilane (CAS 5089-70-3; EC 225-805-6) was tested in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 in accordance with OECD Test Guideline 471 and in compliance with GLP. The results obtained with and without metabolic activation in either the initial plate incorporation assay or the subsequent pre-incubation assay were in agreement. The solvent (ethanol) and positive controls were included and gave the expected results. The test substance was therefore concluded to be negative for mutagenicity to bacteria under the conditions of the test (Laboratory of Pharmacology and Toxicology KG, 2002).

(3-Chloropropyl)triethoxysilane has been tested for mutagenicity in bacteria according to a protocol that is similar to OECD Test Guideline 471 and  in compliance with GLP using the pre-incubation method and Salmonella strains (TA98, TA100, TA1535, TA1537) and E.coli strains (WP2 uvrA and WP2), in the presence and absence of metabolic activation.

No increase in the number of revertants was detected in any strain with or without metabolic activation when tested using DMSO as the vehicle. When the study was repeated using ethanol as solvent, there were difficulties with dosing levels, so parts of the experiment had to be repeated. An increase in the number of revertants (5.6-fold) was observed in strain TA 1535 in the presence of activation only. The experiment was not repeated to confirm if the positive result was reproducible. It is concluded that the test substance is positive for mutagenicity to bacteria in the presence of metabolic activation when ethanol is used as solvent (Dow Corning Corporation, 1995).

(3-Chloropropyl)triethoxysilane has been tested for mutagenicity in bacteria according to a protocol that is similar to OECD Test Guideline 471 and in compliance with GLP using the pre-incubation method and Salmonella strains (TA98, TA100, TA1535, TA1537) and E.coli strains (WP2 uvrA and WP2), in the presence and absence of metabolic activation.

No increase in the number of revertants was observed in any strain at any test concentration in the presence or the absence of metabolic activation. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for mutagenicity under the conditions of the test (Dow Corning Corporation, 1994).

(3-Chloropropyl)triethoxysilane has been tested for ability to cause chromosome aberrations in human lymphocytes cells according to OECD Test Guideline 473 and in compliance with GLP. Three treatment conditions were used for the study; 4-hour exposure in the absence of metabolic activation with a 20-hour expression period; 4-hour exposure in the presence of metabolic activation at a 2% final concentration with cell harvest after a 20-hour expression period and a 24-hour continuous exposure in the absence of metabolic activation. No statistically significant increase in the number of cells with aberrations was observed with or without activation. Vehicle and positive controls gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations in peripheral human lymphocytes under the conditions of the test (Harlan Laboratories Ltd, 2010a).

(3-Chloropropyl)triethoxysilane has been tested for mutagenicity to mammalian cells in mouse lymphoma L5178Y cells according to OECD Test Guideline 476 and in compliance with GLP. The cells were treated with the test material at eight dose levels, in duplicate, together with vehicle (Acetone) and positive controls for 4 hours both with and without metabolic activation, and for 24 hours without metabolic activation. No increase in the number of revertants was observed with or without metabolic activation. The vehicle and positive controls gave the expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the test (Harlan Laboratories, 2010b).

Justification for classification or non-classification

Based on the in vitro available data, (3-chloropropyl)triethoxysilane (CAS 5089-70-3) does not require classification for germ cell mutagenicity in accordance with Regulation (EC) No 1272/2008.