4,4,13,13-tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key study for gene mutation conducted according to OECD test guideline 471 (Bacterial reverse mutation assay / Ames test) and in compliance with GLP was negative with and without metabolic activation in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA (OECD TG 471) (BioReliance, 2000). This test was conducted on low purity 4,4,13,13-tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane (low purity "S2").

The key study for cytogenicity in mammalian cells conducted according to OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) and in compliance with GLP was negative with and without metabolic activation in Chinese hamster lung fibroblasts (V79) (ASTA Medica, 1998a). This study was conducted using the registered substance S2 (CAS No. 56706-10-6, EC No. 260-350-7).

The key study for mutagenicity in mammalian cells conducted according to OECD test guideline 476 and in compliance with GLP used read-across from bis[3-(triethoxysilyl)polysulfides (“polysulfides”; CAS No. 211519-85-6, EC No. 915-673-4) and was negative in L5178Y mouse lymphoma cells (Harlan, 2010).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-08-03 to 1998-08-12

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Exp 1:7.5-120 µg/ml -S9, 10-500 µg/ml +S9; exp 2: 5-80 µg/ml -S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification: recommended by Sponsor

Untreated negative controls:

yes

Remarks:

test medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without activation

Untreated negative controls:

yes

Remarks:

test medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours (with metabolic activation); 4, 18 and 28 hours (without metabolic activation)
- Expression time (cells in growth medium): 18 and 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Duplicate cultures; independent repeat experiment

NUMBER OF CELLS EVALUATED: 200 metaphases per experimental group per experiment were examined for structural chromosomal abnormalities. 1000 cells were scored to determine the mitotic index.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS
- Determination of polyploidy: yes
- Determination of endoreplication: no data

METABOLIC ACTIVATION:
- Aroclor induced rat liver S9 prepared from Wistar rats with 43.7 mg/ml protein.
- S9 mix included 10% S9 and cofactor solution including MgCl2, KCl, Glucose-6-phosphate and NADP. Final concentration of S9 in cultures was approximately 1%.

Evaluation criteria:

A substance was considered negative when there was no statistically significant, biologically relevant and reproducible positive response at any test point, and no concentration related statistically significant and biologically relevant increase in structural chromosomal aberrations compared with the solvent control group.

Statistics:

Frequencies of metaphases with structural aberrations of test substance and positive control groups were compared with those of the solvent control group. A Chi squared test was applied.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>50 % reduction in mitotic index at 40 μg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>50 % reduction in mitotic index at 40 μg/ml (without metabolic activation); 100 μg/ml (with metabolic activation)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant effect
- Effects of osmolality: no significant effect
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: non-interfering precipitate noted at 100 μg/ml and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: preliminary range finding test was carried out and cytotoxicity determined: 40 μg/ml (without metabolic activation) and 100 μg/ml (with metabolic activation

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

Table 1 Chromosome aberrations Experiment 1

Without metabolic activation 4 h treatment, 18 h sampling
Treatment (μg/ml)	Number of cells analysed	Mitotic Index (mean of two cultures %)	Number of cells with Aberrations		% of cells with aberrations
			inc Gaps	excl Gaps	inc Gaps	excl Gaps
0^1	100+++	31.0	7	7	7.0	7.0
0^2	200	51.0	15	11	7.5	5.5
15	200	29.0	16	10	8.0	5.0
30	200	37.0	11	8	5.5	4.0
60	192++	6.5	11	9	5.7	4.7
Positive control	200	22.5	114**	110**	57.0	55.0
With metabolic activation 4 h treatment, 18 h sampling
Treatment (μg/ml)	Number of cells analysed	Mitotic Index (mean of two cultures %)	Number of cells with Aberrations		% of cells with aberrations
			inc Gaps	excl Gaps	inc Gaps	excl Gaps
0^1	200	78.5	16	12	8.0	6.0
0^2	200	106.5	13	9	6.5	4.5
31.6	200	70.5	12	8	6.0	4.0
100^P	200	106.0	22	16	11.0	8.0
60^P	200	28.5	21	18	10.5	9.0
Positive control	200	56.0	49**	47**	24.5	23.5
With metabolic activation 4 h treatment, 28 h sampling
Treatment (μg/ml)	Number of cells analysed	Mitotic Index (mean of two cultures %)	Number of cells with Aberrations		% of cells with aberrations
			inc Gaps	excl Gaps	inc Gaps	excl Gaps
0^1	100+++	77.0	5	7	5.0	4.0
0^2	200	90.5	18	15	9.0	7.5
31.6	200	75.0	8	7	4.0	3.5
100^P	200	99.0	11	8	5.5	4.0
60^P	200	52.5	22	19	11.0	9.5
Positive control	200	109.0	27	21	13.5	10.5

 

¹Negative control with culture medium

²Negative control with solvent (DMSO)

++ maximum number of evaluable metaphases on the slides examined

+++ determined from one culture only

* p<0.05

* p <0.01

^PPrecipitation Table 2 Chromosome aberrations Experiment 2

Without metabolic activation 18 h treatment, 18 h sampling
Treatment (μg/ml)	Number of cells analysed	Mitotic Index (mean of two cultures %)	Number of cells with Aberrations		% of cells with aberrations
			inc Gaps	excl Gaps	inc Gaps	excl Gaps
0^1	89+++	46.0	5	3	5.6	3.3
0^2	190++	32.5	11	7	5.8	3.7
10	142++	15.0	11	7	7.7	4.9
20	153++	16.0	10	7	6.5	4.6
40	200	8.0	11	7	5.5	3.5
Positive control	108++	12.5	12.5	26**	24.0	19.4
Without metabolic activation 28 h treatment, 28 h sampling
Treatment (μg/ml)	Number of cells analysed	Mitotic Index (mean of two cultures %)	Number of cells with Aberrations		% of cells with aberrations
			inc Gaps	excl Gaps	inc Gaps	excl Gaps
0^1	37+++	2.0	4	4	10.8	10.8
0^2	200	42.0	15	13	7.5	6.5
10	200	37.0	25	17	12.5	8.5
20	151++	43.5	6	4	4.0	2.6
40	88++	6.0	5	4	5.7	4.5
Positive control	158++	27.5	5.**	47**	31.6	29.7

¹Negative control with culture medium

²Negative control with solvent (DMSO)

++ maximum number of evaluable metaphases on the slides examined

+++ determined from one culture only

* p<0.05

* p <0.01

Conclusions:

S2 has been tested according to OECD 473 and in compliance with GLP. No test substance-induced structural or numerical chromosomal aberrations were observed in Chinese hamster V79 fibroblasts when tested up to cytotoxic concentrations, with and without metabolic activation. The experiment was repeated and the results confirmed. Appropriate solvent and positive controls were included and gave expected results. It is considered that the substance is negative for cytogenicity under the conditions of the test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-11-18 to 2000-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test substance was similar to the registered substance but contained a higher proportion of an S3 isomer.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

75-5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Sponsor's request and compatibility with target cells.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All Salmonella Strains, WP2 uvrA (with 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 activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 (without 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 activation)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA (without activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar, preincubation


DURATION
- Preincubation period: 60 minutes

- Expression time (cells in growth medium): 48 to 72 hours

- Fixation time (start of exposure up to fixation or harvest of cells): 12 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration; study repeated

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn monitoring

Evaluation criteria:

For the test article to be positive, it must cause a dose-related increase in mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations per test article.

A result is positive if the number of revertants is significantly increased compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and WP2 uvrA, and 3-fold of the solvent control for TA 1535 and TA 1537.

Cytotoxicity is defined as a reduction in the number of colonies by >50% compared with the solvent control and/or a sparse background lawn.

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:

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data

Table 2: Experiment 1 Preliminary toxicity assay Number of revertants per plate

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	—  MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	NC***	30	No	128	172	No	15	8	No
6.7	14	29	No	145	124	No	19	14	No
10	18	22	No	150	171	No	19	11	No
33	12	19	No	142	177	No	12	13	No
67	21	28	No	170	193	No	16	10	No
100	9	19	No	136	189	No	19	13	No
333	17	24	No	138	181	No	23	23	No
667	23	23	No	147	162	No	15	15	No
1000	14**	24**	No	132**	169**	No	19**	25**	No
3333	15**	17**	No	167**	184**	No	24**	16**	No
5000	21**	26**	No	139**	199**	No	23**	15**	No

*solvent control with DMSO

**Non-Interfering Precipitate

***No count due to procedural error in which plate did not receive an aliquot of tester strain

Table 2: Experiment 1 Preliminary toxicity assay Number of revertants per plate

	TA1537			WP2 uvrA
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	56	45	No	14	16	No
6.7	49	42	No	19	15	No
10	37	41	No	12	19	No
33	49	43	No	12	17	No
67	47	40	No	10	10	No
100	47	46	No	13	17	No
333	37	48	No	9	12	No
667	48	39	No	10	16	No
1000	37**	67**	No	11**	12**	No
3333	45**	52**	No	8**	18**	No
5000	44**	55**	No	15**	14**	No

*solvent control with DMSO

**Non-Interfering Precipitate

***No count due to procedural error in which plate did not receive an aliquot of tester strain

Table 3: Experiment 2 Preincubation mutagenicity assay, Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	16	21	No	174	203	No	13	15	No
75	9	25	No	161	217	No	13	16	No
200	14	24	No	166	213	No	17	16	No
600	10	18	No	159	212	No	17	18	No
1800	13	17	No	169	232	No	14	16	No
5000	9	17	No	140	209	No	16	18	No
Positive control	981	1350	No	626	1197	No	370	140	No

*solvent control with DMSO

Table 3: Experiment 2 Preincubation mutagenicity assay, Number of revertants per plate (mean of 3 plates)

	TA1537			WP2 uvrA
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	8	9	No	12	14	No
75	4	6	No	9	12	No
200	5	5	No	8	13	No
600	4	9	No	9	11	No
1800	4	5	No	9	15	No
5000	5	8	No	9	6	No
Positive control	827	149	No	495	462	No

*solvent control with DMSO

Table 4: Experiment 2 Preincubation (Repeat) mutagenicity assay, Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	—  MA	+ MA	Cytotoxic (yes/no)	—  MA	+ MA	Cytotoxic (yes/no)
0*	17	16	No	152	160	No	11	10	No
75	14	14	No	145	171	No	7	13	No
200	15	19	No	135	189	No	7	15	No
600	14	10	No	137	159	No	10	9	No
1800	16	14	No	143	156	No	9	12	No
5000	15	12	No	115	173	No	8	7	No
Positive control	409	785	No	677	759	No	207	68	No

*solvent control with DMSO

Table 4: Experiment 2 Preincubation (Repeat) mutagenicity assay, Number of revertants per plate (mean of 3 plates)

	TA1537			WP2 uvrA
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	5	6	No	10	10	No
75	4	4	No	12	6	No
200	4	6	No	9	8	No
600	5	5	No	12	10	No
1800	2	7	No	9	12	No
5000	4	5	No	11	8	No
Positive control	719	71	No	317	75	No

*solvent control with DMSO

Conclusions:

S2 has been tested a reliable assay conducted according to OECD TG 471 and in compliance with GLP. The test substance did not cause a positive response in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with or without metabolic activation in either the initial or repeat tests using the preincubation method. Appropriate solvent and positive controls were included and gave expected results. The test substance is negative for mutagenicity to bacteria 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:

2009-08-11 to 2009-09-14

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

0.31 to 20 μg/ml (4 and 24 h -S9); 5 to 60 μg/ml (4 h +S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: none
- Exposure duration: 4 h (with and without activation) 24 hours (without activation
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 5-trifluorothymidine

NUMBER OF REPLICATIONS: cell cultures treated in duplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: relative suspension growth; 2 day viability

OTHER: microwells used

ACTIVATION: 20% S9 mix contained S9, 8 mM of MgCl2, 33 mM of KCl, 5 mM of glucose-6-phosphate and 5 mM of NADP. The final concentration of S9 was 25 throughout the study.

Evaluation criteria:

A substance is judged to be positive if it produces a reproducible, dose-dependent, statistically significant increase in the mutant frequency relative to the vehicle control, by a factor that equals or exceeds the global evaluation value for the microwell method of 126 E-06.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

25 µg/ml (4 and 24 h without activation)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at or above 194.19 µg/ml in all exposure groups

COMPARISON WITH HISTORICAL CONTROL DATA: control values were within historical control data

Table 1 Preliminary toxicity test

Dose (μg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0*	100	100	100
12.4	74	96	72
24.27	90	96	26
48.55	83	84	0
97.09	100	68	0
194.49	95	82	1
388.38	94	102	1
776.75	108	98	1
1553.5	100	102	4
3107	97	82	39

* solvent control with ethanol

Table 2 Main experiment Relative suspension growth, relative total growth and mutant frequency

Treatment µg/ml	4 hours - S9			Treatment µg/ml	4 hours + S9
	% RSG	RTG	MF		% RSG	RTG	MF
0*	100	1.00	89.99	0*	100	1.00	114.53
12.5	112	1.29	89.46	12.5	113	1.04	91.57
25	111	1.28	82.23	25	106	1.20	95.58
50	121	1.42	89.78	50	102	1.05	101.72
100**	110	1.28	79.77	100	105	1.09	93.79
150**	109	1.28	86.92	150**	104	1.10	88.74
200**	122	1.38	86.12	200**	106	1.17	98.22
Positive control	94	0.81	710.52	Positive control	65	0.29	935.61

RSG: Relative suspension growth,

RTG: relative total growth and mutant frequency

MF: mutant frequency

*Solvent control with ethanol

**Precipitate observed

Table 3 Main experiment Relative suspension growth, relative total growth and mutant frequency

Treatment µg/ml	24 hours - S9
	% RSG	RTG	MF
0*	100	1.00	96.68
1.56	109	NC	NC
3.13	102	0.95	108.38
6.25	101	1.29	76.85
12.5	104	1.21	113.47
18.75	89	1.02	93.93
25	59	0.69	89.8
37.5	16	0.14	141.03
50	9	NC	NC
Positive control	9	0.69	992.94

RSG: Relative suspension growth,

RTG: relative total growth and mutant frequency

MF: mutant frequency

NC not counted

*Solvent control with ethanol

Conclusions:

Polysulfides (CAS 211519 -85 -9; EC 606-716-5) has been tested for mutagenicity to mouse lymphoma L5178Y cells in a study according to OECD Test Guideline 476 and in compliance with GLP. No increase in the number of revertants was observed. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the mutagenicity in mammalian cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The key study conducted according to OECD test guideline 474 (Mammalian Erythrocyte Micronucleus Test) and in compliance with GLP administered the test substance intraperitoneally in mice. It was concluded that the test substance is negative for the induction of micronuclei in vivo (ASTA Medica, 1998b).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 September 1998 - 3 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Harlan-Winkelmann GmbH, D-33176 Borchen

- Age at study initiation: approximately 7 weeks

- Weight at study initiation: males: 27.4-33.8 g; female: 22.0-26.1g

- Assigned to test groups randomly: yes, under following basis: computerised random number generator.

- Fasting period before study:

- Housing: Macrolon cages type II, 1 animal per cage

- Diet (e.g. ad libitum): ad libitum

- Water (e.g. ad libitum): ad libitum

- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS

- Temperature (°C): 21.0-21.5°C

- Humidity (%): 50-57%

- Air changes (per hr):

- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light


IN-LIFE DATES: From: To: 3 Sept 1998

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: peanut oil
- Lot/batch no. (if required): 1936301

Details on exposure:

Route of exposure: intraperitoneal

Duration of treatment / exposure:

46-48 hours

Frequency of treatment:

2 intraperitoneal administrations of 10 ml/kg bw with an interval of approximately 24 hours (test material and negative control group). Positive control group received a single injection of 10 ml/kg bw

Post exposure period:

Animals were sacrificed 22-24h after second (test and negative control) or single (positive control) administration.

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

- cyclophosphamide
- Justification for choice of positive control(s): standard guideline positive control
- Route of administration: intraperitoneal
- Doses / concentrations: 10 ml/kg bw of 0.9% solution in physiological saline

Tissues and cell types examined:

See table 2

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: limit dose
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): no further information


DETAILS OF SLIDE PREPARATION: centrifuged cells were suspended in a thin layer of serum, and a small drop was smeared on a slide and air-dried overnight. The dried slides were stained using the panoptic stain method of Pappenheim (Queisser W (1978) Das Knochenmark, Georg Thieme Verlag, Stuttgart)


METHOD OF ANALYSIS: microscopic examination. 2000 PCE scored for incidence of polychromatic erythrocytes with micronuclei. Ratio of PCE/NCE was scored based on 1000 erythrocytes (PCE+NCE)

Statistics:

Frequencies of PCE with micronuclei of test material and of positive control group were compared with those of the negative control group. A Poisson test was applied. Data from each treatment group for each sex and for the sexes combined were compared with appropriate negative control using software from ASTA medica AG and an Alpha computer (Digital Equipment Corp)

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See table 3
RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay): negative

- Ratio of PCE/NCE (for Micronucleus assay): see table 3. The PCE/NCE ratio was unchanged in treated animals, so there is no evidence that the test substance reached the target tissue.

- Appropriateness of dose levels and route: appropriate dose and route

- Statistical evaluation: no statistically significant induction of micronuclei occurred

Table 3: Results of in vivo micronucleus test with Silane Si 266

 

		Vehicle Control	Positive control	2000 mg/kg bw
Number of cells evaluated		2000	2000	2000
Sampling time (h)		24	24	24
Number of erythrocytes per animal	normo­chromatic	NR	NR	NR
	poly­chromatic	2000	2000	2000
	polychromatic with micronuclei	3.4	38.1	3.4
Ratio of erythro­cytes	polychromatic / normochromatic	1.9*	1.5*	1.8*
	polychromatic with micro­nuclei / normochromatic	NR	NR	NR

 * Mean calculated from data for individual animals

PCE polychromatic erythrocyte

NR not recorded

Conclusions:

S2 was tested in an in vivo mouse micronucleus assay conducted to OECD 474 and in compliance with GLP. No evidence of genotoxicity was observed under the conditions of the test. It should be noted that the PCE/NCE ratio was unchanged in treated animals, so there is no evidence that the test substance reached the target tissue. It is concluded that the test substance is negative for the induction of micronuclei in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Information is available from reliable studies for all the required in vitro endpoints. The most reliable bacterial mutagenicity study was conducted using low purity S2, which is composed of 65-70% S2, 15-20% S3 and 5-10% S1 as impurity, and therefore does not have sufficient S2 to meet the Substance Identification Profile, but the other components have similar properties and the supporting studies confirmed that the other constituents did not affect the results. Where reliable studies were not available for the test substances, a study for a multi-component substance (polysulfides) including the registration substance was selected. No data are available for mammalian cell gene mutagenicity for the registered substance, however, data are available for polysulfides. The results of all the available genetic toxicity studies were consistent.

Read-across justification

Non-testing methods including read-across from surrogate substances are able to provide information on mutagenic toxicity (REACH Guidance part 07a, R.7.7.3). In the case of genetic toxicity the presence or absence of functional groups that are known to be related to genetic toxicity is considered important, as the presence or absence of reactive groups and molecular substructures is associated with mutagenic and carcinogenic properties of chemicals (Benigni et al., 2008). Consideration is therefore given to the structural similarity, particularly presence or absence of structural alerts for genetic toxicity, when selecting surrogate substances for genetic toxicity endpoints.

Read-across hypothesis

S2, low purity S2 and polysulfides, are closely related substances. S2 includes constituents of polysulfides (S3 and S4), as impurities and polysulfides includes 15-25% S2, with S3 and S4 as the other major constituents. Low purity S2 includes up to 70% S2, with S3 as minor constituent and S1 as impurity. Due to their structural similarity and similar physicochemical properties, the constituents S3 and S4 are considered to be representative of the registered substance.

(a) Structural similarity

The constituents and major impurities of all substances in the analogue have two functional groups in common:

• The triethoxysilane, Si(OEt)₃, group (there are two of these per molecule, therefore each molecule has six reactive ethoxy groups).

• The (poly)sulfide, CH₂S_nCH₂ (n = 1 -4), group.

Only one of the substances, low purity S2, has n=1, as an impurity at a concentration of 5-10%. None of the substances contain functional groups that are not present in the other substances. Clearly the only difference between the constituents/major impurities is the number of sulfur atoms in the sulfide bridge.

The registration and read-across substances are structurally similar, all contain bis[3-(triethoxysilyl)propyl]- structures with the two (triethoxysilyl)propyl groups linked by di- or polysulfide groups. Details on the composition of the substances is given in Section 1.4 but in summary:

- S2 is a monoconstituent substance comprising >80% by weight of disulfide, with approximately 10-20% of the trisulfide as an impurity;

- Polysulfides is multiconstituent substance comprising a mixture of di- (S2, 15-25% w/w), tri- (S3, 30-35% w/w) and tetrasulfides (S4, 20-30% w/w).

- Low purity S2 comprises 65-70% S2, 15-20% S3 and 5-10% S1 as impurity.

(b) Lack of structural alerts for genetic toxicity

S2, low purity S2 and polysulfides do not include structural alerts for genotoxicity Benigni et al. (2008).

Low purity S2 has been tested in an assay conducted according to OECD Test Guideline 471 and in compliance with GLP (BioReliance, 2000). The test substance did not cause a positive response in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with or without metabolic activation in either the initial or repeat tests using the preincubation method. Appropriate solvent and positive controls were included and gave expected results. The test substance is negative for mutagenicity to bacteria under the conditions of the test. The result was supported by two other studies (Hita laboratory, 2000a, reliability score 2; TNO, 1996, reliability score 2) which were conducted to OCED test guideline 471 and in compliance with GLP and which gave negative results.

S2 has been tested according to OECD Test Guideline 473 and in compliance with GLP. No test substance-induced structural or numerical chromosomal aberrations were observed in Chinese hamster V79 fibroblasts when tested up to cytotoxic concentrations, with and without metabolic activation. The experiment was repeated and the results confirmed. Appropriate solvent and positive controls were included and gave expected results. It is considered that the substance is negative for cytogenicity under the conditions of the test.

This study is supported by studies on Low purity S2 which was tested in two assays conducted using Chinese hamster lung fibroblasts (V79) according to OECD test guideline 473 and in compliance with GLP (Safepharm, 2002, reliability score 1; ASTA Medica, 1998a, reliability score 1). The results of these studies were negative.

The surrogate substance, polysulfides has been tested for mutagenicity in mouse lymphoma L5178Y cells according to OECD Test Guideline 176 and in compliance with GLP (Harlan, 2010). No increase in the number of revertants was observed with and without metabolic activation when tested up to cytotoxic concentrations. Appropriate solvent and positive controls were included and gave expected results. It was concluded that the test substance was negative for the mutagenicity in mammalian cells under the conditions of the test.

S2 was tested in an in vivo mouse micronucleus assay conducted according to OECD Test Guideline 474 and in compliance with GLP (ASTA Medica, 1998). No evidence of test substance induced increase in micronuclei was observed under the conditions of the test. It should be noted that the PCE/NCE ratio was unchanged in treated animals, so there is no evidence that the test substance reached the target tissue. It is concluded that the test substance is negative for the induction of micronuclei in vivo.

Benigni et al. (2008). The Benigni/Bossa rule base for mutagenicity and carcinogenicity JR Scientific report EUR 23241 EN

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data, 4,4,13,13 -tetraethoxy-3,14-dioxa-8,9-dithia-4,13-disilahexadecane (CAS 56706-10-6) is not classified for mutagenicity according to Regulation (EC) No 1272/2008.