Reaction products of [3-(2,3-epoxypropoxy)propyl]trimethoxysilane and triacetoxyvinylsilane.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Bacterial reverse mutation assay / Ames test, conducted according to OECD 471 and in compliance with GLP, the Salmonella typhimurium strains TA 100, TA 1535 and E. coli WP2 uvr A, although not TA 98 or TA 1537, were tested. The response was positive with and without activation (Harlan, 2010).

In a Mammalian Cell Gene Mutation Assay, conducted according to OECD Test Guideline 476 and in compliance with GLP, the test substance was positive with and without activation in mouse lymphoma L5178Y cells (Dow Corning Corporation, 1999).

In a Mammalian Chromosome Aberration Test, conducted according to OECD Test Guideline 473 and in compliance with GLP, the test substance was positive in peripheral human lymphocytes (WIL, 2015).

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:

2010-05-19 to 2010-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon (for the Salmonella strains), tryptophan operon (for the E.coli strain)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-napthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 2500, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: methanol
- Justification for choice of solvent/vehicle: solubility properties and relative nontoxicity to the bacteria

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100 (without metabolic activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 1537, TA 98 (without metabolic activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

methanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvr A (without metabolic activation)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

all strains (with metabolic activation)

Details on test system and experimental conditions:

ACTIVATION: The amount of S9 supernatant was 10% v/v in the cultures. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
-After solidification, the plates were incubated upside down for 48-72 hours at 37°C in the dark

SELECTION AGENT (mutation assays): histidine-deficient agar (Salmonella strains); tryptophan-deficient agar (E.coli strain)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: Toxicity of the test item results in a reduction in the mean number of spontaneous revertants (> 50% reduction) or a reduction of the bacterial background lawn, in comparison to the solvent control.

Evaluation criteria:

A test item is considered as a mutagen is a biologically relevant increase in the mean number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvr A) or thrice (strains TA 1535 and TA 1537) the mean colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

Statistics:

No statistical evaluation of the data was required.

Key result

Species / strain:

bacteria, other: TA 1535, TA 100, WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

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, other: TA 1537, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Plate incorporation test: Mean number of revertants per plate (3 plates)

Dose (µg/plate)	TA 1535		TA 1537		TA 98		TA 100		WP2 uvr A
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
3	14	29	10	14	26	36	118	124	39	49
10	18	22	9	16	24	41	101	122	42	46
33	20	24	10	16	27	38	118	129	43	52
100	33	33*	8	15*	28	37	123	128	44	50
333	73	72*	9	18*	27	43*	155	132*	45	56*
1000	133	151*	14	18*	25	37*	214	193*	63	57*
2500	242	270*	11	13*	23	35*	374	283*	83	64*
5000	404	407*	14	15*	25	38*	662	511*	129	105*
Untreated	16	19	9	19	27	45	121	127	44	55
Solvent control (methanol)	14	17	9	19	28	40	117	126	40	48
Positive control (NaN3) - 10	1516	-	-	-	-	-	1608	-	-	-
Positive control (4-NOPD) - 10	-	-	-	-	336	-	-	-	-	-
Positive control (4-NOPD) - 50	-	-	66	-	-	-	-	-	-	-
Positive control (MMS) - 3	-	-	-	-	-	-	-	-	749	-
Positive control (2-AA) - 2.5	-	221	-	455	-	2224	-	3198	-	-
Positive control (2-AA) - 10	-	-	-	-	-	-	-	-	-	270

* Reduced background growth

NaN3 = sodium azide

2 -AA = 2 -aminoanthracene

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

MMS = methyl methane sulfonate

Conclusions:

In a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 and in compliance with GLP, reaction mass of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxyvinylsilane was tested using Salmonella typhimurium TTA 100, TA 1535, TA 98 or TA 1537 and E. coli WP2 uvr A. A substantial and dose dependent increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 100, TA 1535 an d E. coli WP2 uvr A when tested up to limit concentrations. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonel la typhimurium strains TA 98 or TA 1537. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is positive 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

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Without S9-mix: 100, 250, 500, 750, 1000 and 1500 µg/ml culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 500, 1000, 1500, 2000, 2500 and 3000 µg/ml culture medium (3 h exposure time, 24 h fixation time).

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Lymphocytes were cultured for 48 ± 2 h
- Exposure duration: The lymphocytes were exposed in duplicate to selected doses of the test substance for 3 h in the absence and presence of S9-mix
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium)
STAIN (for cytogenetic assays): 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8

NUMBER OF REPLICATIONS: At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of the test substance in the lymphocyte cultures was determined using the mitotic index. No precipitation was observed in the culture medium up to the highest dose level of 5000 µg/ml.

Evaluation criteria:

A test substance is considered positive (clastogenic) in the chromosome aberration test if:

a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.

b)The increase is dose-related when evaluated with the Cochran Armitage trend test.

c)Any of the results are outside the 95% control limits of the historical control data range.

A test substance is considered negative (not clastogenic) in the chromosome aberration test if:

a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided,
p < 0.05) increase compared with the concurrent negative control.

b)All results are inside the 95% control limits of the negative historical control data range.

In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Key result

Species / strain:

lymphocytes: periferal human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Appropriate toxicity was reached at 1000 (42% MI) and 2000 (45% MI) ug/ml for 3h/24h fixation (with and without MA)- omit precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A concentration of 5000 µg/mL showed no precipitation in the culture medium. Therefore, a concentration of 5000 µg/mL was used as the highest concentration of the test substance. In the dose range finding test blood cultures were treated with 52, 164, 512, 1600 and 5000 µg/mL culture medium with and without S9-mix.

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Without S9-mix: 100, 250, 500, 750, 1000 and 1500 µg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 500, 1000, 1500, 2000, 2500 and 3000 µg/mL culture medium (3 h exposure time, 24 h fixation time).

COMPARISON WITH HISTORICAL CONTROL DATA: The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: A concentration of 5000 µg/mL showed no precipitation in the culture medium. Therefore, a concentration of 5000 µg/mL was used as the highest concentration of the test substance. In the dose range finding test blood cultures were treated with 52, 164, 512, 1600 and 5000 µg/mL culture medium with and without S9-mix. The pH was determined for all concentrations.

Table 6: Chromosome aberrations in human lymphocyte cultures treated with the test substance in the absence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Concentration		Solvent control DMSO 1.0 % v/v	Low dose 100µg/ml	Mid dose 500µg/ml	High dose 1000µg/ml	MMC-C 0.5µg/ml
No of cells		150  150  300	150  150  300	150 150   300	150 150 300	75 75  150
Culture		A     B      A+B	A       B    A+B	A     B      A+B	A     B  A+B	A    B   A+B
Chromatid aberrations	gaps	1	1		1      1	1      1
	breaks	3	1       1	4      5	50   48	15   26
	intrachanges				Intra   intra	2intra   3intra
Chromosome aberrations	gaps			1	1      1	1      1
	breaks	3	2	2      3	9       7	12     10
	intrachanges				Intra     intra	2intra   3intra
Mitotic index		100	93	66	42	64
Polyploidy		0	0	0	0	0
Endo reduplication				1	1

 

 Table 7: Chromosome aberrations in human lymphocyte cultures treated with the test substance in the presence of S9-mix in the first cytogenetic assay (3 h exposure time, 24 h fixation time)

Concentration		Solvent control DMSO 1.0 % v/v	Low dose 500µg/ml	Mid dose 1000µg/ml	High dose 2000µg/ml	CP 10µg/ml
No of cells		150  150  300	150  150  300	150 150   300	150 150 300	150 75  225
Culture		A     B      A+B	A       B    A+B	A     B      A+B	A     B  A+B	A    B   A+B
Chromatid aberrations	gaps	1	1		1      3	1
	breaks	1        1	13      8	7      11	53   36	32   36
	intrachanges				Intra intra	2intra
Chromosome aberrations	gaps			1       1	1	1      1
	breaks		5       2	5      2	12     14	8      9
	intrachanges				Intra intra	2intra
Mitotic index		100	81	69	45	56
Polyploidy		0	1	0	0	0
Endo reduplication		0	1	0	0	0

 

 

 

Conclusions:

In a Mammalian Chromosome Aberration Test, conducted according to OECD Test Guideline 473 and in compliance with GLP, the test substance was positive in peripheral human lymphocytes. The test substance induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations both when gaps were included and excluded in the cytogenetic assay. The test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes. Appropriate solvent, negative (cell culture medium) and positive controls were included and gave expected results. It is concluded that Reaction mass of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxyvinylsilane is positive for chromosomal aberrations to mammalian cells 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:

1999-08-18 to 1999-10-20

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

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:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

Without metaboilc activation: 250, 375, 500, 750 μg/ml. With metabolic activation: 500, 750, 1000, 1500 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Chosen on advice from sponsor. Historical data were available to demonstrate that DMSO causes no deleterious or mutagenic effects.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 20-methylcholanthrene

Remarks:

with activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar; in suspension

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 51 hours


SELECTION AGENT (mutation assays): Cloning medium (R30p) with 4 μg/ml TFT (Sigma)

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10(6) cells

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

Evaluation criteria:

A result would be positive if the following were shown:

An increase in mutant frequency in treated cultures of at least 100 relative to the concurrent control.

A statistically significant increase in mutant frequency

Evidence of a dose relationship over at least 2 dose levels

Demonstration of reproducibility in any increase in mutant frequency

An increase in absolute mutant colony numbers in treated cultures

The RTG of cultures showing an increase in mutant frequency should not be less than 10 %

Statistics:

Growth in suspension was calculated as follows:

Cell count 24 hours post treatment / 2 x 105 x Cell count 48 hours post treatment / 2* x 105

* or previous day’s cell count if less than 2 x 105 per ml

Relative total growth over the experimental period was estimated from the following equation:-

RTG = Suspension growth (% control) x Plating efficiency in agar (% control) / 100

Mutant frequency was calculated from:-

MF = 600 / Total no. of viable colonies x Total no. of mutant colonies / 3

Statistical significance was analysed by weighted analysis of variance as described by Arlett et al (1989).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 2: Results of Mammalian Mutagenicity assay (Test 1) with tester strain L5178Y (mean of 3 plates)

Concentration µg/ml	Mutant* Frequency		Relative total growth (mean % of control)		Cytotoxicity (yes/no)
-	— MA	+ MA	— MA	+ MA	— MA	+ MA
Solvent Control**	163	181	100	100	No	No
250	286	-	73	-	No	-
375	469	-	47	-	Yes	-
500	541	272	44	90	Yes	No
750	1304	163	13	50	Yes	Yes
1000	-	649	-	37	-	Yes
1500	-	1328	-	11	-	Yes
Positive Control	601	802	73	45	No	Yes

*Per 10⁶surviving cells

**solvent control with DMSO

Table 3: Results of Mammalian Mutagenicity assay (Test 2) with tester strain L5178Y (mean of 3 plates)

Concentration µl/ml	Mutant* Frequency		Relative total growth (mean % of control)		Cytotoxicity (yes/no)
-	— MA	+ MA	— MA	+ MA	— MA	+ MA
Solvent Control**	148	152	100	100	No	No
187.5	192	-	83	-	No	-
250	246	-	47	-	Yes	-
375	315	-	44	-	Yes	-
500	451	236	35	96	Yes	No
750	767	336	15	71	Yes	No
1000	-	547	-	51	-	Yes
1500	-	962	-	26	-	Yes
Positive Control	754	840	46	40	Yes	Yes

*Per 10⁶surviving cells

**solvent control with DMSO

Conclusions:

In a Mammalian Cell Gene Mutation Assay, conducted according to OECD Test Guideline 476 and in compliance with GLP, the reaction mass of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxyvinylsilane showed a clear, statistically significant evidence of a dose-dependent increase in frequency of revertants with and without activation up to cytotoxic concentrations. Consequently, the test substance is considered mutagenic in mouse lymphoma L5178Y cells under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Information is available from reliable studies for all the required in vitro endpoints. The results of all of the studies were in agreement. There was evidence for mutagenicity and clastogenicity (causing chromosomal aberrations) in the presence and absence of metabolic activation in vitro.

In a valid bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 and in compliance with GLP, Reaction Products of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxy(vinyl)silane was tested using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2 uvr A. A substantial and dose dependent increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 100, TA 1535 an d E. coli WP2 uvr A when tested up to limit concentrations. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98 or TA 1537. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is positive for mutagenicity to bacteria under the conditions of the test (Dow Corning Corporation, 2010).

 

In a Mammalian Cell Gene Mutation Assay, conducted according to OECD Test Guideline 476 and in compliance with GLP, the Reaction Products of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxy(vinyl)silane showed a clear, statistically significant evidence of a dose-dependent increase in frequency of revertants with and without activation up to cytotoxic concentrations. Consequently, the test substance is considered mutagenic in mouse lymphoma L5178Y cells under the conditions of the test (Dow Corning Corporation, 1999).

In a Mammalian Chromosome Aberration Test, conducted according to OECD Test Guideline 473 and in compliance with GLP, the test substance was positive in peripheral human lymphocytes. The test substance induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations both when gaps were included and excluded in the cytogenetic assay. The test substance did not increase the number of polyploid cells and cells with endoreduplicated chromosomes. Appropriate solvent, negative (cell culture medium) and positive controls were included and gave expected results. It is concluded that Reaction Products of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxy(vinyl)silane is positive for chromosomal aberrations to mammalian cells under the conditions of the test (WIL, 2015).

In a supporting bacterial mutagenicity study, conducted according to OECD Test Guideline 471 and in compliance with GLP, reaction products of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxy(vinyl)silane caused dose-related increases in the number of revertants in both the presence and absence of metabolic activation in Salmonella typhimurium strains TA 100 and TA 1535 in the initial and the repeat experiments up to cytotoxic/limit concentrations. No evidence of a test-substance related increase in the number of revertants was observed in the presence or absence of metabolic activation in Salmonella typhimurium strains TA 98, TA 1537 or E.coli strain WP2 uvrA pKM 101. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is positive for mutagenicity to bacteria under the conditions of the test (Dow Corning Corporation, 1999).

In a supporting bacterial mutagenicity study, conducted according to OECD Test Guideline 471 and in compliance with GLP, reaction products of 3-(2,3-epoxypropoxy)propyltrimethoxysilane and triacetoxy(vinyl)silane caused a substantial and dose dependent increase in the number of revertants in the presence and absence of metabolic activation in Salmonella typhimurium strains TA 100, TA 1535 and E. coli WP2 uvrA when tested up to limit concentrations. No evidence of a test-substance related increase in the number of revertants was observed in the presence or absence of metabolic activation in Salmonella typhimurium strains TA 98 or TA 1537. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is positive for mutagenicity to bacteria under the conditions of the test (Dow Corning Corporation, 2010).

The results of all in vitro bacterial mutagenicity studies, in vitro mammalian mutagenicity (MLA) studies and in vitro cytogenicity study were in agreement, and demonstrated clear evidence of mutagenicity. 

For all of the in vitro bacterial mutagenicity studies, positive results were obtained for bacterial strains TA-1535, TA-100 in all studies, and for WP2 uvRA in two out of three studies, which are indicative of base pair substitution (TA-1535, TA-100) and cross-linking mutations (WP2 uvRA). In addition, the test substance was positive in the L5178Y TK+/- MLA test for mutagenicity. The results included an indication of clastogenic potential with the presence of small colonies generated. Furthermore, the test substance was positive in the in vitro cytogenicity study which included significant chromosomal damage to the levels of chromatid breaks, whole chromosome breaks, and exchange-type aberrations. 

 

The results from the above in vitro studies also demonstrate dose-responsiveness, reproducibility, and the assays were conducted with the suitable solvents up to appropriate levels of toxicity.

Also, from a structural alert standpoint, this substance contains a structure with an epoxide equivalent molecular weight present at <1000 which poses a human health concern for carcinogenic potential.  

There is no existing in vivo study or TK data available. In view of the positive results in mammalian cells in vitro, the Lead Registrant proposes to conduct an in vivo micronucleus assay (with bioavailability) combined with a comet assay (OECD 474 treatment option c with OECD 489) as it can detect evidence of chromosome damage/clastogenicity and DNA damage that may lead to somatic or heritable gene mutations.

If the in vivo micronucleus assay results in a positive response, the comet assay would enable it to be demonstrated whether the substance reacts directly with DNA in site of contact tissues and following systemic exposure.

If the micronucleus assay results in a negative response, a conclusion on somatic cell mutagenicity could be reached from the result of the comet assay.

Although it is generally assumed that somatic assays are sufficient to protect the germ cell line, if the results of the micronucleus and comet assays indicate that this is something that needs investigation, then the Transgenic rodent assay will be considered and may be added to the testing regimen.

Justification for classification or non-classification

More information required before final decision can be made for classification.