Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Weight of evidence approach

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Principles of method if other than guideline:

Procedure similar to those described by Kada et al. (1980) in the rec-assay system with B. subtilis and by McCarroll et al. (1981a) with various strains of E. coli.

GLP compliance:

not specified

Type of assay:

other: in vitro DNA damage and/or repair study with E. coli

Species / strain / cell type:

E. coli, other: WP2, WP67, CM871

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

4-nitroquinoline-N-oxide

9,10-dimethylbenzanthracene

benzo(a)pyrene

cyclophosphamide

methylmethanesulfonate

Key result

Species / strain:

E. coli, other: WP2, WP67, CM871

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Ethanol (H3) also induced a direct DNA damage in E. coli, though extremely weak. Clearly, accurate chemical analyses should be performed to rule out the presence of DNA-damaging impurities in the products used, prior to considering ethanol as genotoxic agent.

Conclusions:

Ethanol was not mutagenic to E. coli strains in this DNA repair test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Degussa Hüls AG 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

4 strains used

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

0, 50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION
in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours
- Expression time (cells in growth medium): 72 hours

NUMBER OF REPLICATIONS
3 plates per test concentration

DETERMINATION OF CYTOTOXICITY
relative total growth; background lawn assessment

Evaluation criteria:

For a test compound to be considered positive, it must (in two independant experiments) cause at least a doubling in the mean revertants per plate of at least one tester strain. This increase must be accompanied by a dose response towards increasing concentrations of the test article. Single increases in revertant frequencies, which are not dose-related and not reproducible in two independant tests are considered non-relevant. If however these increases do occur in both tests, this will be taken as an indication of mutagenic effect.

Statistics:

Mean number of revertants per plate and the standard deviation around the mean were calculated.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: > 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: Numbers of spontaneous revertants were within acceptable range

Table 2a: Experiment 1 Plate incorporation assay; number of revertants/plate (mean of 3)

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	14	30	No	139	151	No	8	9	No
50	16	37	No	147	144	No	8	11	No
160	16	28	No	138	143	No	8	10	No
500	14	29	No	136	150	No	11	9	No
1600	22	41	No	136	150	No	9	13	No
5000	21	27	No	10	171	Yes	10	9	No
Positive Control	104.2	1490	No	536	1592	No	316	153	No

*solvent control with DMSO

Table 2b: Experiment 1 Plate incorporation assay; number of revertants/plate (mean of 3)

	TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)
0*	5	13	No
50	0	11	No
160	2	15	No
500	2	10	No
1600	2	9	No
5000	0	3	Yes
Positive Control	84	109	No

*solvent control with DMSO

Table 3a: Experiment 2 Preincubation assay; number of revertants/plate (mean of 3)

	TA98			TA100			TA1535
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)	— MA	+ MA	Cytotoxic (yes/no)
0*	19	25	No	121	116	No	12	9	No
250	21	25	No	97	102	No	7	11	No
500	20	30	No	98	91	No	13	11	No
1000	17	32	No	100	99	No	11	15	No
2000	18	31	No	101	110	No	8	10	No
4000	16	30	No	100	103	No	9	14	No
Positive Control	103	1628	No	608	1384	No	341	216	No

*solvent control with DMSO

Table 3b: Experiment 2 Preincubation assay; number of revertants/plate (mean of 3)

	TA1537
Conc. (µg/plate)	— MA	+ MA	Cytotoxic (yes/no)
0*	15	16	No
250	17	10	No
500	17	13	No
1000	17	15	No
2000	13	15	No
4000	12	13	No
Positive Control	263	131	No

*solvent control with DMSO

Conclusions:

It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Executive summary:

3-aminopropyltriethoxysilane has been tested in a reliable study according to OECD TG 471 and under GLP. The test substance did not show any reproducible mutagenic activity in any of the tester strains with or without metabolic activation in either the plate incorporation or the repeat preincubation assay. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 Nov - 17 Dec 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon for S. typhimurium strains
trp operon for the E. coli strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB; WP2: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix prepared from Wistar rats treated with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally, each on three consecutive days.

Test concentrations with justification for top dose:

First experiment (standard plate test, with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, 3 plates/dose or control): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure water
- Justification for choice of solvent/vehicle: good solubility of the test item in the vehicle

Untreated negative controls:

yes

Remarks:

sterility control

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2.5 µg/plate in DMSO for TA 1535, TA 1537, TA 100, TA 98; 60 µg/plate in DMSO for E. coli WP2 uvrA

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

5 µg/plate in DMSO for TA 1535 and TA 100

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

9-aminoacridine

Remarks:

100 µg/plate in DMSO for TA 1537 Migrated to IUCLID6: (AAC)

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

5 µg/plate in DMSO for E. coli WP2 uvrA Migrated to IUCLID6: (4-NQO)

Positive controls:

yes

Remarks:

(without S9-mix)

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

10 µg/plate in DMSO for TA 98

Details on test system and experimental conditions:

STANDARD PLATE TEST (SPT)
According to Ames et al., Mut Res 31: 347-364 (1975) and Maron & Ames, Mut Res 113: 173-215 (1983)

In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42 to 45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.

PREINCUBATION TEST (PIT)
According to Yahagi et al. Mut Res 48: 121-129 (1977) and Matsushima et al., In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens, Springer Verlag Berlin, Heidelberg, New York (1980)

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of either S9 mix or phosphate buffer were incubated at 37°C for 20 minutes. After addition of 2 mL soft agar, samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.

Evaluation criteria:

An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+8/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no increase in number of revertants was observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: sterility control, yes

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

other: sterility control, yes

Positive controls validity:

valid

Additional information on results:

No precipitation was detected.
No cytotoxic effects were seen in either the standard plate test (SPT) or the preincubation test (PIT) with and without metabolic activation.
Negative and positive controls were as expected and confirmed the validity and sensitivity of the test method and system.

Remarks on result:

other: Standard plate test

Experiment 1: Standard plate-incorporation test

SPT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	14	48	8	21	103
33	10	51	8	15	100
100	12	49	9	19	103
333	12	53	7	20	106
1000	13	47	7	18	100
2500	15	52	9	21	104
5000	12	55	6	18	104
Respective positive control	1354	949	331	723	1048
SPT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	17	57	9	28	111
33	16	60	8	27	104
100	21	57	10	27	105
333	16	63	10	28	108
1000	16	62	11	32	112
2500	18	66	8	26	104
5000	17	61	9	29	113
Respective positive control	461	1225	664	776	196
Experiment 2: Preincubation test PIT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	10	47	6	34	98
33	12	46	9	31	97
100	12	42	8	34	101
333	10	48	9	32	107
1000	10	52	6	31	103
2500	10	52	7	31	99
5000	13	45	7	37	109
Respective positive control	1639	1345	868	890	986
PIT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	13	51	11	49	108
33	12	53	10	47	107
100	11	60	9	44	106
333	12	56	11	47	107
1000	11	48	13	47	108
2500	12	51	10	48	103
5000	12	58	13	46	105
Respective positive control	512	1440	313	1224	301

Conclusions:

The test item diethylene glycol was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

A test for bacterial gene mutagenicity was conducted with diethylene glycol according to the OECD TG 471 (1997), using the following strains: Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA. The test concentrations were 0, 33, 100, 333, 1000, 2500, and 5000 µg/plate for the standard plate test with and without S9 mix, and for the preincubation test with and without S9 mix, respectively. Negative (sterility and solvent) and positive controls were considered.

Under the experimental conditions chosen, the test item diethylene glycol was not mutagenic in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Plate incorporation test acc. to Ames et al. (1975)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

4-nitroquinoline-N-oxide

9,10-dimethylbenzanthracene

benzo(a)pyrene

cyclophosphamide

methylmethanesulfonate

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Ethanol was not mutagenic to bacteria in this Ames reversion test.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: various strains

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Remarks:

Controls not specified, but cited studies meet the requirements of current test guidelines

Key result

Species / strain:

S. typhimurium, other: various strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

other: weak positive effect at far in excess concentrations (>= 160 mg/plate)

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

It can be concluded that ethanol is not mutagenic to bacteria.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Preincubation as per method of Haworth (Env. Mutagen, 5 suppl 1, 3-142, 1983)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Analytical purity approx. 91%

Species / strain / cell type:

S. typhimurium, other: TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

mitomycin C

other: 4-nitro-o-phenylenediamine TA98, TA1538 (-S9) / 2-aminoanthracene all strains (+S9)

Key result

Species / strain:

S. typhimurium, other: TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It can be concluded that ethanol is not mutagenic to bacteria.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Degussa Hüls AG 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

50, 100, 200, 400, 600, 1000, 1800, 3000, 5000 µg/plate

Vehicle / solvent:

culture medium

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:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: none
- Exposure duration: 3 hours (expt 1 and 3); 18 hours (expt 2)
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures at each concentration

NUMBER OF CELLS EVALUATED: 2000

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

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

Evaluation criteria:

Criteria for evaluating results (e.g. cell evaluated per dose group):
The test chemical is to be considered clastogenic in this assay if
1. it induces chromosomal aberrations (excl. gaps) in statistically significant manner in one or more concentrations
2. the induced proportion of aberrant cells at such test substance concentrations exceeds the normal range of the test system (i.e.>5%)
3. positive results can be verified in an independent experiment

Statistics:

Chi-square test

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH increased to an unphysiological range so the stock solution was neutralised by addition of HCl
- Other confounding effects: the test substance hydrolyses with water to form( 3-aminopropyl) silane triol. It is the opinion of the reviewer that although hydrolysis is expected to occur within the course of the experiment, this does not invalidate the result as hydrolysis would be expected to occur after ingestion or absortion of the substance.

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

Cytotoxicity determination: nine concentrations of the test substance (50-5000 µg/ml) in the presence and absence of metabolic activation were used to determine cytotoxicity and to set doses for the chromosome aberration study. Only after 18 hours exposure without metabolic activation was cytotoxicity observed.

The frequency of polyploid cells in both parts of the experiment was within the expected range (<10%) of historical control.

Table 1: Results of chromosome analysis Experiment 1 without activation (3 hour exposure, 200 cells scored)

		Solvent Control	Low dose 600 µg/ml	Mid dose 3000 µg/ml	High dose 5000 µg/ml	Positive Control
Proportion of cells with:	exchanges	0	0	0.5	1.0	6.5
	aberrations inc gaps	5.0	5.0	5.0	9.0	26.5
	aberrations excluding gaps	0	0	0.5	1.5	16.0
Mitotic index		7.3	7.8	9.2	8.0	4.9

Table 2: Results of chromosome analysis Experiment 1 with activation (3 hour exposure, 200 cells scored)

		Solvent Control	Low dose 600 µg/ml	Mid dose 3000 µg/ml	High dose 5000 µg/ml	Positive  Control
Proportion of cells with:	exchanges	0.5	0.5	1.0	0	21.0
	aberrations inc gaps	7.0	11.0	8.5	13.0	36.5
	aberrations excluding gaps	0.5	1.5	3.5	1.5	26.0
Mitotic index		5.1	5.2	7.1	7.1	4.0

 Table 3: Results of chromosome analysis Experiment 2 without activation (20 hour exposure, 200 cells scored)

		Solvent Control	Low dose 600 µg/ml	Mid dose 3000 µg/ml	High dose 5000 µg/ml	Positive  Control
Proportion of cells with:	exchanges	0	0.5	0	0.5	17.0
	aberrations inc gaps	6.5	3.5	7.0	5.5	41.5
	aberrations excluding gaps	0.5	0.5	0.5	2.0	34.0
Mitotic index		8.4	8.3	7.2	5.4	3.7

Table 4: Results of chromosome analysis Experiment 2 with activation (3 hour exposure, 200 cells scored)

		Solvent Control	Low dose 600 µg/ml	Mid dose 3000 µg/ml	High dose 5000 µg/ml	Positive  Control
Proportion of cells with:	exchanges	0.5	0.5	1.0	1.0	10.0
	aberrations inc gaps	8.5	7.0	7.5	8.0	32.0
	aberrations excluding gaps	1.0	1.0	2.0	1.0	17.0
Mitotic index		5.3	4.8	5.0	4.3	4.8

Conclusions:

It is concluded that the test substance is negative for the induction of chromosome aberrations (not clastogenic) in vitro under the conditions of the test.

Executive summary:

3-aminopropyl-triethoxysilane has been tested in a very reliable in vitro cytogenetic assay according to OECD TG 473 and under GLP. The test substance did not induce statistically and biologically significant increases in the chromosomal aberration frequency of V79 Chinese hamster cells in either the initial or the repeat assay.

It is concluded that the test substance is negative for the induction of chromosome aberrations (not clastogenic) in vitro under the conditions of the test.

Reference 8

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented, acceptable study report

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Target gene:

CHO cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

<= 50 mg/ml

Vehicle / solvent:

Test chemical was added directly into the cell culture medium of the test systems.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: dimethylnitrosamine and ethylmethanesulfonate

Details on test system and experimental conditions:

Selection of Test Concentrations: Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would not produce excessive inhibition of the mitotic index. Test results wíth diethylene glycol indicated that concentrations up to 50 mg/ml, tested respectively with and without S9 metabolic activation, produced no detectable cytotoxic effects upon cell culture growth or mitotic indices. The 50 mg/ml (5.°G w/v) dose is the maximum dose used in the standard BRRC protocol and it was the highest dose tested with and without S9 activation in this study.
Test Procedures: For evaluations of direct clastogenic potential, CHO cells were exposed to diethylene glycol and appropriate controls for the complete 12 hour period without S9 activation. Determination of indirect genotoxic potential, requiring metabolic activation by liver S9-homogenate, was studied with a 2-hour exposure period to the test chemical and S9 activation system. Cells were sampled at 8 hr and at 12 hr after starting exposure to the test agent and chromosomes were prepared by standard procedures. A total of fifty cells/culture/harvest interval was examined for chromosome damage using duplicate cultures for the test agent and solvent controls. At least 5 dose levels were tested both with and without metabolic activation. Incidence of chromosome damage was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division (mitosis). The number of chromatid and chromosome-type aberrations, the total number of aberrations per 50 cells examined (with and without including gaps in the total) and the level of statistical significance of the increases of aberrant cells are presented in tabular form.
Control Agents: Positive and solvent control materials were tested concurrently with the test sample to assure the sensitivity of the test system. Cyclophosphamide (CAS 50-18-0) and triethylenemelamine (CAS 51-18-3) were used as the positive control agents to assure the reliability and sensitivity of the test system for detecting metabolic activation dependent and independent clastogens, respectively. Cell culture medium was used as the vehicle and solvent control agent for this test chemical. The positive control agents were assessed for chromosome aberration frequencies only at 8 hr after treatment initiation using single cultures.
Metabolic Activation: S9 liver homogenate, prepared from Aroclor l254-induced, Sprague-Dawley male rats, as purchased from Microbiological Associates (MBA), Bethesda, MD. The S9 preparation was screened for metabolic activity by the supplier and at BRRC prior to use in our general testing program. Data from MBA showed that the S9 preparation was active with three different activation dependent mutagens in Salmonella bacterial strains TA98 and TA100.

Evaluation criteria:

The criteria for evaluation of a positive or negative response depend both on the level of statistical significance and subjective analyses of concurrent and historical control data. The key determinant is whether a dose-dependent increase in SCEs is induced by the test agent. When no clear dose-response relationship is evident and when one or more responses of marginal indications of statistical differences are obtained, a careful examination of the data in comparison to the concurrent controls and the historical data base is necessary to determine the biological significance of the statistical indicators. Testing may be repeated to clarify unusual responses, if data for the concurrent positive or negative controls suggest a defect in the original experiment. Overall assessment will also rely on corroborating data from the other tests in the testing battery. Clearly positive responses will include any of the following: (1) Doubling in the SCE frequency by any single concentrations with both of the duplicate cultures/dose; (2) statistically significant responses of p < 0.05 with two or more consecutive concentrations and (3) induction of a statistically significant, dose-related increase in the number of SCE. Repeatability of effects in the two individual cultures/dose will be used as an important determinant in the classification of a biologically significant effect.

Statistics:

The data were analyzed after transformation of the mutation frequencies (MF) and SCE values according to the conversion method of Box and Cox (1964) . For CHO mutation studies with a concurrent control frequency of zero mutants, the variance of recent historical controls was used for the statistical analyses. For SCE data, statistical analyses of historical data at BRRC indicate that an exponent of 0.15 is the appropriate value for transformation of SCE values. Positive controls for the CHO mutation test were run concurrently to assess the sensitivity of the assays in comparison to historical experience with the test system. Data for positive control agents were not compared statistically whenever differences were at least 5 times the concurrent negative control value and results were within the historical positive control range.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: > 50 mg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The relative cytotoxicity of the various concentrations, tested both in the presence and absence of an S9 metabolic activation system, was determined by measuring the relative growth of treated and control cells incubated overnight following removal of the test chemical. The authors observed that diethylene glycol was not highly cytotoxic when tested either with or without S9 metabolic activation. A concentration of 50 mg/ml produced 16% inhibition of growth without S9 and 27% inhibition with S9. For the definitive tests, a concentration range between 30 to 50 mg/ml was tested in the mutagenicity tests with and without S9. The 50 mg/ml (5% w/v) dose is the usual maximum dose for non-cytotoxic chemicals tested by the BRRC Standard Protocol to avoid possible artefacts produced by non-physiological cell-culture conditions at higher doses.

Conclusions:

Interpretation of results: negative

Diethylene glycol was neither genotoxic nor cytotoxic to CHO cells under the conditions of this in vitro test system.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

other: in vitro cytogenicity / chromosome aberration study in mammalian cells

Species / strain / cell type:

lymphocytes: human

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Species / strain / cell type:

mammalian cell line, other: Chinese hamster Lung (CHL)

Metabolic activation:

with and without

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

mammalian cell line, other: Chinese hamster Lung (CHL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

It can be concluded that ethanol is not clastogenic in vitro.

Reference 10

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Degussa Hüls AG 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and β-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

200, 600, 1800, 3000, 5000 µg/ml

Vehicle / solvent:

culture medium

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:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (with and without activation)
- Expression time (cells in growth medium): 9 days
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):


SELECTION AGENT (mutation assays): H 10 medium

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

NUMBER OF CELLS EVALUATED: approx 5x10 E05

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test compound is considered positive in this assay if it causes a statistically significant, dose related increase in mutant frequency at concentrations of test substance resulting in >20% survival, at a level which is significantly above the maximum spontaneaous mutant frequency.

Statistics:

t-test

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Cytotoxicity test: Concentrations in the range of 50-5000 ug/ml were tested for induction of cytotoxicity. 
There was no effect of the test substance on cloning efficiency of the CHO cells in  the presence or absence of metabolic activation at any concentration  tested.

 

The frequency of mutations was low in all groups.  In the absence of metabolic activation mutant frequencies of test substance treated cells were within the range of historical negative controls 
(i.e., 0-21 mutants per 10 E06 viable cells. 

The test substance did not produce a significant mutant frequency at any concentration tested in the absence of metabolic activation.  
In the presence of metabolic activation, the test substance did induce significant increases of the mutant frequencies at the concentration of 600 ug/ml.  
The observed mutant frequencies were within the range of the historical negative control (0-23 mutants per 10(6)  viable cells) 
and did not show a dose response relationship, the  statistical significance is the result of normal assay variation rather  than indicative of a mutagenic effect of the test substance.

Table 1 Mutant frequency (x10E-06) (mean of 5 flasks)

Concentration µg/ml	Experiment 1			Experiment 2
	- MA	+ MA	cytotoxicity	- MA	+ MA	cytotoxicity
0 *	3	1	no	4	3	no
200	2	1	no	5	3	no
600	2	5***	no	0	8****	no
1800	0	0	no	0	5	no
3000	1	1	no	5	3	no
5000	0	3	no	1	2	no
Positive control	265**	290**	no	342**	230**	no

* Solvent control with culture medium

** Highly significant, no statistical evaluation

*** Statistically significant, P<0.001 but within the range of the historical negative control

**** Statistically significant 0.1<P<0.05 but within the range of the historical negative control

Conclusions:

It is concluded that the test substance in not mutagenic to mammalian cells under the conditions of the test.

Executive summary:

3-aminopropyltriethoxysilane has been tested in a very reliable assay according to OECD TG 476 and under GLP. The results from the repeat experiment were consistent with those from the initial test. Expected results were obtained from medium and positive controls. No increase in the mutant frequency was observed in the absence of activation. In the presence of activation, a statistically significant increase was observed at a single concentration. As this result was within the range of the mutant frequencies of the historical negative controls, and no dose response relationship was observed, the increases are a result of normal assay variation rather than a mutagenic effect of the test substance. It is the opinion of the reviewer that although the test substance hydrolyses in water, and so the use of aqueous cell culture medium may have led to exposure of the test organisms to the products of hydrolysis rather than the test substance itself, this does not invalidate the test, as hydrolysis would occur in exposed organisms.

 

It is concluded that the test substance in not mutagenic to mammalian cells under the conditions of the test.

 

Reference 11

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented, acceptable study report

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

other: HGPRT assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

up to 50 mg/mL

Vehicle / solvent:

Cell-culture medium was used as the solvent as the test material was added directly into the cell-culture test system.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Diethylene glycol was neither genotoxic nor cytotoxic to CHO cells under the conditions of this in vitro test system.

Executive summary:

A HGPRT assay with CHO cells tested in absence and presence of S9 mix was conducted with the test item equivalent or similar to OECD TG 476. The relative cytotoxicity of the various concentrations, tested both in the presence and absence of an S9 metabolic activation system, was determined by measuring the relative growth of treated and control cells incubated overnight following removal of the test chemical. It was observed that diethylene glycol was not highly cytotoxic when tested either with or without S9 metabolic activation.

A concentration of 50 mg/mL produced 16% inhibition of growth without S9 and 27% inhibition with S9. For the definitive tests, a concentration range between 30 to 50 mg/mL was tested in the mutagenicity tests with and without S9.

In the main test, diethylene glycol was neither genotoxic nor cytotoxic to CHO cells under the conditions of this in vitro test system.

Reference 12

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

other: in vitro gene mutation study in mammalian cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Untreated negative controls:

yes

Remarks:

ethanol, methanol, acetone, DMSO, saline

Negative solvent / vehicle controls:

yes

Remarks:

ethanol, methanol, acetone, DMSO, saline

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Test validation included several known positive control substances

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at concentrations of 1%

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Ethanol was not miutagenic in this mouse lymphoma TK+/- assay.

Reference 13

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

not specified

Type of assay:

other: in vitro gene mutation study in mammalian cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The results of gene mutation assays with ethanol are universally negative.

Reference 14

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

peer reviewed publication

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Method acc. to Clive et al. (Muta Res., 59, 61, 1979) with some minor modifications

GLP compliance:

not specified

Type of assay:

other: in vitro gene mutation study in mammalian cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

benzo(a)pyrene

cyclophosphamide

ethylmethanesulphonate

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Conclusions:

Ethanol did not produce significant mutagenic activity.

Reference 15

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented, acceptable study report

Justification for type of information:

For detailed justification see summary

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

CHO cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S-9 mix

Test concentrations with justification for top dose:

30 - 50 mg/ml

Vehicle / solvent:

Cell-culture medium was used as the solvent as the test material was added directly into the cell-culture test system.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: cyclophosphamide and triethylenemelamíne

Details on test system and experimental conditions:

Selection of Test Concentrations: Preliminary experiments were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill, no more than (approximately) 90% of the treated cells. Test concentrations for the SCE test were chosen so that sufficient numbers of cells in the second division (M2) would be available for determination of SCEs. Test results with diethylene glycol indicated that concentrations up to 50 mg/ml, the maximum concentration used for evaluation of test chemicals following the BRRC standard test protocol, were not cytotoxic to CHO cells. For the definitive tests, 50 mg/ml was also selected as the maximum dose and additional concentrations below this dose were evaluated. In this tests, doses between 30 and 50 mg/ml were evaluated for genotoxic potential.
Control Agents: Positive and solvent control materials were tested concurrently with the test sample to assure both the sensitivity of the test systems and the concurrence of the results to historical test performance at BRRC. For this assays, dimethylnitrosamine (DMN)-CAS 62-75-9 and ethylmethanesulfonate (EMS)-CAS 62-50-0 were used as positive control agents to assure the sensitivity and reliability of the test system for detecting metabolic activation dependent and independent mutagens, respectively. Cell culture medium was used as the negative control.
Metabolic Activation: S9 liver homogenate, prepared from Aroclor 1254-induced, Sprague-Dawley male rats, was purchased- from Microbiological Associates, Bethesda, MD. The S9 preparation used for the CHO test was screened for appropriate metabolic activity by the supplier with three activation-dependent mutagens and Salmonella strains TA98 and TA100. A volume of 50 ul of S9 homogenate was used for each 1.0 ml of the S9 activation mixture.

Evaluation criteria:

For evaluations of direct clastogenic potential, CHO cells were exposed to diethylene glycol and appropriate controls for the complete 12 hour period without S9 activation. Determination of indirect genotoxic potential, requiring metabolic activation by liver S9-homogenate, was studied with a 2-hour exposure period to the test chemical and S9 activation system.

Statistics:

Statistical analyses of the test data employed the Fisher's Exact Test (one-tailed) to determine significance of differences between the test and control populations. This statistical test was considered appropriate for the analysis of the data because it is a distribution independent test and cytogenic data often vary from a normal distribution required for parametric analyses. A positive test result was interpreted by the attainment of differences from the control at the p < 0.05 level of significance for at least one test concentration, together with an indication of a concentration-related effect of exposures or
reproducibility between the duplicate cultures. Rounding of data to either two decimal places or to the appropriate number significant figures was performed for presentation on tables. Although statistically significant decreases in genotoxicity indices can occur because of cytotoxic responses, only statistically significant increases ín responses above control values are indicated on tables for simplicity. All statistical tests were performed to determine whether the treatment with the test agent produced a response statistically different from the value(s) obtained with the concurrent solvent control cultures.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: > 50 mg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Diethylene glycol did not produce dose-related, statistically significant increases in the incidence of chromosome aberrations in CHO cells in tests conducted with and without the addition of a rat-liver homogenate, S9 metabolic activation system. A single statistical indication of an increase above control values was obtained for the lowest dose sampled at 12 hr after test initiation in the test with S9 activation. This result was not repeated at higher doses and the level of the increase was essentially the same as the variation in the concurrent negative control values. For these reasons, the statistical indication was not considered biologically relevant. The positive control agents, cyclophosphamide and triethylenemelamine, both produced significant increases in chromosome aberrations indicative of the appropriate, reliability of the test system. The control cultures employing only the culture growth medium had low and acceptable levels of chromosome aberrations typical for these cultured cells. Diethylene glycol was concluded to lack significant genotoxic activity under the conditions employed for this in vitro test system.

Conclusions:

Interpretation of results: negative

No statistically significant increase in the number of SCEs was observed with any of the test cultures treated with the test agent. The incidence of SCEs in all of the cultures exposed to the test agent was within the historical negative control range for this test at BRRC (Methods, Section G1). Diethylene glycol was considered inactive under the non-activation conditions of this sensitive in vitro assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine (CAS 152261-43-3) is a complex reaction mixture (UVCB) of 3-aminopropyltriethoxysilane (CAS 919-30-2) and diethylene glycol (CAS‎ 111-46-6). Diethylene glycol is the main component besides smaller amounts of ethanol (CAS 64-17-5). No free 3-aminopropyltriethoxysilane is detectable, because it is covalently bound to the oligomeric structures of the reaction product.

Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine is unstable upon contact with moisture. lt undergoes further condensation reactions that form highly polymerized poly silicic acids while liberating the diethylene glycol, that was initially bound to the oligomeric structures. The underlying chemistry is commonly known as sol-gel reaction. The poly silicic acid moieties are not stable and prone to further condensation generating waterinsoluble, resinous polymers. The molecular weight of the resulting polymers is predicted to be over 1000. These polymers are stable and not bioavailable.

Because 3-aminopropyltriethoxysilane is completely consumed into polymer matrix the toxicological profile of Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine will be mainly determined by diethylene glycol and to a lesser extent by ethanol.

Several in vitro genotoxicity studies have been conducted with diethylene glycol. Diethylene glycol was not genotoxic in CHO cells in the cell chromosomal aberration, sister chromatid exchange as well as HGPRT mutation assay and the Ames test. Furthermore, diethylene glycol was assessed within the Community rolling action plan (CoRAP). It was concluded that diethylene glycol has no mutagenic potential (Substance evaluation conclusion and report for 2,2’-oxydiethanol, 2016). Furthermore, diethylene glycol is not classified for genotoxic effects in accordance with Annex VI to Regulation (EC) No 1272/2008.

The balance of evidence is that ethanol is not genotoxic in vitro. The results of bacterial mutagenicity assays have generally been negative for ethanol. Positive results are only found at ethanol concentrations higher than recommended for guideline testing. In mammalian cell mutation assays a weak mutagenic effect in mouse lymphoma cells were observed only at test substance concentrations higher than recommended. Data from use of ethanol as a vehicle control show, that it is not mutagenic or clastogenic in vitro. In vivo tests for chromosome aberrations gave mostly negative results (Phillips and Jenkinson, 2001). Some degree of genotoxicity may result from excessive alcohol drinking, but this is not considered relevant to any conceivable exposure obtainable by either inhalation or dermal exposure. In addition, ethanol is not classified for genotoxic effect in accordance with Annex VI to Regulation (EC) No 1272/2008.

3-Aminopropyltriethoxysilane has been submitted to an Ames assay, in vitro V79 hamster lung cell chromosome aberration assay and Chinese hamster ovary cell gene mutation assay. Existing in vitro studies have not revealed any evidence of genotoxic potential.The tests are executed under conditions, which lead to formation of silanols and other oligomeric compounds, therefore these structures are covered as well.

Existing data on the main components of Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine clearly show that they do not exhibit genotoxic properties. Furthermore, Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine will react upon contact with water to form poly silicic acids by releasing diethylene glycol. The chemically stable reaction product is water-insoluble, highly polymerized, not bioavailable and therefore unlikely to produce genotoxic effects. For the aforementioned reasons, Ethanol, 2,2'-oxybis-, reaction products with 3-(triethoxysilyl)-1-propanamine is not classified for genotoxic effects based on a Weight of Evidence assessment.