Reaction mass of [3-(trimethoxysilyl) propyl]urea, [3-(dimethoxyethoxysilyl) propyl]urea, [3-(methoxydiethoxysilyl) propyl]urea and [3-(triethoxysilyl) propyl]urea.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471, GLP): negative with and without metabolic activation.
Chromosome aberration assay (OECD TG 473, GLP): positive with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro cytogenicity study in mammalian cells

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct - 08 Nov 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test (migrated information)

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Peripheral lymphocytes from healthy human males were cultured within 4 h after blood sampling. The cells were cultured in Ham´s F10 without thymidine and hypoxanthine supplemented with fetal calf serum, L-glutamine, P/S, sodium bicarbonate and heparin.

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of Aroclor 1254 induced male Wistar rats

Test concentrations with justification for top dose:

Without S9-mix: 100, 180, 333, 420, 560 µg/ml
With S9-mix: 333, 560, 1000, 1300, 1800 µg/ml

Vehicle / solvent:

Dimethylsulphoxide (final concentration 0.9%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: Cyclophosphamide, 15 µg/ml; -S9: Mitomycin C, 0.5, 0.2 and 0.1 µg/ml for 3, 24 and 48 h treatment, respectively.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µg/ml) was added 3 h before fixation of cells.
STAIN (for cytogenetic assays): Giemsa (5% in tap water)

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (metaphases per 1000 cells)

OTHER EXAMINATIONS:
- Determination of polyploidy: no data
- Determination of endoreplication: no data
- Range finding test: cells were treated for 3 (fixation at 24 h, ± S9), 24 (fixation at 24 h, -S9), and 48 h (fixation at 48 h, -S9) for determination of mitotic index.

Evaluation criteria:

A test substance was considered positive (clastogenic) if:
- induction of a dose-related statistically significant increase in the number of cells with chromosomal aberrations.
- a statistically significant increase in the frequencies of the number of cells with chromosome aberrations as observed in the absence of a clear dose-response relationship.

Statistics:

- induction of a dose-related statistically significant increase in the number of cells with chromosomal aberrations: Chi-square test, p<0.05

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Mitotic index < 50% at the highest concentration ± S9

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 7.54 was measured at the highest concentration of 5000 µg/ml (pH 7.67 in the solvent control)
- Effects of osmolality: 385 mOsm/kg was measured at the highest concentration of 5000 µg/ml (428 mOsm/kg in the solvent control)

RANGE-FINDING/SCREENING STUDIES: for details see table 1

COMPARISON WITH HISTORICAL CONTROL DATA: Solvent control data were within the historical control data

Table 1: Results determined in the range-finder assay:

Test item	Concentration	Mitotic Index
	in µg/ml	in %
Exposure period 3 h, fixation time 24 h, without S9 mix
Test substance	100	93
	333	66
	1000	7
	3333	0
	5000	0
Exposure period 24 h, fixation time 24 h, without S9 mix
Test substance	100	110
	333	28
	1000	0
	3333	0
	5000	0
Exposure period 48 h, fixation time 48 h, without S9 mix
Test substance	100	79
	333	15
	1000	0
	3333	0
	5000	0
Exposure period 3 h, fixation time 24 h, with S9 mix
Test substance	100	120
	333	104
	1000	56
	3333	0
	5000	0

Table 2: Results of the cytogenicity assay:

Test item	Concentration	Mitotic Index	Aberrant cells in %
	in µg/ml	in %	with gaps	without gaps
Exposure period 3 h, fixation time 24 h, without S9 mix
DMSO	0.9% (v/v)	100	1	1
MMC	0.5	113	9	8
Test substance	180	101	7	6
	333	69	16	13
	420	39	18	15
Exposure period 3 h, fixation time 24 h, with S9 mix
DMSO	0.9% (v/v)	100	1	1
CP	15	31	32	32
Test substance	333	99	2	2
	560	84	4	3
	1000	48	19	15

Conclusions:

Interpretation of results: positive

In a chromosomal aberration assay according to OECD 473 and GLP, a genotoxic effect was observed for the test substance tested in a 3 h treatment in two independent experiments without and with metabolic activation. Appropriate solvent and positive controls were included and gave expected results. The test substance is genotoxic under the test conditions applied.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-09-01 to 1999-09-13

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

Type of assay:

bacterial reverse mutation assay

Target gene:

"his operon" (for S. typhimurium strains) and "trp operon" (for 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:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

First and second experiment - TA1535, TA1537, TA98 - 100, 333, 1000, 3330 and 5000 µg/plate First experiment - TA100, WP2uvrA - 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate. Second experiment - 100, 333, 1000, 3330, 5000 µg/plate.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors. In the first experiment, 0.5 ml S9-fraction to 9.5 ml S9 mix components to give 5% v/v S9 fraction. In the second experiment, 1.0 ml S9 fraction was added to 9.0 ml to give 10% v/v S9 fraction. 0.5 ml S9 mix was added to 3 ml top agar, 0.1 ml bacterial culture and 0.1 ml of test or control substance, giving a final S9 concentration of approximately 1.4% in the first experiment and approximately 0.7% in the second experiment.

DURATION
- Exposure duration: 48 hrs at 37ºC

NUMBER OF REPLICATIONS: triplicate plates, experiment repeated

DETERMINATION OF CYTOTOXICITY
- Method: condition of background lawn

DETAILS ON CONTROLS:
-S9: Daunomycin (TA 98, 4 µg/plate); 9-aminoacridine (TA 1537, 50 µg/plate); sodium azide (TA 1535, 1 µg/plate); methylmethanesulfonate (TA 100, 650 µg/plate); 4-nitroquinoline-N-oxide (WP2 uvrA, 10 µg/plate); +S9: 2-aminoanthracene(TA 1537, 2.5 µg/plate; TA 1535, TA 98, TA 100, 1 µg/plate; WP2uvrA, 5 µg/plate)

Evaluation criteria:

A test substance is considered positive in the test if it induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. The positive response should be reproducible.

Statistics:

None

Species / strain:

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

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

Table 1: Number of revertants per plate (mean of three plates) in experiment 1.

Dose (µg/plate)	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent control	12	11	7	6	12	26	106	106	13	13
3	-	-	-	-	-	-	93	93	10	10
10	-	-	-	-	-	-	91	91	11	11
33	-	-	-	-	-	-	87	87	13	13
100	7	9	5	6	18	23	105	105	7	7
333	8	9	4	6	13	18	91	91	9	9
1000	9	8	5	4	11	18	93	93	13	13
3330	7	8	4	5	16	20	88	88	10	10
5000	9	11	4	3	14	11	93	93	8	8
Positive control	250	260	563	619	394	1417	739	739	477	477

Table 2: Number of revertants per plate in experiment 2.

Dose (µg/plate)	TA 1535		TA 1537		TA 98		TA 100		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent control	13	10	4	4	19	25	96	6	11	3
100	15	14	5	4	19	23	91	11	13	3
333	14	12	5	4	19	26	91	2	8	4
1000	10	15	3	5	17	25	94	11	8	1
3330	8	12	6	4	14	24	101	25	8	2
5000	10	13	5	3	15	17	71	13	6	1
Positive control	300	267	237	153	504	537	514	66	538	45

Conclusions:

Interpretation of results: negative with and without metabolic activation

Ureudopropyltrialkoxysilane, mixed methoxy and ethoxy esters has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD 471, and in compliance with GLP. No evidence of a test substance related increase in the number of revertants was observed with or without metabolic activation in the initial or repeat experiments. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus test (OECD TG 474, GLP): negative

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:

03 Apr 2001 - 04 Jul 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Males: 27.4 – 31.0 g; Females: 24.6 – 29.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: five per sex per polycarbonate cage
- Diet: standard pelleted laboratory diet (Altromin, code VRF-1, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Test substance was given undiluted.

Duration of treatment / exposure:

Not applicable

Frequency of treatment:

Single treatment

Post exposure period:

24 and 48 h after treatment

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 per sex per dose at any sampling time point

Control animals:

other: yes (0.9% NaCl)

Positive control(s):

- cyclophosphamide
- Justification for choice of positive control(s): none given in report - standard control
- Route of administration: by oral gavage
- Doses / concentrations: 50 mg salt/kg bw

Tissues and cell types examined:

Bone marrow smear of the femurs (polychromatic erythrocytes with and without micronucleus, normochromatic erythrocyes)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on a range finding assay in 4 animals with a dose of 2000 mg/kg bw

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
500, 1000, 2000 mg/kg bw: 24 h post-dose
2000 mg/kg bw: 48 h post-dose

DETAILS OF SLIDE PREPARATION: cells were washed, resuspended, fixed on glass slides with methanol, stained using the "Wright-stain procedure"

METHOD OF ANALYSIS: light microscope - area selected at x100, analysis performed at x 1000
2000 PCEs scored for presence of micronuclei. The ratio of PCE to NCEs, ( normochromatic erythrocytes) was determined by counting and differentiating the first 1000 erythrocytes.

Evaluation criteria:

The test article would have been considered to induce a positive response if a biologically and statistically significant increase in the frequency of micronucleated polychromatic erythrocytes is observed at one or more doses (Wilcoxon Rank Sum Test, two-sided test at p≤ 0.05) at any sampling time in the combined data for both sexes or in the male or female animals separately.
The test article was judged negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control values was observed at any sampling time.

Statistics:

Wilcoxon Rank Sum Test

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:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: no abnormalities were observed within 3 days.
- Evidence of cytotoxicity in tissue analyzed: no abnormalities were observed.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no induction was observed.
- Ratio of PCE/NCE: No change of the ratio was observed after treatment with test substance. A decrease of the PCE/NCE ratio was observed after treatment with CP.

Summary of results of bone marrow micronuleus analysis following single ip administration

Treatment (10 ml/kg bw)	Sex	Time (hr)	No. of animals	PCE/total erythrocytes	Micronuclei/1000 PCE
0.9% NaCl	M	24	5	1.00 ± 0.06	1.2 ± 1.6
	F	24	5	1.02 ± 0.04	3.8 ± 0.8
500	M	24	5	0.96 ± 0.05	1.6 ± 1.8
	F	24	5	0.93 ± 0.01	2.4 ± 1.8
1000	M	24	5	0.98 ±0.08	0.8 ± 1.1
	F	24	5	0.99 ± 0.07	1.6 ± 1.1
2000	M	24	5	0.98 ± 0.07	2.6 ± 1.5
	F	24	5	0.96 ± 0.04	1.6 ± 1.8
Positive control	M	48	5	0.53 ± 0.11	*23.8 ± 9.3
	F	48	5	0.408 ± 0.01	*18.0 ± 4.2
2000	M	48	5	0.99 ± 0.04	3.8 ± 3.6
	F	48	5	0.98 ± 0.06	2.2 ± 1.9

*Statistically significant increase, p < 0.05

Conclusions:

Interpretation of results: negative
Ureidopropyltrialkoxysilane, mixed methoxy and ethoxy esters has been tested according to OECD 474 and under GLP conditions. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed in the bone marrow of male or female rats 24 or 48 hours after a single oral dose of up to and including 2000 mg/kg bw. A reduction in the PCE/total erythrocyte ratio was only observed in the 48 hour positive control group. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Two in-vitro and one in vivo study are available for assessment of the genetic toxicity of ureidopropyltrialkoxysilane, mixed methoxy and ethoxy esters.

In vitro:

Ureidopropyltrialkoxysilane, mixed methoxy and ethoxy esters has been tested for mutagenicity to bacteria, in a study which was conducted according to OECD 471, and in compliance with GLP. S. typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 and E. coli WP2 uvrA were treated with the test substance up to the limit dose of 5000 µg/plate. No evidence of a test substance related increase in the number of revertants was observed with or without metabolic activation in the initial or repeat experiments. No cytotoxicity was observed. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (NOTOX, 1999f).

In a second in vitro test, performed according to OECD TG 473 and in compliance with GLP, the test substance was investigated on its ability to induce chromosome aberrations (NOTOX, 2000d). Cultured peripheral human lymphocytes were treated for 3 hours (main test) with 100, 180, 333, 420, 560 µg/ml (without S9 -mix) and 333, 560, 1000, 1300, 1800 µg/ml (with S9 -mix) and were fixed after 24 h. A genotoxic effect was observed for the test substance tested in a 3 h treatment in two independent experiments without and with metabolic activation. Appropriate solvent and positive controls were included and showed expected results. The test substance is genotoxic under the test conditions applied.

In vivo:

For assessment of genetic toxicity in vivo a micronucleus test with Wistar rats according to OECD TG 474 and in compliance with GLP is available (NOTOX, 2001). Five male and female animals were treated once per gavage with 500, 1000, 2000 mg/kg bw test substance. After 24 or 48 h (high dose group only) the animals were killed and the bone marrow was prepared for investigation of erythrocytes. No significant increase in the incidence of micronucleated polychromatic erythrocytes was observed in the bone marrow of male or female rats 24 or 48 hours after a single oral dose of up to and including 2000 mg/kg bw. A reduction in the PCE/total erythrocyte ratio was only observed in the 48 hour positive control group. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the study. Although no bioavailability of the test substance was proven, the study was evaluated as valid, because it was tested up to the limit concentration of 2000 mg/kg bw.

In conclusion, the test substance should not be classified in the category "genetic toxicity", as there is no indication for mutagenicity from an Ames test, and no chromosome aberration was observed in the in vivo micronucleus test (according to ECHA, guidance on information requirements and chemical safety assessment, chapter R.7a, May 2012).

Justification for classification or non-classification

Based on the available in vitro and in vivo data on mutagenicity of the test substance, ureidopropyltrialkoxysilane, mixed methoxy and ethoxy esters is not classified for germ cell mutagenicity according to Regulation (EC) 1272/2008 or Directive 67/548/EEC.