Bis(trimethoxysilylpropyl)amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): S. typhimurium TA 98, TA 100, TA 1535, TA 1537, and E. coli WP2 uvrA: negative with and without metabolic activation (according to OECD 471)
Mammalian cytogenicity (Chromosome Aberration): CHO: negative with and without metabolic activation (similar to OECD 473)
Gene Mutation (Mammalian Cells, WoE: RA from CAS 919-30-2 and CAS 1760-24-3): CHO: negative with and without metabolic activation (according to OECD 476)

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:

16 August 2005 to 26 August 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

(no data on test material purity given, slightly modified data evaluation)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BAYERISCHES LANDESAMT FÜR ARBEITSSCHUTZ, ARBEITSMEDIZIN UND SICHERHEITSTECHNIK, Munich, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon, trp operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria

Untreated negative controls:

yes

Remarks:

with aqua destillata

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Remarks:

with aqua destillata

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD: 10 µg/plate for TA 98 and 40 µg/plate for TA 1537 TA 98, TA 1537 (without metabolic activation)

Untreated negative controls:

yes

Remarks:

with aqua destillata

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

with aqua destillata

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA (2.5 µg/plate)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 independent experiments with 3 plates each strain and dose level

DETERMINATION OF CYTOTOXICITY
- Method: reduced background lawn (25% and above) or a reduction in the number of revertants down to a mutation factor of approx. up to 0.5 in relation to solvent control

Evaluation criteria:

A test item was considered mutagenic if:
- a clear and dose related increase in the number of revertants over at least two increasing concentrations occurs without concomitant cytotoxicity in at least one of these dose groups, and/or
- a biologically relevant positive response for at least one of the non cytotoxic dose groups in at least one tester strain with or without metabolic activation.

Cytotoxicity is measured by clearing of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <0.5 in relation to the solvent control.

Statistics:

The mutation factor was calculated by dividing the mean number of revertants in the test item by the mean number of revertants in the solvent control.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- none stated in the study report

COMPARISON WITH HISTORICAL CONTROL DATA
The results were within range of the historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tabl.1: Experiment 1 Plate incorporation Number of revertants per plate(mean out of 3 plates)

	TA98			TA100			TA1535
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Negative control**	44±5.5	41±2.3	No	122±9.9	127±21.9	No	18±4 .9	9±3.2	No
solvent control*	34±10.1	42±10	No	114±8.5	139±24.8	No	20±5.3	8±2.6	No
31.6	34±9.6	46±57	No	133±14.0	126±28.2	No	20±2.6	10±1.5	No
100	29±3.2	37±3.8	No	120±8.1	122±8.1	No	21±5.0	10±4.9	No
316	28±1.5	37±8.1	No	120±10.1	109±5.7	No	25±8.9	13±6.1	No
1000	28±1.5	48±1.7	No	116±5.8	97±11.0	No	18±5.3	11±2.5	No
2500	26±2.6	43±4.9	No	102±7.6	105±13.5	No	11±3.0	8±2.5	No
5000	24±1.7	49±3.1	No	118±11.4	87±11.6	Yes	12±2.3	8±3.5	Yes
Positive control	1234±173.1	2636±123.7	No	1116±28.9	2327±94.9	No	1138±27.7	108±7.6	No

*solvent control with DMSO

**negative control with water

Tab. 1: Experiment 1 Plate incorporation Number of revertants per plate (mean of 3 plates) (ctd.)

	TA1537			E. coli WP2 uvrA
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Negative control**	17±1.7	20±1.0	No	52±5.0	43±9.2	No
solvent control*	15±5.0	22±0.5	No	40±9.5	38±8.6	No
31.6	18±2.1	20±5.1	No	47±7.1	45±8.0	No
100	16±2.5	18±0.0	No	45±2.1	48±6.6	No
316	17±1.5	28±3.0	No	42±5.6	53±10.0	No
1000	12±0.8	17±5.1	No	44±0.6	57±9.3	No
2500	7±3.6	18±3.1	Yes	48±4 .9	50±14.5	No
5000	5±1.0	6±1.5	Yes	32±7.5	55±8.8	No
Positive control	165±12.6	343±44.0	No	573±107.7	194±25.6	No

*solvent control with DMSO

**negative control with water

Tab. 2: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates)

	TA98			TA100			TA1535
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Negative control**	29±5.6	45±3.1	No	130±10.2	132±10.6	No	20±2.6	11±2.6	No
solvent control*	34±3.6	40±8.1	No	126±12.0	115±8.3	No	25±7.2	12±3.5	No
31.6	31±3.6	44±1.7	No	128±6.4	117±10.8	No	21±5.3	13±4.0	No
100	33±2.1	45±8.4	No	137±16.9	131±20.4	No	20±6.1	11±2.6	No
316	28±3.5	46±7.6	No	114±2.0	114±4.6	No	20±1.5	12±1.2	No
1000	27±6.1	43±4.5	No	98±6.7	123±10.0	No	19±2.9	15±5.0	No
2500	23±2.6	29±6.1	No	89±8.3	117±8.3	No	12±1.2	5±2.3	Yes
5000	19±2.3	15±3.2	Yes	78±25.5	57±6.7	Yes	0±0	4±1.5	Yes
Positive control	1768±191.4	1277±102.8	No	1278±35.9	1583±71.1	No	1353±27.6	68±13.7	No

*solvent control with DMSO

**negative control with water

Tab. 2: Experiment 2 Preincubation Number of revertants per plate (mean of 3 plates) (ctd.)

	TA1537			E. coli WP2 uvrA
Conc. µg/plate	- MA	+ MA	Cytotoxic (yes/no)	- MA	+ MA	Cytotoxic (yes/no)
Negative control**	8±1.2	9±1.2	No	49±3.6	61±4.5	No
solvent control*	13±3.5	8±1.5	No	38±5.5	61±6.1	No
31.6	12±4.6	8±3.5	No	48±2.9	47±5.7	No
100	19±1.2	7±1.7	No	35±11.2	48±9.9	No
316	9±1.5	9±2.3	No	38±5.0	59±9.0	No
1000	5±2.5	9±1.7	Yes	41±12.3	57±16.5	No
2500	5±1.2	3±2.5	Yes	24±11.8	41±4.9	Yes
5000	6±1.0	2±0.0	Yes	22±8.1	24±5.0	Yes
Positive control	266±46.6	131±24.6	No	654±22.3	139±6.1	No

*solvent control with DMSO

**negative control with water

Conclusions:

Interpretation of results: negative

The test item was investigated for mutagenicicty to bacteria according to the OECD TG 471, and in compliance with GLP. In two independent experiments (plate incorporation and preincubation) the test material was tested in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and in E. coli WP2 uvrA up to precipitation and cytotoxic concentrations with and without a metabolic activation system. No significant increase in the number of revertants was observered in any of the tester strain with and without metabolic activation. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 1990 to 13 April 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The restrictions were that only 50 cells per culture were scored.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

(only 50 cells per culture were scored)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(as stated in the study report, which seems to be a writing error)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: The cells were subcultured in antibiotic-free, Ham's Modified F12 Medium (hypoxanthine-free) supplemented with 10% (v/v) heat inactivated foetal bovine serum (F12-10). For the experiments with metabolic activation serum free medium was used.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

other: CHO-Kl-BH4 (Subclone D1)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 homogenate from Aroclor-1254 induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

- Range-finding study: 0.78 - 50 mg/ml
- Repeated range-finding study: 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, and 1 mg/ml
- Main Study (without metabolic activation): 0.16, 0.24, 0.36, 0.53, and 0.8 mg/ml
- Main study (with metabolic activation): 0.53, 0.8, 1.2, 1.8, and 2.7 mg/ml

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

triethylenemelamine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): approximately 6 h
- Fixation time (start of exposure up to harvest of cells): approximately 10 h

SPINDLE INHIBITOR (cytogenetic assays): 0.5 µg/ml colchicine for the last two hours of incubation
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 50 cells each slide

DETERMINATION OF CYTOTOXICITY
- Method: relative growth (in the range-finding study for the dose-selection of the main study); mitotic index (in the main study for the selection of test concentrations, which were finally microscopically evaluated)

Evaluation criteria:

50 metaphase cells were scored from slides prepared from each culture/dose/sample time. Only the highest three test concentrations that yield a sufficient number of mitotic cells was evaluated microscopically. Slides were coded prior to evaluation to to prevent bias. Only metaphase cells containing 19-21 chromosomes were evaluated. Each cell was evaluated for chromosome number, chromosome- or chromatid-type aberrations and further classified for deletions and exchanges. Gaps were noted and tabulated but were not included in the proportion of aberrant cells or in statistical analyses. Severely damaged cells (>= 10 breakage events) and pulverised cells were recorded as severely damaged, but no attempt was made to classify the types of damage.

A statistically significant, dose-related increase in the frequency of structural chromosome aberrations was considered a positive effect. Production of statistically significant increase for one dose level which was reproducible in in cultures may have been considered a positive effect, even if there was no evidence of a a dose-related response. A single positive effect in only one of the two cultures per dose level may have been considered an equivocal effect. A test substance, which did not produce positive increases as describe above was considered inactive as a clastogenic agent in vitro. The positive control agents must produce significant responses in this test system. The final evaluation of the data was based upon scientific judgement as well as statistical significance.

Statistics:

Statistical analyses of the data were performed using Fisher's Exact test. The percentage of aberrant cells at each concentration was compared to the respective solvent control value. A difference between treated and control cells was considered significant when p<= 0.05 (1-tailed). A positive response that appeared to be dose-related was tested for significance using regression analysis.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.53 mg/ml and above without metabolic activation and at 1.8 mg/ml and above with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the test substance preperation was checked for 3 concentrations rangind from 0.36 to 2.7 mg/ml. All pH readings were within the range of 7.3 to 8.0. Hence, no no adjustments of the culture medium pH were performed.
- Precipitation: In the range-finding test precipitation was observed for the 5 top doses. Hence, the experiment was repeated with lower concentrations.

RANGE-FINDING/SCREENING STUDIES
In the range-finding study cytotoxicity of the test item was evaluated for the purpose of dose-selection by determination of the relative growth. Due to high toxicity at 25 and 50 mg/ml and precipitation, this test was repeated with lower concentrations. Based on the results of the second range-finding test, which revealed toxicity at a concentration of 1 mg/ml, the doses determined in the main study were selected.

Conclusions:

Interpretation of results: negative

The test item was evaluated for clastogenicity in a study conducted similar to the OECD TG 473, and in compliance with GLP. CHO cells treated with the test material up to cytotoxic concentrations with and without metabolic activation, did not exhibit any significant changes of chromosomal aberrations as compared to the control. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not clastogenic under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

weight of evidence

Justification for type of information:

see endpoint summary for read across justification

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

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 examined

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

4.5 mg/ml with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: CAS 919-30-2

Conclusions:

Two in vitro mammalian mutagenicity assays are available from the structural analogues CAS 919-30-2 and CAS 1760-24-3. In both tests the structural analogue substances did not show any mamallian genotoxic potential and thus this is also estimated for the target substance. As explained in the endpoint summary, the differences in molecular structure between the target and the source are unlikely to lead to differences in genetic toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Chromosome aberration (micronucleus assay): negative (similar to OECD 474; 3 concentrations (50, 100, and 160 mg/kg bw) single intraperitoneal injection to mice)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

other: General recommendations in the Environmental Protection Agency - Health Effect Test Guidelines, EPA Report 560/6-83-001

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboraties, Portage, MI
- Age at study initiation: 5 weeks
- Weight at study initiation: 20.7-24.9 g (males of the first main study); 15.5-17.5 g (females of the first main study); 21.4-24.8 g (males of the second main study); 19.2-22.0 g (females of the second main study)
- Assigned to test groups randomly: yes, by weight (only animals with a body weight within a range of 2 standard deviations of the mean body weight were used)
- Housing: 5 mice per sex per cage were housed in shoe-box type plastic cages, measuring 30x20x12.5 cm. Ab-Sorb-Dri bedding (Garfield, NJ) was placed in the cages and changed weekly.
- Diet: basic diet of Agway PROLAB Animal Diet (Rat/Mouse/Hamster 3000, Agway Inc., Syracuse, NY), ad libitum
- Water: supplied by Municipal Authority of Westmoreland County (Greensburg, PA), ad libitum
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature: continously controlled by a Cole Parmer monitoring apparatus
- Humidity: continously controlled by a Cole Parmer monitoring apparatus
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil (CAS 8001-30-7)
- Justification for choice of solvent/vehicle: The test item is reactive in water, and hence, the use of a non-aqueous vehicle was recommended. A test was conducted using propylene glycol as vehicle, but sacrifice and examination of the peritoneal cavaties revealed a dense, white precipitate, leading to the assumption that the test item is not sufficiently distributed to the blood stream. Hence, propylene glycol was considered not suitable as a vehicle in this study.
- BRRC Sample No.: 50-248

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions were prepared fresh on the morning of each test day and the accuracy of dilution was verified by gravimetric analysis.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

none

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

- Range-finding study (toxicity testing): 5
- Main studies: 5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name of the positive control substance: triethylenemelamine (TEM, CAS 51-18-3)
- Doses / concentrations: 0.3 mg/kg bw
- Sampling time for positive control: 30 h
- Justification for sampling time: Previous tests have shown that blood sample preperation of the TEM treated animals at the 30 sample period was the optimum time interval for detecting a positive response.

Tissues and cell types examined:

Polychromatic erythrocytes of the pripheral blood were examined for micro nuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The dose selection was based on the results of the preliminary toxicity testing.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Samples were taken 30, 48, and 72 h after test material injection.

DETAILS OF SLIDE PREPARATION:
One or two blood smear slides were prepared and stained with Giemsa for each animal per sampling time.

METHOD OF ANALYSIS:
A minimum of 1000 polychromatic erythrocytes was examined microscopically for each animal per sampling time, unless cytotoxicity of the test material prevented this goal. The polychromatic : normochromatic erythrocyte ratio for approximately 1000 total cells was calculated and recorded and these data were summarised as an estimate of cytotoxicity of the test material. Micronuclei were identified as darkly-stained, spherical, inclusions in polychromatic erytrhrocytes. Polychromatic erythrocytes were identified by the pale-blueish staining of the cytoplasm in contrast to the lack of blue stain for normochromatic erythrocytes.

Evaluation criteria:

Data were compared for significant differences from the vehicle control frequencies using the Fisher's Exact Test. Data for males and female mice at each sample period were combined for statistical analyses when Analysis of Variance tests showed that there were no significant differences in micronuclei frequencies between sexes at each sample period.

Statistics:

A positive result in the micronucleus test was concluded if at least one statistically significant (p = 0.01) increase above the vehicle control was observed with an indication of a dose-related effect of treatment. A test was considered to be inconclusive if only one dose produced effects statistically different from the control (0.0.5 = p > 0.01) and a dose-relationship was not apparent. A test result was considered to be negative if no statistically significant differences were apparent between the vehicle control and groups of animals treated with the test item.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 10-2430 mg/kg bw
- Solubility: poor solubility at high dose level (2430 mg/kg bw) and, as a consequence, precipitation in the peritoneal cavities leading to survival of the test animals and low toxicity
- Clinical signs of toxicity in test animals: All animals of the 810 mg/kg bw dose group died within 3 days after dosing. At 90 mg/kg bw all animals survived, but treatment-related toxicity (significant reduction in body weight gain in both males and femles) was observed.
- Evidence of cytotoxicity in tissue analyzed: PCE/NCE ratios were calculated for vehicle control animals and animals of the highest dose with 3 or more survivers. Although 3 females survived dosing with 2430 mg/kg bw, also animals of the 90 mg/kg bw dose group were evaluated since it was likely that he 2430 mg/kg bw dose level was poorly distributed because of precipitation within the peritoneal cavity. At 48 h after dosing the PCE/NCE ratios of both males and females were similar to the vehicle control values, thus indicating the absence of bone marrow toxicity.
- Rationale for exposure: A combined LD50 of 194 mg/kg bw for both males and females was evaluated in the range-finding study. Based on these results the doses applied in the main study were set at 50, 100, and 160 mg/kg bw, referring to 25- 50, and 80% of the determined LD50 value.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Statistically significant increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes were observed at 30 h post injection with mice treated with the lowest dose level (50 mg/kg bw) and at 72 h post injection with mice treated with the highest dose level (160 mg/kg bw).
- Ratio of PCE/NCE (for Micronucleus assay): Only males treated with 100 mg/kg bw and sampled 72 h after dosing showed significant decrease in PCE/NCE ratios relative to control values. However, significant increases in in the number of PCEs were observed with various dose groups of both males and females at all three sampling periods. Increased levels of PCEs in the peripheral blood can occur as a recruitment response to excessive blood loss or haemorrhage. PCE/NCE ratios of animals treated with TEM (positive control) were lower than the concurrent negative control values, which is an expected typical finding because of cytotoxicity and clastogenicity of this agent.

Tab. 1: Results of the in vivo micronucleus assay in male animals (Experiment 1)

			Mean PCEs / 1000 NCEs at sampling time			Total micronuclei per 1000 PCEs at sampling time
Exp group	Number of animals	Dose	30 h	48 h	72 h	30 h	48 h	72 h
Vehicle control (corn oil)	5	10 ml/kg	22.4±6.80	21.8±5.72	26.0±9.70	14	8	12
Positive control (triethylmelamine)	5	0.3 mg/kg	18.6±4.77	n. d.	n. d.	177	n. d.	n. d.
Test substance	5	50 mg/kg	31.6±14.54	26.8±7.05	22.8±6.61	28	12	11
Test substance	5	100 mg/kg	25.6±8.76	28.4±10.45	17.2±6.46	11	17	11
Test substance	5	160 mg/kg	44.0±5.57	41.4±9.07	44.6±13.22	16	17	27

n. d. = not determined

  

Tab. 2: Results of the in vivo micronucleus assay in female animals (Experiment 1)

			Mean PCEs / 1000 NCEs at sampling time			Total micronuclei per 1000 PCEs at sampling time
Exp group	Number of animals	Dose	30 h	48 h	72 h	30 h	48 h	72 h
Vehicle control (corn oil)	5	10 ml/kg	39.0±16.09	34.2±2.59	26.8±6.57	16	17	8
Positive control (triethylmelamine)	5	0.3 mg/kg	22.6±9.86	n. d.	n. d.	107	n. d.	n. d.
Test substance	5	50 mg/kg	55.2±10.13	41.8±9.09	36.0±4.30	19	13	16
Test substance	5	100 mg/kg	37.4±6.73	42.6±9.34	24.2±6.67	7	11	7
Test substance	5	160 mg/kg	46.2±15.34	44.4±11.74	39.7±8.50	11	17	6

n. d. = not determined

 

Tab. 3: Results of the in vivo micronucleus assay in male animals (Experiment 2)

			Mean PCEs / 1000 NCEs at sampling time			Total micronuclei per 1000 PCEs at sampling time
Exp group	Number of animals	Dose	30 h	48 h	72 h	30 h	48 h	72 h
Vehicle control (corn oil)	5	10 ml/kg	52.0±7.38	48.4±3.91	52.4±4.88	20	16	26
Positive control (triethylmelamine)	5	0.3 mg/kg	45.2±13.77	n. d.	n. d.	191	n. d.	n. d.
Test substance	5	50 mg/kg	56.8±5.76	47.8±11.26	37.4±10.50	19	31	18
Test substance	5	100 mg/kg	60.2±12.93	52.6±11.84	47.6±24.53	29	36	14
Test substance	5	160 mg/kg	53.6±6.69	36.8±5.36	39.2±9.09	26	38	10

n. d. = not determined

  

Tab. 4: Results of the in vivo micronucleus assay in female animals (Experiment 2)

			Mean PCEs / 1000 NCEs at sampling time			Total micronuclei per 1000 PCEs at sampling time
Exp group	Number of animals	Dose	30 h	48 h	72 h	30 h	48 h	72 h
Vehicle control (corn oil)	5	10 ml/kg	36.0±3.54	37.4±3.58	34.4±7.89	15	9	10
Positive control (triethylmelamine)	5	0.3 mg/kg	37.6±8.96	n. d.	n. d.	205	n. d.	n. d.
Test substance	5	50 mg/kg	41.0±6.82	48.8±11.30	41.8±16.62	14	24	11
Test substance	5	100 mg/kg	52.0±12.31	52.4±19.15	33.8±16.25	15	18	11
Test substance	5	160 mg/kg	51.7±9.79	59.0±18.77	65.4±20.63	47	38	36

n. d. = not determined

Conclusions:

Interpretation of results: negative expert judgement
The test item was tested for chromosome aberration in vivo in the mouse micronucleus assay. The study was conducted similar to the OECD TG 474, and in compliance with GLP. The test item was administered by a single injection to both male and female Swiss Webster mice at doses of 50, 100, and 160 mg/kg bw, based on a previous toxicity evaluation. Samples from peripheral blood were taken 30, 48, and 72 h after test material injection. Appropriate vehicle and positive controls were included into the study and gave the expected results. No sex-related differences in the response were observed. Hence, for statistical analysis the results obtained for both males and females were combined. In the first experiment, statistically significant increases in the frequencies of micronuclei were observed in the low dose animals (50 mg/kg bw) at 30 h sampling time and in the high dose group (160 mg/kg bw) at the 72 h sampling time. However, the increase in micronuclei observed never reached values two times higher than the control values. Increased ratios of polychromatic erythrocytes (PCE) to normochromatic erthrocytes (NCE) were also observed at various dose levels and sample periods, which may be indicative of excessive blood loss or haemorrhage. Since either of these factors can artificially elevate the incidence of micronucleated erythrocytes, additional testing was performed to verify the reproducibility of the first test and to evaluate the possibility of concurrent internal haemorrhage. In the repeat experiment, significant levels of PCE with micronuclei were observed with the highest dose level (160 mg/kg bw) sampled at 30 h and with all three dose levels sampled 48 h post-injection. The results indicate that the increases observed had no dose-related pattern. In addition, PCE/NCE ratios for the female mice were significantly higher than concurrent control values. A gross necropsy of the gastrointestinal organs and peritoneal cavity was performed 72 hr after dosing. All animals dosed with the highest two dose levels of the test item had darkened and distended intestines and/or caecums indicative of internal haemorrhage.
Based on these results, the authors concluded the test material to be questionably active as a clastogenic agent in vivo under the conditions of this micronucleus test. Nevertheless, since no dose dependency was observed and haemorrhage occurred, a biologically relevance cannot be assumed. Hence, the test item can be considered to be not clastogenic to mammals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Several Ames assays and an in vitro chromosome aberration assay as well as an in vivo micronucleus assay are available that were performed with the test substance bis(trimethoxysilylpropyl)amine (CAS 82985-35-1). For assessment of mammalian mutagenicity two hypoxanthine-guanine phosphoribosyl transferase (HPRT) assays are available that were performed with the analogue substances 3-aminopropyltriethoxysilane (CAS 919-30-2) and N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3).

The silicon-containing products of hydrolysis are close structural analogues: bis(trimethoxysilylpropyl)amine (CAS 82985-35-1), 3-aminopropyltriethoxysilane (CAS 919-30-2), and N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3) hydrolyse to bis(trihydroxysilylpropyl)amine, 3-aminopropylsilanetriol, and N-(3-(trihydroxysilyl)propyl)ethylenediamine. Bis(trimethoxysilylpropyl)amine (CAS 82985-35-1), 3-aminopropyltriethoxysilane (CAS 919-30-2), and N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3) hydrolyse very rapidly: hydrolysis is expected to occur during testing and following exposure. The further products of hydrolysis are methanol and ethanol, respectively. Since neither methanol nor ethanol would contribute to general systemic toxicity effects in rodents at the dose levels tested, it is considered that the observed toxicological effects are due to the action of the silicon-containing moiety, although the specific toxicological mechanism cannot be determined from the available information. Further information is given in a supporting report (PFA, 2013aa) attached in Section 13 of the IUCLID 5 dossier.

Genetic toxicity in vitro:

In the available key study (BSL Bioservice, 2005) the test item was investigated for mutagenicity to bacteria according to the OECD TG 471, and in compliance with GLP. In two independent experiments (plate incorporation and pre-incubation) the test material was tested in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and in E. coli WP2 uvrA up to precipitation and cytotoxic concentrations with and without a metabolic activation system. No significant increase in the number of revertants was observed in any of the tested strains with and without metabolic activation. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not mutagenic to bacteria under the conditions of the test.

This result is further supported by several studies (Biotoxtech, 2010; Huntingdon Research Center Ltd., 1989b; Bushy Run Research Center, 1982b and 1988a) evaluating the genotoxic potential of the test item in the bacterial reverse mutation assay (Ames) with and without metabolic activation. The test item was not mutagenic in any of these studies.

In the available key study (Bushy Run Research Center, 1990), the test item was evaluated for cytogenicity to mammalian cells in a study conducted similar to the OECD TG 473, and in compliance with GLP. CHO cells treated with the test material up to cytotoxic concentrations with and without metabolic activation, did not exhibit any significant changes of chromosomal aberrations as compared to the control. Appropriate negative, solvent, and positive controls were included into the test and gave the expected results. Hence, the test item was considered to be not clastogenic under the conditions of the test.

No studies are available on mutagenicity in mammalian cells that were performed with the test substance bis(trimethoxysilylpropyl)amine (CAS 82985-35-1) itself. Nevertheless, two studies are available with the analogue substances 3-aminopropyltriethoxysilane (CAS 919-30-2) and N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3) where the mutagenicity in mammalian cells was investigated. These studies were used in a weight-of-evidence approach. In the first study Chinese Hamster Ovary cells were treated (4-hour treatment) with 200, 600, 1800, 3000, 5000 µg/ml (with and without metabolic activation) 3-aminopropyltriethoxysilane (CAS 919-30-2), according to OECD 476 and in compliance with GLP (Hüls AG, 1998). No cytotoxicity was observed up to the limit concentration of 5000 µg/ml. Expected results were obtained from solvent and positive controls. The results from the repeat experiment were consistent with those from the initial test. No increase in the mutant frequency was observed in Chinese hamster Ovary (CHO) cells 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 concluded that the test substance is not mutagenic to mammalian cells under the conditions of the test.

In the second study Chinese Hamster Ovary cells were treated (5-hour treatment) with 2.5-4.0 mg/ml (without S9) and 2.0-4.5 mg/ml (with S9) N-(3-(trimethoxysilyl)propyl)ethylenediamine (CAS 1760-24-3), equivalent to OECD 476 and in compliance with GLP (Bushy Run Research Center, 1988b). Cytotoxicity was observed after treatment with 4.5 mg/ml test substance in the presence of metabolic activation. An increase in mutant frequency was observed in one of two duplicate cultures with metabolic activation at one concentration in the initial experiment. No other increase in mutant frequency was observed at any concentration with and without activation. The experiment was repeated (with metabolic activation): no increase in mutant frequency was observed. The appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutations in CHO cells under the conditions of the test.

 

Genotoxicity in vivo:

In the available key study (Bushy Run Research Center, 1988c) the test item was evaluated for genotoxicity to mammals in the in vivo mouse micronucleus assay in a study conducted similar to the OECD TG 474, and in compliance with GLP. The test item was administered by a single injection to both male and female Swiss Webster mice at doses of 50, 100, and 160 mg/kg bw, based on a previous toxicity evaluation and referring to 25, 50, and 80% of the LD50 determined in the pre-test of this study. Samples from peripheral blood were taken 30, 48, and 72 h after test material injection. Appropriate vehicle and positive controls were included into the study and gave the expected results. No sex-related differences in the response were observed. Hence, for statistical analysis the results obtained for both males and females were combined. In the first experiment, statistically significant increases in the frequencies of micronuclei were observed in the low dose animals (50 mg/kg bw) at 30 h sampling time and in the high dose group (160 mg/kg bw) at the 72 h sampling time. However, the increase in micronuclei observed never reached values two times higher than the control values. Increased ratios of polychromatic erythrocytes (PCE) to normochromatic erthrocytes (NCE) were also observed at various dose levels and sample periods, which may be indicative of excessive blood loss or haemorrhage. Since either of these factors can artificially elevate the incidence of micronucleated erythrocytes, additional testing was performed to verify the reproducibility of the first test and to evaluate the possibility of concurrent internal haemorrhage. In the repeat experiment, significant levels of PCE with micronuclei were observed with the highest dose level (160 mg/kg bw) sampled at 30 h and with all three dose levels sampled 48 h post-injection. The results indicate that the increases observed had no dose-related pattern. In addition, PCE/NCE ratios for the female mice were significantly higher than concurrent control values. A gross necropsy of the gastrointestinal organs and peritoneal cavity was performed 72 h after dosing. All animals dosed with the highest two dose levels of the test item had darkened and distended intestines and/or caecum indicative of internal haemorrhage.

Based on these results, the authors concluded the test material to be questionably active as a clastogenic agent in vivo under the conditions of this micronucleus test. Nevertheless, since no dose dependency was observed and haemorrhage occurred, a biologically relevance cannot be assumed. Hence, the test item can be considered to be not clastogenic to mammals.

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on this data, classification for genetic toxicity according to 67/584/EEC and EC/1272/2008 is not warranted.