Bis(trimethoxysilylpropyl)amine

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

Bis(trimethoxysilylpropyl)amine

EC Number:

280-084-5

EC Name:

Bis(trimethoxysilylpropyl)amine

Cas Number:

82985-35-1

Molecular formula:

C12H31NO6Si2

IUPAC Name:

3,3,11,11-tetramethoxy-2,12-dioxa-7-aza-3,11-disilatridecane

Details on test material:

- Name of test material (as cited in study report): Organofunctional Silane Y-9492
- Physical state: clear, colourless nonviscous liquid
- Impurities (identity and concentrations):
- BRRC Sample No: 51-A
- Stability: reactive in water
- Storage condition of test material: room temperature
- Date of receiption: 08 January 1988

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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.

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.