[3-(2,3-epoxypropoxy)propyl]triethoxysilane

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

Study period:

13th of January 2015 to 16th of March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 26th of Sep 2014

Deviations:

yes

Remarks:

Use of peanut oil that was not anhydrous

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

[3-(2,3-epoxypropoxy)propyl]triethoxysilane

EC Number:

220-011-6

EC Name:

[3-(2,3-epoxypropoxy)propyl]triethoxysilane

Cas Number:

2602-34-8

Molecular formula:

C12-H26-O5-Si

IUPAC Name:

triethoxy[3-(oxiran-2-ylmethoxy)propyl]silane

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Hsd:ICR (CD-1)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, Maryland, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: 31.5 - 37.0 grams
- Assigned to test groups randomly: yes, a randomised procedure with a weight variation that did not exceed ± 20% of the mean weight.
- Fasting period before study: not specified
- Housing: Up to 5 male animals were housed in Micro-Barrier cage.
- Diet: Harlan 2018C Certified Global Rodent Diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C (72 ± 3°F)
- Humidity (%): 50 ± 20%
- Air changes (per hr): ⩾10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: January 2015 To: 16th of March 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Peanut oil
- Justification for choice of solvent/vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics.
- Concentration of test material in vehicle: 20 mL/kg
- Amount of vehicle: 20 mL/kg for the test and vehicle control and 10 mL/kg for the positive control cyclophosphamide.
- Type and concentration of dispersant aid: N/a
- Lot/batch no.: MKBN5294V
- Purity: not specified

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Single administration

Post exposure period:

N/a

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5 males for each treatment group and 5 additional males for the control and positive control, respectively.

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide monohydrate
- Justification for choice of positive control(s): The standard OECD test guideline requires a positive control.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw/day

Examinations

Tissues and cell types examined:

Bone marrow was collected from all treatment groups. Polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) were examined for micronuclei.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The doses were based on the results of a previous dose range finding study. The highest dose level was selected with the aim of inducing toxic effects, however, not death or severe suffering. The dose levels tested were 500, 1000 or 2000 mg/kg bw/day. Clinical signs including piloerection was observed at the highest tested dose, however, there were no other toxicological relevant observations. Consequently, the maximum tolerated dose for the definite study was set to 2000 mg/kg bw/day which is also the highest dose that the guideline recommends for this type of assay.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): no further information available.

DETAILS OF SLIDE PREPARATION: Following euthanasia, approximately 24 or 48 hours after the final dose, the femurs were exposed (cut just above the knee) and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow was centrifuged and the supernatant drawn off. The cell pellet was re-suspended and a small drop of the bone marrow suspension was spread onto a clean glass slide (at least 4 slides per mouse). The slides were air-dried, fixed in methanol and stained with acridine orange, the latter was done for one of two sets of slides to enable a set of backup. Slides were thereafter randomly coded.

METHOD OF ANALYSIS: Bone marrow cells were evaluated utilising a fluorescent microscope. At least 4000 polychromatic erythrocytes were scored for micronuclei per animal. To determine the proportion of PCEs as an index of bone marrow cytotoxicity, at least 500 erythrocytes (PCEs and NCEs) were scored per animal.

Evaluation criteria:

To be considered as inducing a positive response, at least one of the test substance doses would exhibit a statistical significant, dose related increase compared to the concurrent negative control as well as with results of the group mean or of the individual animals in at least one group being outside the 95% control limit of the historical negative control data.

Statistics:

Statistical analysis including mean and standard deviation, was performed on the micronucleus frequency and PCE, using the animal as the unit. Moreover, parametric or non-parametric statistical methods were used depending on the variation between groups. Levene’s test was used for determining the group variances for micronucleus frequency for the vehicle and test substance groups. This variation was found to not be significant why a parametric one-way ANOVA was performed followed by a Dunnett post-hoc analysis to compare each dose group to the concurrent vehicle control.
To assess dose responsiveness in the treated groups, a linear regression analysis was conducted. Additionally, a pair-wise comparison (Studen't T-test) was used to compare the positive control group to the concurrent vehicle control group.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

There was evidence for exposure of the target tissue in the reported cytotoxicity to bone marrow, where a statistically significant increase in the ratio of PCEs to total erythrocytes (EC) was observed. However, this increase was not dose-related.

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 1000 or 2000 mg/kg bw/day
- Solubility: not specified
- Clinical signs of toxicity in test animals: Clinical signs included piloerection occurring in the 2000 mg/kg bw/day dose group for both males and females.
- Evidence of cytotoxicity in tissue analysed: Not specified
- Rationale for exposure: The guideline recommends the highest dose do not exceed 2000 mg/kg bw/day.
- Harvest times: not specified
- High dose with and without activation: N/a

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No statistically significant in the incidence of micronucleated polychromatic erythrocytes (MnPCEs) in the test substance-treated groups was observed relative to the negative control group (p<0.05, One-Way ANOVA).
- Ratio of PCE/NCE: There was evidence for exposure of the target tissue in the reported cytotoxicity to bone marrow, where a statistically significant increase in the ratio of PCEs to total erythrocytes (EC) was observed. However, this increase was not dose-related.
- Appropriateness of dose levels and route: The dose levels were based on the dose range finder study which was performed prior to the definitive study. The recommended route in OECD test guideline (474) is oral gavage.
- Statistical evaluation: A statistically significant increase in the incidence of MnCPEs was observed for the positive control. All other criteria for a valid study were also met.

Any other information on results incl. tables

Table 1: Summary of Bone Marrow Micronucleus Analysis

Treatment (20 mL/kg)	Sex	Time (h)	No of Animals	PCE% (Mean ± SD	Change from Control (%)	MnPCE% (Mean ± SD)		Number of MnPCE/PCE Scored
Peanut oil	M	24	5	42.5 ± 1.0	-	0.01 ± 0.01		1/20 000
500 mg/kg bw/day	M	24	5	45.8 ± 0.7	8	0.02 ± 0.02		3/20 000
1000 mg/kg bw/day	M	24	5	44.5 ± 0.7	5	0.01 ± 0.01		1/20 000
2000 mg/kg bw/day	M	24	5	45.5 ± 0.9	7	0.00 ± 0.00		0/20 000
Cyclophosphamide 50 mg/k	M	24	5	50.6 ± 2.0	19	1.84 ± 0.08	**	367/20 000
Peanut oil	M	24	5	42.3 ± 0.6	-	0.01 ± 0.01		2/20 000
2000 mg/kg	M	24	5	46.0 ± 0.7	9	0.03 ± 0.03		6/20 000

* p<0.05 or ** p<0.01, One-Way ANOVA with Post-Hoc analysis or T-Test

PCE – polychromatic erythrocytes; MnPCE – micronucleated polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:

In an in vivo micronucleus study with the test substance [3-(2,3-epoxypropoxy)propyl]triethoxysilane, conducted according to OECD TG 474 and in compliance with GLP, no induction of micronucleated polychromatic erythrocytes occurred. There was evidence for exposure of the target tissue in the reported cytotoxicity to bone marrow where a statistically significant increase in the ratio of PCEs to total erythrocytes (EC) was observed. However, this increase was not dose-related. The positive control provided expected results. It is concluded that the test substance is negative under the conditions of the test.