Trisilicon tetranitride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 May to 12 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trisilicon tetranitride

EC Number:

234-796-8

EC Name:

Trisilicon tetranitride

Cas Number:

12033-89-5

Molecular formula:

N4Si3

IUPAC Name:

1Si-hexacyclo[3.1.1.0¹,⁴.0²,⁵.0³,⁶.0³,⁷]trisilazane

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Batch number: A126325
Purity: >98%

Method

Target gene:

hemizygous hypoxanthine phoshoribosyl transferase (HPRT)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Preliminary toxicity test: 10.9, 21.9, 43.8, 87.5, 175, 350, 700 and 1400 μg/mL
Mutation tests:
Test 1: 87.5, 175, 350, 700 and 1400 μg/mL
Test 2: 87.5, 175, 350, 700 and 1400 μg/mL

Vehicle / solvent:

Sodium chloride (0.9% w/v)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

In the presence of S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

In the absence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: Duplicate cultures were used for each test substance concentration and positive control, and four cultures for the vehicle control.

NUMBER OF CELLS EVALUATED: For each culture, three 60 mm tissue culture plates were seeded with 200 cells in H10, to determine cloning efficiency and five 100 mm tissue culture plates with 2E+05 cells in selective medium to determine the number of mutant colonies.

DETERMINATION OF CYTOTOXICITY
- Method: Colonies were counted and the Day 1 relative survival was calculated.

No additional data

Evaluation criteria:

The statistical significance of the data was analysed by weighted analysis of variance, weighting assuming a Poisson distribution following the methods described by Arlett et al. (1989). Tests were conducted for a linear concentration-response relationship of the test substance, for non-linearity and for the comparison of positive control to vehicle control. The criteria for a positive response will be:
The demonstration of a statistically significant increase in mutant frequency following exposure to the test substance;
Evidence of a relationship, over at least two dose levels, in any increase in mutant frequency;
Demonstration of reproducibility in any increase in mutant frequency;
The mean mutant frequency should fall outside the upper limit of the historical vehicle control of 20 mutants per 1E+06 survivors with a corresponding survival rate of 20% or greater.

Statistics:

None stated

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
In the preliminary toxicity test, a four-hour exposure to 1400 μg/mL the test substance, in the absence or the presence of S9 mix, resulted in Day 1 Relative Survivals of 101 to 78% and 130 to 80 % respectively. Concentrations for the main test were based upon these data.

Main mutation test 1 - 4 hour treatment in the absence of S9 mix:
Cultures were exposed to the test substance at concentrations from 87.5 to 1400 μg/mL. Precipitate was seen at 700 μg/mL and above post dosing and at 175 μg/mL and above at the end of treatment. Exposure to the test substance resulted in Day 1 Relative Survivals of between 101 and 21%. All cultures exposed were plated out for determination of Cloning Efficiency and mutation induction. The Day 8 Cloning Efficiencies were not reduced relative to the vehicle controls. No significant increases in Mutant Frequency were observed after exposure to the test substance. EMS, the positive control substance, induced significant increases in Mutant Frequency.

Main mutation test 1 - 4 hour treatment in the presence of S9 mix:
Cultures were exposed to the test substance at concentrations from 87.5 to 1400 μg/mL. Precipitate was seen at 700 μg/mL and above post dosing and at 175 μg/mL and above at the end of treatment. Exposure to the test substance resulted in Day 1 Relative Survivals of between 102 and 43 %. All cultures exposed were plated out for determination of Cloning Efficiency and mutation induction. The Day 8 Cloning Efficiencies were reduced from 106 to 49% relative to the vehicle control. No significant increases in Mutant Frequency were observed after exposure to the test substance. 3MC, the positive control substance, induced significant increases in Mutant Frequency.

Main mutation test 2 - 4 hour treatment in the absence of S9 mix:
Cultures were exposed to the test substance at concentrations from 87.5 to 1400 μg/mL. Precipitate was seen at 700 μg/mL and above post dosing and at 175 μg/mL and above at the end of treatment. Exposure to the test substance resulted in Day 1 Relative Survivals of between 94 and 17%. All cultures exposed were plated out for determination of Cloning Efficiency and mutation induction. The Day 8 Cloning Efficiencies were 102 to 74% relative to the vehicle control. No significant increases in Mutant Frequency were observed after exposure to the test substance. EMS, the positive control substance, induced significant increases in Mutant Frequency.

Main mutation test 2 - 4 hour treatment in the presence of S9 mix:
Cultures were exposed to the test substance at concentrations from 87.5 to 1400 μg/mL. Precipitate was seen at 700 μg/mL and above post dosing and at 175 μg/mL and above at the end of treatment. Exposure to the test substance resulted in Day 1 Relative Survivals of between 103 and 44%. All cultures exposed were plated out for determination of Cloning Efficiency and mutation induction. The Day 8 Cloning Efficiencies were 99 to 85% relative to the vehicle control. No significant increases in Mutant Frequency were observed after exposure to the test substance. 3MC, the positive control substance, induced significant increases in Mutant Frequency.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that the test substance did not demonstrate mutagenic potential in this in vitro HPRT cell mutation assay, under the experimental conditions described.