SILATRIZOLE

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 November 1995 to 06 March 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 471 without any deviation

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Germany GLP Compliance Programme (inspected on 05/06/07 April 1995/signed on 1995-08-02)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-940-4

EC Name:

-

Cas Number:

155633-54-8

Molecular formula:

C24H39N3O3Si3

IUPAC Name:

2-(2H-1,2,3-benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-(2,2,4,6,6-pentamethyl-3,5-dioxa-2,4,6-trisilaheptan-4-yl)propyl]phenol

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): G4375
- Physical state: whitish solid
- Analytical purity: > 99%
- Impurities (identity and concentrations): Methanol (<100 ppm) and Isopropanol (<100 ppm)
- Purity test date: certificate of analysis 20/10/1995
- Lot/batch No.: DEF/C 95003/B
- Expiration date of the lot/batch: September 1996 (retest date)
- Stability under test conditions: No data
- Storage condition of test material: room temperature, avoid UV irradiation

Method

Target gene:

Histidine gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: see table 7.6.1/1

Species / strain 2

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

15% S9 mix; S9 fraction prepared from liver homogenates of Wistar male rats induced with Aroclor 1254

Test concentrations with justification for top dose:

PRELIMINARY CYTOTOXICITY ASSAY:
Experiment with and without S9 mix (plate incorporation method): 3.3, 10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate (TA 98, TA 100)

MUTAGENICITY ASSAY:
Experiment 1 with and without S9 mix (plate incorporation method): 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate (TA 1535, TA 1537, TA 98, TA 100, WP2 and WP2 uvrA)
Experiment 2 with and without S9 mix (Pre-incubation method): 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate (TA 1535, TA 1537, TA 98, TA 100, WP2 and WP2 uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

yes

Remarks:

untreated

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Experiment 1:in agar (plate incorporation)
Experiment 2: preincubation

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

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: 1 plate/dose
- Mutation studies: 3 plates/dose

NUMBER OF CELLS EVALUATED: no data

DETERMINATION OF CYTOTOXICITY
- Method: reduction the number of spontaneous revertants or clearing of the bacterial background lawn.:

OTHER:
- The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS Gmbh, D-61184 Karben, FRG).

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or reproducible increase for at least one test concentration is induced.

A test article is considered mutagenic if in the strains TA98, TA100, WP2, and its uvrA derivate, the number of reversions will be at least twice as high, and in the strains TA1535 and TA1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

None

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: no toxic effects, evidenced by a reduction in the number of revertants, occurred in the groups with and without metabolic activation in all strains used.

MUTATION STUDIES:
- No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with the test item at any dose level, either in the presence or absence of metabolic activation.
- There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
- Positive controls showed distinct increase of induced revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA: not applicable

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation

Under these test conditions, Silatrizole was found to be not mutagenic in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia Coli stains WP2 and WP2 uvrA with and without metabolic activation according to the Regulation (EC) N° 1272-2008 (CLP) and according to the Directive 67/548/EEC.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100) and of Escherichia Coli (WP2 and WP2 uvrA) were exposed to Silatrizole diluted in acetone at the following concentrations for mutagenicity assays (direct incorporation method in experiment 1 and pre-incubation method in experiment 2): 33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate in the presence or in the absence of metabolic activation. Metabolic activation system used in this study was 15 % S9 mix. S9 fraction was prepared from liver homogenates of Wistar male rats induced with Aroclor 1254. Vehicle and positive control groups were also included in this test.

 

No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with the test item at any dose level, either in the presence or absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Positive controls showed distinct increase of induced revertant colonies. No cytotoxicity was observed in any of the strains although it has been tested up to limit concentrations required by the OECD Guideline 471 (5000 µg/plate).

 

Under these test conditions, Silatrizole was found to be not mutagenic in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and in Escherichia Coli stains WP2 and WP2 uvrA with and without metabolic activation, according to the criteria of the CLP regulation (EC) N°1272/2008.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.