Y-513

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:

07 December 2007 - 11 January 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Y-513
- Purity test date: no data
- Lot/batch No.: F-1570
- Expiration date of the lot/batch: 19 February 2013
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

histidine gene in Salmonella typhimurium
tryptophan gene in Escherichia coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

uninduced male Golden Syrian Hamster liver S9-mix

Test concentrations with justification for top dose:

Preliminary test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate +/- S9 mix
Main study: 3, 10, 33, 100, 333 µg/plate +/- S9 mix

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: test substance is soluble in DMSO. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S-9 mix

Migrated to IUCLID6: 5 μg/plate in saline for TA1535

Controls 2

Positive control substance:

2-nitrofluorene

Remarks:

without S-9 mix

Migrated to IUCLID6: 15 μg/plate in Milli-Q water for TA1537

Controls 3

Positive control substance:

2-nitrofluorene

Remarks:

without S-9 mix

Migrated to IUCLID6: 10 μg/plate in DMSO for TA98

Controls 4

Positive control substance:

methylmethanesulfonate

Remarks:

without S-9 mix

Migrated to IUCLID6: 650 μg/plate in DMSO for TA100

Controls 5

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S-9 mix

Migrated to IUCLID6: 10 μg/plate in DMSO for WP2uvrA

Controls 6

Positive control substance:

other: 2-aminoanthracene (2AA) 1 μg/plate in DMSO for TA1535

Remarks:

with S-9 mix

Controls 7

Positive control substance:

other: 2-aminoanthracene (2AA) 2.5 μg/plate in DMSO for TA1537

Remarks:

with S-9 mix

Controls 8

Positive control substance:

other: 2-aminoanthracene (2AA) 1 μg/plate in DMSO for TA98

Remarks:

with S-9 mix

Controls 9

Positive control substance:

other: 2-aminoanthracene (2AA) 5 μg/plate in DMSO for TA100

Remarks:

with S-9 mix

Controls 10

Positive control substance:

other: 2-aminoanthracene (2AA) 5 μg/plate in DMSO for WP2uvrA

Remarks:

with S-9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:preincubation


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 h
- Expression time (cells in growth medium): 10^9 cells/ml


NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.



DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies



OTHER: precipitation of test substance

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment

Statistics:

none

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

(precipitation at 333 µg/plate and above at the end of the incubation period)

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 33 µg/plate and upwards and at 333 µg/plate and above at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed. No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

In an AMES test, performed according to OECD guideline and GLP principles, Y-513 was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to the OECD guideline and GLP principles. All bacterial strains showed negative responses up to precipitating concentrations (precipitation was observed at 333 µg/plate and above at the end of the incubation period), i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that Y-513 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.