GASIR1

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:

16/2/2005-1/3/2005

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): GASIR1
- Physical state: red solid
- test material was ground prior to testing.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Substance used: S9, organ: liver, origin: male Sprague Dawley rats.

Test concentrations with justification for top dose:

Preliminary toxicity test: 0; 0.15; 0.5; 1.5; 5; 15; 50; 150; 500; 1500; 5000 μg/plate
Range-finding test: 50, 150, 500, 1500, 5000 μg/plate
Main test: 50 - 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: polyethylene glycol
- Justification for choice of solvent/vehicle:
1 Polyethylene glycol 400 was screened for compatibility in the Salmonella and Escherichia coli mutagenicity test at four dose volumes, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver 89 in standard co- factors). The method used conforms to the guidelines for bacterial mutagenicity testing.
2. Four dose volumes were used in the assay: 100, 200, 300 and 400 μ1. In addition the standard positive controls used routinely in-house were suspended in PEG 400 and dosed onto base agar plates at 200 and 400 μ1. The methodology used in the test was performed as described in the general text of the report.
3. PEG 400 caused no visible reduction in the growth of the bacterial lawn or to the frequency of revertant colonies at any of the dose volumes tested either with or without metabolic activation.
4. PEG 400 did not inhibit the mutagenic effects of any of the standard positive controls used in the assay and gave revertant frequency counts within the acceptable ranges for each strain.

CONCLUSION
All results (both vehicle and positive control counts) were within acceptable ranges and therefore we consider PEG 400 to be satisfactory for use in the reverse mutation assay. The test material formed the best dosable suspension in polyethylene glycol 400 at 50mg/ml.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

using a solvent treatment group

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: + 4-nitroquinoline-1-oxide, 9-aminoacridine (without metabolic activation), 2-aminoanthracene and benzo(a)pyrene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:/
- Exposure duration:48h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration:48h

SELECTION AGENT (mutation assays):



NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration:48h


SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period:
- Exposure duration:48h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:3

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression (5)) significant increase in the revertant count in at least one strain of bacteria.

Statistics:

/

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:done, not discussed

COMPARISON WITH HISTORICAL CONTROL DATA:not done

ADDITIONAL INFORMATION ON CYTOTOXICITY:

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

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the USA, EP A (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TAI 535, TAI537, TA98 and TAI00 and Escherichia coli strain WP2 uvrA" were treated with suspensions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (polyethylene glycol 400) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A slight pink colour was observed at and above 1500 μg/plate with an associated blue precipitate observed at 5000 μg/plate. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.