Registration Dossier

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:
Between 05 Jan 1995 and 03 March 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
The positive controls for the S9 series of plates with salmonella strains TA100, TA98 and TA1537 have been changed for Benzo(a)prene (BP) at 5µg/plate to 2-aminoantracene for Bacterial strains TA100-1µg/plate, TA98-0.5µg/plate and TA1537-2µg/plate.
GLP compliance:
yes (incl. certificate)
Remarks:
Date of Inspection 31st Jan 1994 and Date of signing 16th March 1994
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Alkane 2 (C1527-04-2)
- Substance type: Clear colourless liquid
- Physical state: Liquid
- Storage condition of test material: At room temperature in a silver coloured metal cannister.

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
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:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in house from the livers of male Sprague Dawley. These had received a single i.p injection of Aroclor 1254 at 500mg/kg.
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 0,15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 0,15,50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test material was fully soluble in Acetone in solubility checks performed in-house.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix

Migrated to IUCLID6: WP2uvrA- TA100 TA1535
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix

Migrated to IUCLID6: TA1537
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: TA 98
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene WP2uvrA-, TA100, TA1535, TA 1537 and TA 98
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth and thinning of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
1. Tester strain Integrity

The tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate when plated along with the vehicle under selective conditions. These ranges may increase by as much as 25% if S9 concentrations of greater than 10% are used in the mutagenicity assay.

2. Tester Strain Culture Density

To demonstrate that appropriate numbers of viable bacteria have been plated, the viable count for each tester strain culture must be greater than or equal to 0.1 x 10^9 bacteria per ml.

3. Positive control Values.

All positive control chemicals must exhibit positive responses within the historical range for the dose levels used. Acceptable positive control values demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and of the integrity of the S9 mix.

4.Cytotoxicity

A minimum of four non toxic dose levels will be required to evaluate assay data.

For a substance to be considered positive in this test system, it should have induced a dose related and statistically(5) significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels.

To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000µg/plate.


Statistics:
All data are statistically analysed using the methods recommended by the UKEMS (5) and normally Dunnetts method of linear regression is used to evaluate this result.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 micro.g/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was fully soluble in Acetone at 50 mg/ml in solubility checks performed in-house.
- Precipitation: A precipitate was observed at and above 500µg/plate, this however did not interfere with the scoring of revertant colonies.



RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The numbers of revertant colonies for the toxicity assay are presented in Table 1 in Appendix 2.

The reults of the S9 optimisation study are presented in Table 2 of Appendix 2. No marked difference was observed between the four levels of S9 and therefore the default level of 10% S9 was selected for use in the mutation studies.





COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.


ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 500µg/plate, this however did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies was recorded for any of the strains of bacteria used, at any dose level, either with or without metabolic activation.

The positive control substances all produced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

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:

1. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escerichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at six dose levels. In triplicate both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EEC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 15 to 5000µg/plate in the first experiment. A second experiment was performed on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh chemical formulations. The optimum concentration of S9 in the S9 -mix was also determined in a preliminary assay and was 10% for both experiments.

2. The vehicle (acetone) and untreated control plates produced counts of revertant colonies within the normal range.

3. All of the positive control chemicals used in the study induced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

4. The test material caused no visible reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. The test material was therefore, tested up to the maximum recommended dose level of 5000µg/plate, this however did not interfere with the scoring of revertant colonies.

5. No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test.

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