Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 26 August 1997 and 11 September 1997.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Gel All DX
- Batch number: 4073003
- Description: white powder
- Date received: 20 August 1997
- Storage conditions: ambient temperature, stored under artificial light.

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:
Aroclor induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Study: 50, 150, 500, 1500 and 5000 µg/plate
Mutation study (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of bacterial strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Positive controls (ENNG, 9AA and 4NQO) used without metabolic activation (S9)
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of bacterial strains
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
2AA used with metabolic activation (S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plaing

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



Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in revertant count in one or more strains of bacteria in the presence and/or absence of S9 in both experiments at sub-toxic dose levels.
To be considered negative, the number of revertants at each dose level should be less than twofold that of the vehicle control frequency.
Statistics:
All of the data was analysed using the statistical methods recommended by the UKEMS with Dunnett's method of linear regression used to evaluate the result.

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Manual counts were performed at and above 1500 µg/plate because of test material precipitation.


PRELIMINARY TOXICITY STUDY:
The dose range of the test material used in the preliminary toxicity study was 0, 50, 150, 500 and 5000 µg/plate. The test material was non-toxic to the strains of bacterial used (TA100 and WP2uvrA-).

MUTATION STUDY:
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) are presented in Table 1 (attached background material) and were considered to be acceptable. The data are for concurrent untreated control plates performed the same day as the Mutation Study.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls both with and without metabolic activation are presented in Tables 2 and 3 for Experiment 1 and Tables 4 and 5 for Experiment 2 (see attached background material).

No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 1500 µg/plate, but this did not prevent scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the stains of bacteria, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction was shown to be satisfactory.












Remarks on result:
other: A precipitate was observed at 1500 µg/plate

Any other information on results incl. tables

Preliminary Toxicity Study

The mean number of revertant colonies for the toxicity assay were:

Strain

Dose (µg/plate)

0

50

150

500

1500

5000

TA100

137

130

139

139

134P

130P

WP2uvrA-

18

18

16

18

21P

19P

P = precipitate.

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of the test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- 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 was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (dimethyl sulphoxide) 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 and without metabolic activation.

The test material caused no visible reduction in the growth of the bacterial lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose of 5000 ug/plate. A precipitate was observed at and above 1500 ug/plate, this 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.

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