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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid

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:
Aroclor-induced rat-liver S9 mix
Test concentrations with justification for top dose:
preliminary toxicity test without metabolic activation: 20 - 5000 µg/plate
main test (with and without metabolic activation): 313, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "Details on test system"
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS: 3METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: three Petri dishes are prepared per strain and per group

DETERMINATION OF CYTOTOXICITY
- Method: ; relative total growth

- POSITIVE CONTROLS
WITHOUT S9
1) for strain TA 98: 2-nitrofluorene, 10 µg/plate in dimethylsulfoxide
2) for strains TA 100 and TA 1535: sodium azide, 2.0 µg/plate in bidistilled water
3) for strain TA 1537: 9 (5)-aminoacridine hydrochloride monohydrate, 150 µg/plate in dimethylsulfoxide
4) for strain E. coli: 4-nitroquinoline-N-oxide, 1 µg/plate in dimethylsulfoxide.

WITH S9
1) for strains TA 98 and TA 100: 2-aminoanthracene, 2.5 µg/plate in dimethylsulfoxide
2) for strain TA 1535: cyclophosphamide monohydrate, 400 µg/plate in bidistilled water
3) for strain TA 1537: 2-aminoanthracene, 5µg/plate in dimethylsulfoxide;
4) for strain E. coli WP2uvrA: 2-aminoanthracene, 50 µg/plate in dimethylsulfoxide
.
Evaluation criteria:
Criteria for a positive response:
The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535, TA 1537 and E. coli
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.
Assay acceptance criteria:
A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.
In either case the final decision has to be based on scientific judgement.

Results and discussion

Test results
Key result
Species / strain:
other: S.typhimurium TA 98, TA 100, TA 1535, TA 1537; Escherichia coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Nine concentrations of the test item ranging from 20 to 5000 µg/0.1 ml were tested to determine the highest concentration to be used in the mutagenicity assay. From the results obtained, the highest concentration suitable for the mutagenicity test was found to be 5000 µg/0.1 ml.

Any other information on results incl. tables

EXPERIMENTAL RESULT

Experiment 1

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 20 13 116 130 12 11 29 35 26 33
313 13 16 108 121 8 13 23 52 22 31
625 13 17 114 124 9 12 18 43 13 33
1250 13 13 99 123 9 15 21 39 16 26
2500 17 15 114 115 10 13 18 43 26 37
5000 13 9 98 142 9 11 26 50 17 36
positive control 568 451 606 1781 1225 307 1225 815 443 1223

Experiment 2

TA 1535 TA 100 TA 1537 TA 98 WP2uvrA
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
0 8 12 138 120 8 11 23 34 16 23
312.5 9 11 132 147 8 13 18 40 23 27
625 12 6 162 148 10 12 24 36 17 24
1250 9 12 133 129 9 13 16 34 27 17
2500 11 8 148 128 10 11 20 46 16 27
5000 11 10 140 134 7 11 20 48 26 19
positive control 455 477 667 1481 1585 126 1520 1184 379 546

Applicant's summary and conclusion

Conclusions:
In the experiments performed without and with microsomal activation none of the tested concentrations of the test item led to an increase in the incidence of both histidine- or tryptophan-prototrophic mutants by comparison with the negative control.