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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
Study period:
15.10. - 11.12.1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study, GLP

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991
Reference Type:
other: Summary Report
Title:
Unnamed
Year:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1984
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
1987
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
THE MINISTRY OF HEALTH AND WELFARE, Japan, 1984
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

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:
S-9 mix prepared from male RAI rat liver five days after i.p. application of 500 mg/kg bw Aroclor 1254.
Test concentrations with justification for top dose:
- Range finding test in WP2 uvrA and TA 100: 0, 20.58, 61.73, 185.19, 555.56, 1666.67, 5000 µg/plate; with and without S9-mix.
- Mutagenicity test: 0, 312.5, 625, 1250, 2500, 5000 µg/plate; with and without S9-mix tested with all tester strains.
Vehicle / solvent:
The test substance was completely dissolved in acetone at a concentration of 50 mg/mL. No precipitates or aggregates were noted at any concentration.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
see under "Details on test system and conditions"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hrs

NUMBER OF REPLICATIONS: three plates for each dose level

NUMBER OF INDEPENDENT EXPERIMENTS: 2

DETERMINATION OF CYTOTOXICITY
- Method: colony counting

POSTIVE CONTROLS
Without S9 mix:
TA 100, TA 1535: sodium azide (5 µg/plate in bidest water)
WP2 uvrA: 4-nitroquinoline-N-oxide (2 µg/plate in DMSO)
TA 98: 2-nitrofluorene (20 µg/plate in DMSO)
TA 1537: 9-aminoacridine (150 µg/plate in DMSO)
with S9 mix:
TA 100, TA 98, TA 1537: 2-aminoanthracene (2.5 µg/plate in DMSO)
TA 1535: cyclophosphamide (400 µg/plate in bidest water)
WP2 uvrA: 2-aminoanthracene (50 µg/plate in DMSO)
Evaluation criteria:
Reaction to a substance is regarded as positive if all of the following criteria are met
- doubling of spontaneous control mutation rate
- dose-response relationship
- reproducibility of results
Statistics:
Not performed.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitating of test substance at 1666.67, 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test article was observed at concentrations of 2500 µg/plate and higher

RANGE-FINDING/SCREENING STUDIES:
From the results obtained the highest concentration suitable for the mutagenicity test was selected to be 5000 µg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical values of negative and positive controls obtained with each strain without and with microsomal activation, which contained mean values of controls obtained from separate experiments over the period of 15.01.1990 to 02.01.1991.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Results:

Main Experiment

TA 100 TA 1535 WP2 uvra TA 98 TA 1537
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Solvent Control 115 86.7 15.3 14.7 26 29 45 61.3 8.3 9
312.5 100.3 87.3 15 13.7 24.7 27 45.3 58.7 7.7 10
625 119 76.3 14.7 13 24.3 30 43.7 67.3 9 6.7
1250 99 76.7 12.3 16 29.7 32.7 41.7 64 6.3 8.7
2500 107.3 85 13.3 14.7 25.7 19.3 45.7 68.3 9.7 10
5000 110 80.3 14.7 16.3 30 28 44.3 62.7 9 11.7
positive control 1250.7 1763 972.3 460.7 876.3 1102.7 1941.3 1839.3 294 2100.7

Confirmatory Experiment

TA 100 TA 1535 WP2 uvra TA 98 TA 1537
Dose (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Solvent Control 146.3 131.3 15.7 16 17.7 20.7 21.3 35 8 6.3
312.5 132.7 129.7 15.7 18.7 13.7 20.3 15.7 37 6.7 11.7
625 134 136 16 14 15 13.7 21.7 42 8.7 10
1250 129.7 113.7 16.3 15 16 16 17.7 31 10.7 7.7
2500 139 143.7 15 17.7 17.7 17.7 24.7 42.3 8.3 8.7
5000 127.3 129.3 15.3 16.3 20.7 16.3 23.3 35.3 10.3
positive control 1919 2613 698 373.7 1121.7 894.7 1333.7 2524.7 1189.7 137

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of these experiments and on standard evaluation criteria, it is concluded that the test article and its metabolites did not induce gene mutations in the strains of S. typhimurium and E. coli used.
Executive summary:

The test substance was tested in the Salmonella typhimurium reverse mutation assay with Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and with Escherichia coli WP2 uvrA. In the dose range finding test, the test article was tested up to concentrations of 5000 µg/plate in the absence of S9-mix in the strains TA100 and in E.coli WP2 uvrA. At this dose level no toxicity was observed. Consequently, the main test was performed with concentrations of up to 5000 µg/plate in the absence and presence of S9 -mix. In the experiments performed without and with microsomal activation, treatment with the test article did not lead to an increase in the incidence of either histidine- or tryptophanprototrophic mutants in comparison with the negative control. In the confirmatory experiment performed without and with microsomal activation, again after treatment of the bacteria with the test substance no increase in the incidence of either histidine- or tryptophan-prototrophic mutants was observed in comparison with the negative control. Precipitation of the test material occurred at concentrations of 2500 µg/plate and up. Based on the results of this study it is concluded that the test article is not mutagenic in the Salmonella typhimurium reverse mutation assay.