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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1977
Report Date:
1977

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No information on the purity of the substance and number of cells used. No individual plate counts, no statistical analyses. Only four strains tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Aroclor 1254 plus co-factors
Test concentrations with justification for top dose:
without microsomal activation : 10, 25, 50, 100, and 250 μg/0.1 mL
with microsomal activation: 5, 10, 25, 50, and 100 μg/0.1 mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Remarks:
with and without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3 out of 6 plates per dose with activation were preincubated

NUMBER OF REPLICATIONS:
In the experiments without the addition of microsomal activation mixture, three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In the experiments with activation mixture six Petri dishes were used per strain and per group; three of each set of six were pre-incubated

POSITIVE CONTROLS:
Without S9:
Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 2 µg/0.1 ml DMSO;
Strain TA 1537: 9(5)-aminoacridine hydrochloride monohydrate, 100 µg/0.1 ml DMSO
Strain TA 98: daunoblastin, 50 µg/0.1 ml DMSO;
Strain TA 100: 4-nitroquinoline-n-oxide, 10 µg/0.1 ml DMSO.
With S9:
Strain TA 100: cyclophosphamide, 100 and 250 µg/0.1 ml physiological saline
Evaluation criteria:
The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation: at 100 and 250 μg/0.1 mL in soft agar
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Salmonella mutagenicity test: reverse mutation without microsomal activation. Numbers represent the arithmetic mean of histidine-prototrophic colonies.

Test substance Strain of S. typhi-murium used
TA 98 TA 100 TA 1535 TA 1537
control 28 115 11 8
10 μg/0.1 mL 24 118 11 8
25 μg/0.1 mL 24 151 14 5
50 μg/0.1 mL 24 149 17 6
100 μg/0.1 mL 24 136 13 7
250 μg/0.1 mL 24 163 16 7

Table 2. Salmonella/Mammalian microsome mutagenicity test: reverse mutation with microsomal activation. Numbers represent the arithmetic mean of histidine-prototrophic colonies.

n/n: first figures are values without, second figures values with pre-incubation.

Test substance Strain of S. typhi-murium used
TA 98 TA 100 TA 1535 TA 1537
control 18 / 21 173 / 165 18 / 20 8 / 7
5 μg/0.1 mL 23 / 25 179 / 202 17 / 17 9 / 9
10 μg/0.1 mL 22 / 25 181 / 188 21 / 20 8 / 5
25 μg/0.1 mL 23 / 21 190 / 217 26 / 20 7 / 8
50 μg/0.1 mL 23 / 23 177 / 189 21 / 19 7 / 7
100 μg/0.1 mL 23 / 22 157 / 155 17 / 18 7 / 7

Applicant's summary and conclusion

Conclusions:
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment revealed no significant differences.