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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:
1990-09-18 to 1990-09-27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study. No data were available on analytical inveatigations on the stability of the test substance in solution. However, this deviation did not limit the assessment of the results. Only four strains tested.
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Directive 84/449/EEC
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 26 May 1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Method

Target gene:
Histidine-auxotrophic Salmonella typhimurium strains TA 1535, TA 100, TA 1537 and TA 98.
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:
Aroclor 1254-induced rat liver S9 fraction
Test concentrations with justification for top dose:
With S9 mix: 8, 40, 200, 1000, 5000 µg/plate
Without S9 mix: 8, 40, 200, 1000, 5000 µg/plate
Vehicle / solvent:
The solvent employed for the test substance was ethanol and for the positive controls DMSO.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
in strain TA1535, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: nitrofurantoin
Remarks:
in strain TA100, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene diamine
Remarks:
in strains TA1537 and TA 98, without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
all strains, with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

The following criteria were used for the acceptance of an assay:
a) The negative controls have to be within the expected range, as defined by literature data and the laboratories' own historical data.
b) The positive controls have shown sufficient effects as defined by the laboratories' experience.
c) The titer determinations had to demonstrate sufficient bacterial density in the suspension.
An assasy which was not in agreement with at least one of the above criteria was not used for assessment.
Furthermore the data generated in this assay have to be confirmed by two additional independent experiments. Even, if the criteria for points a, b and c were not met, an assay was accepted if it showed mutagenic activity of the test substance.
Evaluation criteria:
A reproducable and dose-related increase in mutant counts for at leat one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice the amount of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement. In case of questionable results, inveatigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
not applicable

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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no indication of a bacteriotoxic effect for the test substance at doses up to and including 200 µg per plate. The total bacteria counts consictently produced results comparable to the neagtive control, or differed only insignificantly. Likewise an inhibition of growth was not detected.
Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only be used to a limited extent up to 5000 µg per plate.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the test with and without S9 mix and was confirmed by the results of the repeated tests.


Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, the test substance showed no mutagenic activity in this bacterial reverse mutation test on Salmonella typhimurium.
Executive summary:

The test substance was investigated in the Salmonella / microsome test for point-muagenic effects in doses up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 200 µg per plate did not cause any bateriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

At higher doses, the substance had a strain-specific bacteriotoxic effect, so that this range could only be used to a limited extend up to 5000 mg per plate for assessment purposes. Evidence of mutagenic activity for the test substance was not seen. No biologically-relevant increase of the mutant count, in comparision with the negative controls, was observed. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2 -phenylene-diamine and 2 -aminoanthracene had a marked mutagenic effect, as was seen by a biologically-relevant increase in mutant colonies compared to the corresponding negative controls.