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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:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: New and Revised Health Effects Test Guidelines October 1984, (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295). HG - Gene Muta - S. typhimurium, October 1984
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
semi-solid (amorphous): gel
Remarks:
migrated information: paste
Details on test material:
Name of test substance: Metanilic acid
Order number: BALK 90/068
Product number: 037354
Batch number: K 195
Content: 70.2% Metanilic acid, 30.9% H2O (analytical result dated February 18, 1991)
Chemical name: Benzenesulfonic acid, 3-amino or Aniline-3-sulfonic acid
Approved: until September 19, 1992
Appearance: white to light-grey paste
Storage: refrigerator

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Checked for Christal Violet and UV sensitivity.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 mix
Test concentrations with justification for top dose:
First test: 0, 8, 40, 200, 1000, 5000 µg/plate
Repeat test: 0, 150, 300, 600, 1200, 2400, 4800 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water. The solvent used was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
(Solvent employed for the positive controls: DMSO)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Strain TA 1537, TA 98, TA 100 and TA 1535, with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene diamine
Remarks:
Strain TA 1537 and TA 98, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Nitrofurantoin
Remarks:
Strain TA 100, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strain TA 1535, without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);

For the mutant count, four plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained four plates per strain. The amount of solvent for the test substance and for the controls was 0.1 mL/plate.

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 μg or 5 μl per plate were used as the highest dose. At least four additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
The toxicity of the substance was assessed in three ways. The first method was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37°C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers. The results of these determinations may be seen in the negative controls.
The dilution of bacterial suspensions used for the determination of titers was 1 :1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased from 0.5 mM to 2.5 mM to permit the complete growth of bacteria.
The tests were performed both with and without S9 mix.
The count was made after the plates had been incubated for 48 hours at 37°C. If no immediate count was possible, plates were temporarily stored in a refrigerator.
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density-in the suspension.
An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed 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 compound.

No "untreated” negative control was set up for deionized water, since sufficient evidence was available in the literature ( e.g. Maron and Ames, 1983) and from our own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the bacterial strains used.

Evaluation criteria:
Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that 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, investigations should continue, possibly with modifications, until a final evaluation is possible.

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
Remarks:
≥ 300µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
There was no indication of a bacteriotoxic effect of the test substance at doses of up to and including 200 μg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a weak, strain-specific bacteriotoxic effect. Nevertheless, these doses could be used for assessment up to and including 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 tests with and without S9 mix and was confirmed by the results of the repeat tests.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.
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