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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:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
1987
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Direct Orange 39

Method

Target gene:
Histidine-prototrophic
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Test concentrations with justification for top dose:
- Range in the cytotoxicity test*: 20.6-5000 μg /plate
- Range in the orig. mutagenicity test*: 61.7-5000 μg/plate
- Range in the 1st conf. mutagenicity test**: 228.6 - 18519 μg/plate
- Range in the 2nd conf. mutagenicity test**: 228.6 - 18519 μg/plate

* The purity of the tested batch is 27%. Related to 100% purity the highest concentration in these parts of the assay was 1350 μg/plate active ingredient.
** Related to 100% purity the highest concentration in this part of the assay was 5000 μg / plate active ingredient.
Vehicle / solvent:
Bidistilled water (suspension)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
Colonies were counted electronically with an Artek counter. The results were sent on line to a computer. They were checked on a random basis by the operator. Observations indicating precipitates of the test substance in the top agar or a reduced or absent bacterial background lawn were registered additionally. Means and standard deviations for all mutagenicity assays were calculated by a previously validated computer program and included in the Results section.
Evaluation 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.

Criteria for a positive responses:
At least a reproducible meaningful increase of the mean number of revertants per plate above that of the negative control at any concentration for one or more of the following strains:
S. typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537.
Generally a concentration-related effect should be demonstrable.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
The highest concentration applied was 5000 ug/plate. The five lower concentrations decreased by a factor of 3
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Based on the results of these experiments and on standard evaluation criteria, it is concluded that the substance exerted a marginal mutagenic action on strain S. typhimurium TA 98. This effect, however, was only observed at the concentration of 18519 µg/plate with a test material of 27% purity. Hence, the test substance was considered not to be mutagenic in the bacteria reverse mutation assay.
Executive summary:

A bacterial reverse mutation test was carried out according to OECD test guideline 471 in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535, and TA 1537.

The concentration range of Direct Orange 39 (purity 27%) to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, Direct Orange 39 was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate based on test material. An independent repetition of the experiments was performed with the concentrations of 228.6 to 18519 µg/plate test material (equals to 62 to 5000 µg/plate active ingredient). In-order to confirm the results obtained in the second assay on strain TA 98 without activation and on strain TA 102 with activation, the experiments on these two strains were repeated once more with the concentrations of 228.6 to 18519 µg/plate (62 to 5000 µg/plate active ingredient).

In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed.

 

Mutagenicitv test, original experiment (61.7 to 5000 µg/plate test mat.)

In the original experiment carried out without and with metabolic activation, none of the tested concentrations of Direct Orange 39 led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

 

Mutagenicitv test, first confirmatory experiment (228.6 to 18519 µg/plate test mat.)

In the first confirmatory experiment performed without metabolic activation on strain TA 98, a marginal increase in the number of revertant colonies was observed at the concentration of 18519 µg/plate. No effect was observed with the other strains. In the experiment performed with activation on strain TA 102, a marginal increase (not reaching two-fold) in the number of revertant colonies was observed at the concentration of 685.9 µg/plate only. No effect was seen with the other strains.

 

Mutagenicitv test, second confirmatory experiment (228.6 to 18519 µg/plate test mat.)

In the second confirmatory experiment performed without metabolic activation on strain TA 98, again, a marginal increase in the number of revertant colonies was observed at the concentration of 18519 µg/plate. In the experiment performed with activation on strain TA 102, none of the tested concentrations of Direct Orange 39 led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control.

In the mutagenicity tests without and with metabolic activation, normal background growth was observed. The number of revertant colonies was not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Direct Orange 39 exerted a marginal mutagenic action on strain S. typhimurium TA 98. This effect, however, was only observed at the concentration of 18519 µg/plate.

The test was performed under Good Laboratory Practice conditions and was subjected to a periodical quality assurance evaluation.

No circumstances, which may have affected the quality or integrity of the data, have been noted.