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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:
March 21, 2000 - July 19, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Aqueous solution
Details on test material:
- Physical state: Aqueous solution
- Appearance: Yellowish liquid
- Composition of test material, percentage of components: see section confidential details on test material

Method

Target gene:
Histidine (S. typhimurium) and tryptophan (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by phenobarbitone and betanaphthoflavone
Test concentrations with justification for top dose:
Concentrations were expressed in terms of material as received (aqueous solution)
Toxicity test: 50, 158, 500, 1580 and 5000 µg/plate
Experiment 1, plate incorporation method: 313, 625, 1250, 2500 and 5000 µg/plate (TA1535, TA1537, TA98 and WP2 uvrA) and 156, 313, 625, 1250 and 2500 µg/plate (TA100)
Experiment 2, preincubation method: 313, 625, 1250, 2500 and 5000 µg/plate (TA1535, TA1537, TA98 and WP2 uvrA) and 156, 313, 625, 1250 and 2500 µg/plate (TA100)
Experiment 3, preincubation method: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate (TA1535 and TA1537) and 39.1, 78.1, 156, 313 and 625 µg/plate (TA100 without S9-mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Justification for choice of solvent/vehicle: Water is regarded as compatible with the Ames test. The minimum required solubility for the test article of 5% (corresponding with 5000 μg/plate) was reached.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: See section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: First experiment: in agar (plate incorporation); second and third experiment: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: Toxicity test single plate, main experiments three replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: the number of revertants and the background growth

Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose-level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels. The effect must be reproduced in an independent experiment.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions.

Results and discussion

Test resultsopen allclose all
Key result
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:
See section Additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See section Additional information on results
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING STUDY:
Moderate toxicity, as indicated by reduction in revertant numbers and thinning of the background lawn, was observed in TA1535 and TA1537 at the highest dose-level tested, both in the absence and presence of S9 metabolic activation. A more pronounced effect was observed in TA100. Reduction in revertant colonies was observed at the two highest concentrations in WP2uvrA, in the absence of S9 metabolism.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: slight toxicity, as indicated by thinning of the background lawn, was observed at the highest dose level in strain TA1535, TA1537 and TA100.
Experiment 2: moderate thinning of the background lawn was observed at the highest dose-level with TA98 and WP2uvrA tester strains, while marked toxicity was observed with TA1535, TA1537 and TA100 tester strains at most of the concentrations tested.
Experiment 3: toxicity, as indicated by reduction in revertant numbers and thinning of the background lawn, was observed at the highest dose-level only in strain TA1535, TA1537 and TA100, both in the absence and presence of S9 metabolism.

Any other information on results incl. tables

PRECIPITATION CONCENTRATION: none

Applicant's summary and conclusion

Conclusions:
The mutagenic potential of a related member of MIRANOL ULTRA L32 (amphoacetates C12) was assessed in Salmonella typhimurium bacterial strains and Escherichia coli , according to EC method B.13-B.14 and modified OECD guideline 471, and in compliance with Good Laboratory Practice. It was concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The mutagenic potential of a related member of MIRANOL ULTRA L32 (amphoacetates C12) was assessed in Salmonella typhimurium bacterial strains and Escherichia coli , according to EC method B.13-B.14 and modified OECD guideline 471, and in compliance with Good Laboratory Practice.

The histidine-requiring S. typhimurium mutants TA 1535, TA 1537, TA 98, TA 100 and tryptophan-requiring E.coli mutant WP2uvrA were used in the presence and the absence of metabolic activation (+/- S9 -mix). All determinations were made in triplicate. The first experiment was conducted using a plate-incorporation method at 5 doses levels up to 5000 µg/plate. The second and third experiment were performed using a pre-incubation method. Simultaneously negative (solvent) and positive controls were used in all experiments.

In a preliminary range-finding assay, toxicity was seen.

In the plate-incorporation assay, 4 strains were treated at dose levels 313 to 5000 µg/plate, while TA100 was treated at 156 to 2500 µg/plate. Slight toxicity was observed at the highest dose level with TA1535, TA1537 and TA100, but there was no increase in the revertant numbers in any of the strains tested.

The second assay was performed with a pre-incubation step and using the same dose-range. Moderate thinning of the background lawn was observed at the highest dose level with TA98 tester strain, while marked toxicity was observed with TA1535, TA1537 and TA100 at the 3 highest dose levels. A third experiment was then performed with these 3 strains using a lower dose-range (39.1- 625 µg/plate without S9-mix, and 78.1-1250 µg/plate only for TA1535 and TA1537 in the presence of S9 -mix).

The test material did not cause any two-fold or greater increase in the mean number of revertant colonies in the test plates, both in the absence and the presence of metabolic activation. Positive controls gave the expected increases in the number of revertants, with and without S9 mix.

Under the conditions of this study, the test material did not demonstrate any in vitro mutagenic activity in these bacterial test systems.