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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Key study: OECD 471.GLP study. The test item had no mutagenic activity in the bacterium tester strains.
Link to relevant study records
Reference
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:
29 October 2013 - 7 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD 471. GLP study.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). The test item was partially soluble in Distilled water. However the formulations at 100 mg/mL concentration using DMSO or DMF as vehicle were suitable for the test. Due to the better biocompatibility to the test system, DMSO was selected as vehicle of the study.
Solvent controls:
yes
Remarks:
DMSO and distilled water
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
sodium azide
Remarks:
TA100, TA1535, -S9
Positive controls:
yes
Remarks:
4 µg/plate
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
TA98, -S9
Positive controls:
yes
Remarks:
50 µg/plate
Positive control substance:
9-aminoacridine
Remarks:
TA1537, -S9
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA, -S9
Positive controls:
yes
Remarks:
2 µg/plate
Positive control substance:
other: 2-aminoantracene
Remarks:
all Salmonella strains, +S9
Positive controls:
yes
Remarks:
50 µg/plate
Positive control substance:
other: 2-aminoanthracene
Remarks:
WP2 uvrA, +S9
Details on test system and conditions:
METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item (or controls) and other components (top agar, overnight culture of test strain, phosphate buffer (pH 7.4) or S9 mix) were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.

Pre-incubation (confirmatory mutation test):
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were incubated at 37°C for 48 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines [5][6][7], statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.
Criteria for a Negative Response:
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The observed numbers of revertant colonies were in the normal range and within the historical control range in all cases. No precipitate, inhibitory or cytotoxic effect of the test item was detected.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

In the Initial Mutation Test and Confirmatory Mutation Test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect. There were no signs of inhibitory, cytotoxic effect of the test item in the Initial Mutation Test and the Confirmatory Mutation Test in any examined bacterial strain at any concentration with or without metabolic activation. Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (vehicle/solvent) and positive control plates were within the historical control range. The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.

Conclusions:
Interpretation of results (migrated information):
negative (with and without metabolic activation)

The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the solvent control and were within the normal biological variability of the test system. There were no signs of inhibitory, cytotoxic effect of the test item in the Initial Mutation Test and the Confirmatory Mutation Test in all examined bacterial strains at all concentrations with or without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid. In conclusion, the test item had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Key study: The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471 (GLP study). The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats) up to 5000 μg/plate. None of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. There were no signs of inhibitory, cytotoxic effect. The tests were considered to be valid. In conclusion, the test item had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.


Justification for selection of genetic toxicity endpoint
Only one study avalaible.

Justification for classification or non-classification

Based on the available data, the substance is not classified for mutagenicity according to CLP Regulation (EC) no. 1272/2008.