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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:
02 December 2014 - 18 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
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

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

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia 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:
S9 mix from rat liver.
Test concentrations with justification for top dose:
Salmonella typhimurium strains:
Initial mutation test: 1581, 500, 158.1; 50; 15.81; 5; 1.581 and 0.5 µg/plate with metabolic activation and 500, 158.1; 50; 15.81; 5; 1.581; 0.5 and 0.1581 µg/plate without metabolic activation.
Confirmatory mutation test: 500, 158.1; 50; 15.81; 5; 1.581; 0.5 and 0.1581 µg/plate with metabolic activation and 158.1; 50; 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate without metabolic activation.

E.coli strain:
Initial and Confirmatory mutation test: 5000, 1581, 500, 158.1; 50; 15.81, 5 and 1.581 µg/plate with and without metabolic activation.
Complementary Confirmatory Mutation Test: 158.1; 50; 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate without metabolic activation.
Vehicle / solvent:
- 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 Acetone. Emulsion was achieved using Distilled water as vehicle at 100 mg/mL concentration. However, the test item was soluble at the same concentration using DMSO or Acetone as vehicles. Due to the better biocompatibility to the test system, DMSO was selected as vehicle (solvent) for the study.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO and distilled water.
Positive controls:
yes
Remarks:
4 µg/plate.
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine
Remarks:
Salmonella TA 98, without metabolic activation.
Positive controls:
yes
Remarks:
2 µg/plate.
Positive control substance:
sodium azide
Remarks:
Salmonella TA 100 and TA 1535, without metabolic activation.
Positive controls:
yes
Remarks:
50 µg/plate.
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA 1537, without metabolic activation.
Positive controls:
yes
Remarks:
2 µL/plate.
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA, without metabolic activation.
Positive controls:
yes
Remarks:
2 µg/plate.
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Salmonella strains, with metabolic activation.
Positive controls:
yes
Remarks:
50 µg/plate.
Positive control substance:
other: 2-aminoanthracene
Remarks:
E.coli WP2 uvrA, with metabolic activation.
Details on test system and experimental 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 and other components 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:
- in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA strains the number of reversion was more than two times higher than the spontaneous reversion rate of the negative (vehicle/solvent) control plates,
- the number of reversions was more than three times higher than the reversion rate of the negative (vehicle/solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, 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:
The test item was considered to have shown no mutagenic activity in this study if it produces 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.

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:
at the higher concentration range.
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:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The observed numbers of revertant colonies were in the normal range at the non-cytotoxic concentrations and were within the historical control range. No precipitate of the test item was detected in the Preliminary Range Finding Test.

Any other information on results incl. tables

In the Initial Mutation Test and Confirmatory Mutation Tests 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. No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test in Salmonella typhimurium TA 98 and TA 100 strains at 500 and 158.1 µg/plate concentrations without metabolic activation and in these strains at 1581 and 500 µg/plate concentrations with metabolic activation; in Salmonella typhimurium TA 1535 and TA 1537 strains at 500, 158.1 and 50 µg/plate concentrations without metabolic activation and in these strains at 1581, 500 and 158.1 µg/plate concentrations with metabolic activation. Similar but stronger inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Tests in all tested Salmonella typhimurium strains at 158.1m 50 and 15.81 µg/plate concentrations without metabolic activation; in Salmonella typhimurium TA 98, TA 1535 and TA 1537 strains at 500 and 158.1 µg/plate concentrations with metabolic activation; in Salmonella typhimurium TA 100 strain at 500, 158.1 and 50 µg/plate concentrations with metabolic activation; in Escherichia coli WP2 uvrA strain at 158.1 and 50 µg/plate concentrations without metabolic activation and in this strain at 5000, 1581 and 500 µg/plate concentrations with 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 in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Applicant's summary and conclusion

Conclusions:
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, following the Principles of GLP. The experiments were carried out using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 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 formulated in DMSO. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the examined concentrations in the Initial Mutation Test for all tested Salmonella typhimurium strains with metabolic activation were 1581, 500,158.1; 50; 15.81; 5; 1.581 and 0.5 µg/plate, without metabolic activation were 500,158.1; 50; 15.81; 5; 1.581; 0.5 and 0.1581 µg/plate. Examined concentrations in the Confirmatory Mutation Test for all tested Salmonella typhimurium strains with metabolic activation were 500; 158.1; 50; 15.81; 5; 1.581; 0.5 and 0.1581 µg/plate, without metabolic activation were 158.1; 50; 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate. Examined concentrations in the Initial and Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain with and without metabolic activation were 5000, 1581, 500,158.1; 50; 15.81, 5 and 1.581 µg/plate. Examined concentrations in the Complementary Confirmatory Mutation Test in Escherichia coli WP2 uvrA strain without metabolic activation were 158.1; 50; 15.81; 5; 1.581; 0.5; 0.1581 and 0.05 µg/plate. In the Initial Mutation Test and Confirmatory Mutation Tests 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. Inhibitory, cytotoxic effect of the test item were observed in the Initial Mutation Test in all tested Salmonella typhimurium strains at the higher concentration range.Similar, but stronger inhibitory, cytotoxic effect of the test item were observed in the Confirmatory Mutation Tests at the higher concentration range in all tested bacterial strains. The mean values of revertant colonies of the negative (vehicle) 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 test, the examined concentration range was considered to be adequate. The study was considered to be valid. In conclusion, the test item had no mutagenic activity in the examined bacterial strains under the test conditions of this study.