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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:
12 Oct 2016 to 28 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo Research Limited., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD UK
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat S9
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat S9
Test concentrations with justification for top dose:
EXPERIMENT 1: PLATE INCORPORATION METHOD
- Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
- Justification of top dose
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.

EXPERIMENT 2; PRE-INCUBATION METHOD
- Dose selection:
In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix: Salmonella strain TA98 and E.coli strain WP2uvrA (with S9-mix): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. All other bacterial strains (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate. The eight test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle:
dimethyl sulphoxide
Controls
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

OTHER
- The plates were scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).
Evaluation criteria:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced a stronger toxic response in the second mutation test (pre-incubation method), with weakened bacterial background lawns noted in the absence of S9-mix from 150 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535, TA98 and WP2uvrA). In the presence of S9-mix weakened bacterial background lawns were noted from 500 μg/plate (all Salmonella strains) and 1500 μg/plate (WP2uvrA).

Any other information on results incl. tables

Small, statistically significant increases in TA100 revertant colony frequency were observed in the second mutation test at 15 and 50 μg/plate in the presence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle control.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella Typhimurium and Escherichia Coli reverse mutation assay performed equivalent to OECD 471.
Executive summary:

The mutagenic activity of the test substance was evaluated in a study equivalent to OECD TG 471 (1997) and according to GLP principles. A plate incorporation assay (experiment 1) and a pre-incubation assay (experiment 2) were performed in the absence and presence of S9 mix. The dose levels were selected based on observed cytotoxicity in the plate incorporation dose range finding study. In the pre-incubation assay, the maximum dose level of the test item in the first experiment (plate-incorporation method) was selected as the maximum recommended dose level of 5000 μg/plate. The test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 500 μg/plate in the absence of S9-mix and 1500 μg/plate in the presence of S9-mix. Consequently the maximum recommended dose level of the test item (5000 μg/plate) or the toxic limit was employed in pre-incubation test. Negative, solvent and positive controls were included and gave appropriate responses. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 metabolic activation in both assay formats. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.