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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:
From 2014-05-28 to 2014-07-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity: > 99%
Batch No.: 10181

Method

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
Test concentrations with justification for top dose:
To set the dose levels for the main tests, the 100 mg/mL solution was diluted to 4 times at a common ratio of 4 and a total of 5 dose levels selected (19.5, 78.1, 313, 1250 and 5000 μg/plate) in the dose-finding test.
In the dose-finding test, precipitation of the test article on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration on the plate was not observed at any dose levels irrespective of the presence/absence of metabolic activation. Growth inhibition was observed at 78.1 μg/plate or more in S. typhimurium TA1537 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA 98, TA 100 and TA 1535 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains with or without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: Based on the information of the sponsor, since the test article was insoluble in water, DMSO and acetone, the solubility test was conducted with DMF and 1,4-dioxane. As the result, DMF was used as the vehicle in this study because it was soluble at 100 mg/mL, and 1,4-dioxane was insoluble at 100 mg/mL.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
DMF
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
positive control for TA 100, TA 98, TA 1537 with metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
positive control for TA 1535 without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)
Remarks:
positive control for TA 100, TA 98, WP2 uvrA with metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)- aminopropylamino]acridine.2HCl (ICR-191)
Remarks:
positive control for TA 1537 without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
positive control for TA 1535, WP2 uvrA with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes

Number of Plates: 2 plates at each dose level in the dose-finding test, 3 in the two main tests.
Evaluation criteria:
If a two-fold or more increase in the number of revertant colonies compared to that of spontaneous revertant colonies (the negative control value), dose-response and reproducibility were noted, or even if no clear dose-response was observed but there were at least two-fold increase in the number of revertant colonies in comparison with that of spontaneous revertant colonies and reproducibility in the two main tests, the test article was judged to be positive.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
Dose-finding test:
- Observetion: Precipitation of the test article on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration on the plate was not observed at any dose levels irrespective of the presence/absence of metabolic activation. Growth inhibition was observed at 78.1 μg/plate or more in S. typhimurium TA1537 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA98, TA100 and TA1535 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains with or without metabolic activation.

First main test and second main test:
- Observetion: In both the two main tests, neither precipitation nor coloration of the test article on the plate was observed at any dose levels irrespective of the presence/absence of metabolic activation. Growth inhibition was observed at 39.1μg/plate or more in S. typhimurium TA1537 without metabolic activation, and at 156 μg/plate or more in S. typhimurium TA1535 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA98 and TA100 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.
There was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response for any strains irrespective of the presence/absence of metabolic activation.
Remarks on result:
other: Growth inhibition was observed at 156 μg/plate or more

Applicant's summary and conclusion

Conclusions:
The test item was judged to have no gene mutation inducibility (negative) under the conditions of this study.
Executive summary:

In order to examine the gene mutation inducibility of the test item, a reverse mutation assay was conducted in Salmonella typhimurium (hereinafter referred to as S. typhimurium) TA 100, TA 1535, TA 98 and TA 1537, and Escherichia coli (hereinafter referred to as E. coli) WP2 uvrA with or without metabolic activation by the pre-incubation method according to OECD Guideline 471.

N,N-dimethylformamide (hereinafter referred to as DMF) was used as the vehicle for the test article. A dose-finding test was conducted with dose levels between 19.5 and 5000 μg/plate to set the dose levels for the main test. From the result of the dose-finding test, the minimum dose which showed growth inhibition was selected as the maximum dose for the main test, and the main tests were conducted at 6 dose levels between 2.44 and 78.1 μg/plate in the S. typimurium TA 1537 without metabolic activation, and between 9.77 and 313 μg/plate in the S. typimurium TA 98, TA 100 and TA 1535 without metabolic activation, and between 39.1 and 1250 μg/plate in the E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation. The main test was conducted twice at the same dose levels.

Precipitation on the plate was observed at 5000 μg/plate with or without metabolic activation. Coloration by the test article was not observed at any dose levels irrespective of the presence/absence of metabolic activation.

In the observation of bacterial background lawn using a stereoscopic microscope, growth inhibition was observed at 39.1 μg/plate or more in S. typhimurium TA 1537 without metabolic activation, and at 156 μg/plate or more in S. typhimurium TA 1535 without metabolic activation, and at 313 μg/plate or more in S. typhimurium TA 98 and TA 100 without metabolic activation, and at 1250 μg/plate or more E. coli WP2 uvrA without metabolic activation and other strains with metabolic activation.

In the two main tests, there was neither increase in the number of revertant colonies of two-fold or more in comparison with that of the negative control group nor dose-response in any strains irrespective of the presence/absence of metabolic activation.

In conclusion, the test item was judged to have no gene mutation inducibility (negative) under the conditions of this study.