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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 2017-05-02 to 2017-05-12
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 microsome fraction prepared from Sprague Dawley rat liver homogenate
Test concentrations with justification for top dose:
50, 150, 500 1500 and 5000 μg/plate
Selection of the top dose: The preliminary cytotoxicity test performed on the TA 100 showed that neither original solutions nor dilutions have bacteriostatic effect..
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the test item is highly soluble
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-anthramine, cis-platinum (II) Diammine dichloride
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- First assay: direct incorporation method, +/- S9 mix
- Second assay: pre-incubation test method +/- S9 mix

DURATION
- Preincubation period: 30 min at 37°C when the pre-incubation procedure is used.
- Exposure duration:48-72 hr at 37°C
- Expression time (cells in growth medium): counting completed at the end of the exposure period in each plate


NUMBER OF REPLICATIONS: Triplicates, in parallel with negative and positive controls and the solvent of test material.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies or a clearing or diminution of the background lawn.
Evaluation criteria:
Ensure that the criteria validity of the study are well respected namely :
- The bacteriostatic activity of the highest concentration tested shall be equal or less than 75%
- The spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory
- The spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control
- The mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/ or without metabolic activation shall comply with the historical values of the laboratory
- Negative and positive values should not show significant difference with the historical values of
the laboratory (+/- 2 standards deviation)
Statistics:
not provided

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at the highest dose.

Applicant's summary and conclusion

Conclusions:
The test item induce a mutagenic change in Salmonella typhimurium TA 98 and TA 100, according to the OECD Guideline n°471.
Executive summary:

Assay : Bacterial mutation test using Salmonella typhimurium his-" and "Escherichia coli" WP(uvrA-)(pKM101) according to the OECD guideline n°471 (LEMI operating procedure n°MB08/45).

The test item has been tested for their capacity to induce revese mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvrA-)(pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out.

For assay n° 1, various concentrations were put in contact with the strains in the absence and presence of a metabolic activation system (S9 -mix 10% (v/v)).

For assay n°2, various concentration were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9 -mix 10% (v/v)) and without re-incubation for TA98 strain.

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean corresponding experimental "historical" values obtained in the laboratory.

These results validate the two tests.

There is an increase in the number of revertant colonies in Salmonella typhimurium TA 98:

- without metabolic activation in presence of 150, 500, 1500 and 5000 µg/plate

- with metabolic activation without and with pre-incubation in presence of 500, 1500 and 5000µg/plate

and in Salmonella typhimurium TA 100 without metabolic activation in presence of 5000 µg/plate.

The test item induce a mutagenic change in Salmonella typhimurium TA 98 and TA 100, according to the OECD Guideline n°471.