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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:
14 September - 17 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(2-fluoro-3-methoxyphenyl)boronic acid
EC Number:
609-099-0
Cas Number:
352303-67-4
Molecular formula:
FC6H3(OCH3)B(OH)2
IUPAC Name:
(2-fluoro-3-methoxyphenyl)boronic acid
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
The test material was stored at room temperature and kept in a closed container when not in use

Method

Species / strain
Species / strain / cell type:
other: TA 97a, TA 98, TA 100, TA 102, TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 prepared in-house was used as the metabolic activation system.
Test concentrations with justification for top dose:
Six dose levels were designed using an approximate spacing factor of 3 in the first experiement: 15, 50, 150, 500, 1,500, and 5,000 μg/plate with and without S9 mix.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
sodium azide
other: Dexon, 2-Aminofluorene, 1,8-Dihydroxyanthraquinone, 2-Aminoanthracene
Details on test system and experimental conditions:
Preliminary Test:
A preliminary test for the Bacterial Reverse Mutation Test of FMPBA was performed in the lab. In the test, according to the results of the test item solubility,
DMSO was used as solvent. The standard plate incorporation method was performed at 3 dose levels with 5 intervals between test point, including 5000,
1000 and 200 µg/plate, in 5 tester strains of: TA97a, TA98, TA100, TA102 and TA1535, only without metabolic activation system. Solvent controls (DMSO) in each tester strain were performed at the same time. The dose volume of each dose group and solvent control group were 0.1ml/plate, in duplicate.

Preliminary test results:
About the precipitate of the test item, Monitoring of TA98 showed that there were precipitates at 5000 µg/plate dose level before and after the incubation, but the precipitates did not affect the observation of the background lawn. Moreover, no cytotoxicity was found at all designed dose level in all tester strains.

Dose Selection:
Dose selection is performed based on the results of the preliminary test and the related requirements of PRC.iMEP the Guidelines for Testing of Chemicals (Health Effect, second edition), 471 (2013). Six dose, levels were designed using an approximate spacing factor of 3 in the first experiment, including 5000, 1500, 500, 150, 50 and 15 µg/plate without and with S9mix. Solvent· controls (DMSO) and positive controls in each tester strain were performed at the same time.

Preparation of the test item:
In the experiment DMSO was used as a solvent. Two test items (0.30129g and 0.09051g) were weighed and transfered into calibrated aseptic tubes. 6,000 mL DMSO were added into the tube and mixed thoroughly to the calibrated volume to obtain 50 and 15 mg/mL working solution. Under aseptic condition, the working solution of other concentrations were diluted using the spacing factors of 10. In this study, the test item was prepared just before being applied to the test system.

Enrichment culture:
One day prior to the plate-incorporation, the stored tester strains were thawed and 100 µl of bacterial suspension was cultivated in nutrient broth medium for approximately 16 hours at 37.1-38.1°C.

Viable count:
In order to confum the density of living bacteria in the cultures, some. cultures were taken out for the viable count. The cultures were diluted with sterile water, then
200 µ1 of the dilution of bacteria cultures that had been diluted by a factor of 10^5, 10^6, or 10^7 respectively, 100µ1 of histidine solution supplemented with an excess of
histidine (0.5% L-histidine solution), 100µ1 of 0.01 % d-biotin solution,500 µ1 of PBS and 2000 µl of molten top-layer agar medium kept in a water bath at 45.1-46.8 °C were
added to a sterile tube under aseptic conditions. After vortexing, the mixture was evenly overlaid on the surface of the MGA plate, and each of the above diluted concentrations wereperformed in triplicate. When solidified, the plates were inverted and incubated for about 64 hours at 35.2-37.6°C. Then the number of the colonies in plate with the 10^6 -fold dilutions was counted. The density of viable bacteria in the cultures was calculated based on the mean number of colonies and dilution factor.

Plate-incorporation:
In the absence of S9 mix: 200 µl of histidine-biotin solution,100 µl of bacteria culture, 500 µl of PBS, 100 µl of the test item formulation or DMSO or positive control solution and 2000 µl of molten top-layer agar mediumkept in a water bath at 44.5-46. 7°C were added to a sterile test tube under aseptic conditions. After vortexing, the mixture was evenly overlaid on the surface of MGA plate, all dose levels and the controls were performed in triplicate. After the top-layer agar, was solidified, the plates were inverted and incubated for about 48 hours at 36.3-38.2°C. The test item precipitation was observed by naked Dyes in three parallel plates of each dose group of TA98 before and after incubation. After incubation, the numberof revertant colonies in each plate were counted. As counting the plates of the positive controls (except for TA1535), the back ofa plate was divided into eight sectorson back, and two diagonal parts were chosen randomly and counted, then the result was multiplied by 4 as the number of revertant colonies, and the signs of background lawn were observed microscopically .
In the presence of S9 mix: All procedures were the same as those in the absence of S9 mix except for fBS replaced with S9 mix.
Evaluation criteria:
Criteria of positive result:
When, there is a concentration-related increase over the range (two times equal to or greater than the concurrent solvent control in TA97a, TA98, TA100, TA102 and three times equal to or greater than the concurrent solvent control in TA 1535) in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.

When there is a reproducible increase (two times equal to or greater than the concurrent solvent control in TA97a, TA98, TA100, TA102 and three times equal to or greater than the concurrent solvent control in TA 1535) at one or more concentrations in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.

Criteria of negative result:
The test item should be evaluated as negative if none of the above criteria are met.

Criteria of equivocal result:
Although most tests give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item. Results of this type should be reported as equivocal.
Statistics:
For three plates at each dose and control group, the mean number and the standard deviation of the number of revertant.colonies were calculated: in Excel (2017). At the same time, the ratio of the mean number of revertant colonies between each group and the concurrent solvent control was calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
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:
Test Item Precipitate: There were precipitates at 5,000 µg/plate dose levels on the MGA plates, before and after the incubation, with and without metabolic activation system.

Cytotoxicity: No cytotoxicity was found at any of the designed dose levels in any of the tester strains in the two treated conditions.

Any other information on results incl. tables

All the results of the positive and solvent controls met the requirements of the tests, so the sensitivity of the test and the efficacy of the S9 mix were validated.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the results were positive. Therefore, the test item is considered to be mutagenic in the bacterial reverse mutation assay using histidine requiring tester strains of Salmonella typhimurium.