(1R)-2-[5-(2-halo-3-methoxyphenyl)-3-[2-halo-6-(trihalomethyl)benzyl]- 4-alkyl-2,6-dioxoheterocyclyl]-1-phenylethanaminium salicylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th November 2018 - 10th December 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 476 'In vitro Cell Gene Mutation Test (2013)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Specific details on test material used for the study:

Batch No. BO1801B009
Purity: 97.4%
Physical status: White powder without lumps

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: mouse lymphona cell line L5178Y

For cell lines:
- Absence of Mycoplasma contamination: Confirmed
- Cell cycle and doubling time : Normal
- Periodically checked for karyotype stability: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
temperature 36 - 38 oC,
humidity 4- 6% CO2
Media RPMI plus horse serum (0 - 20%)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 prepared in-house

Test concentrations with justification for top dose:

Based on the preliminary test, the cells were exposed at doses of 30, 20, 10, 5, 2.5 and 1.25ug/ml

Vehicle / solvent:

Dimethyl Sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 6 x 10 5
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours with metabolic treatment, 3 hours without metabolic treatment, 24 hours without metabolic treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
- Method used: microwell plates for the mouse lymphoma assay.
- Selective agent: Trifluorothymidine (TFT), 3ug/ml, incubated for 12 days.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2000 cells/well

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mutant frequency (MF)

Evaluation criteria:

When the IMF (Induced Mutant Frequency) in one or several doses is greater than the GEF of 126 x 10 6 and the increase is concentration related and can be replicated, the result is evaluated as positive.
If this criteria is not met, the result is evaluated as negative.

The increase is dose-related or reproducible.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Additional information on results:

The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 26%, 91%, 97%, 100% and 86%. The  RTG of the cells exposed for 3 hours in the presence of S9 at the doses of 30, 20, 10, 5, 2.5, 1.25μg/ml were 14%, 54%, 85%, 86%, 84% and 103% .The  RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 1%, 22%, 60%, 81% and 91%

The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 10 6.

Applicant's summary and conclusion

Conclusions:

The results of this study were all negative, so it is concluded that the test item, E Stage 3 intermediate is non-mutagenic to the culture L5178Y mouse lymphoma cells under the conditions of this study.

Executive summary:

This study was performed to assess E stage 3 intermediate for its ability to cause gene mutations in vitro cultured mammalian cells L5187Y mouse lymphona cells (TK^± -3.7.2C) after being exposed for 3 hours with and without metabolic action and 24 hours without metabolic action respectively using microwell format method. The method was designed to be compatible wth PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 476 'In vitro Cell Gene Mutation Test (2013)'.

L5178Y cells were exposed at 6 doses of 30, 30, 10, 5, 2.5, and 1.25 μg/ml in 3 treatment conditions. The positive and solvent (DMSO) controls were included at the same time in each treatment. The dose volume of each dose group and solvent control were 5μg/ml medium in duplicate. Plating Efficiency (PE) of the cells after exposure was determined and the Relative Total Growth (RTG) was calculated to evaluate toxicity. The Mutant Frequency (MF), Induced Mutant Frequency (IMF) of each culture and the Induced Mutant Frequency of small clone (IMF_SC) of the highest dose (10 μg/ml ) and all controls were determined after the inhibition with Trifluorothymidine (TFT). In this test, the results of the solvent and positive control met all validity criteria so the sensitivity of the assay and efficacy of the S9 mixture were validated. 

In three treatment conditions, no test item precipitate was observed in any cell culture at all doses before and after incubation. 

The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 26%, 91%, 97%, 100% and 86%. The  RTG of the cells exposed for 3 hours in the presence of S9 at the doses of 30, 20, 10, 5, 2.5, 1.25μg/ml were 14%, 54%, 85%, 86%, 84% and 103% .The  RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 1%, 22%, 60%, 81% and 91%

The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 10 6.

The RTG of the cells exposed for 3 hours in the absence of S9 mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 26%, 91%, 97%, 100% and 86%. The  RTG of the cells exposed for 3 hours in the presence of S9 at the doses of 30, 20, 10, 5, 2.5, 1.25μg/ml were 14%, 54%, 85%, 86%, 84% and 103% .The  RTG of the cells exposed for 24 hours in the absence of S9 mix at the doses of mix at the doses of 20, 10, 5, 2.5, 1.25μg/ml were 1%, 22%, 60%, 81% and 91%

The results of mutant frequency showed that the IMF of all cells cultures at all doses exposed for 3 and 24 hours in the presence and absence of S9 were less than 126 x 10 6.