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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The results from the in vitro mammalian cell gene mutation test, in vitro mammalian chromosome aberration test, and bacterial reverse mutation analysis were negative for genetic toxicity of the test material. Therefore the test material did not meet the GHS criteria to be classified as a genetic toxicant.

Link to relevant study records

Reference

Reference 1

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)

Qualifier:

according to guideline

Guideline:

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

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

Specific details on test material used for the study:

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

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)

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.

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd August 2018 to 3rd September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

other: PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 471 'Bacterial Reverse Mutation Test (2013)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA97a

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

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 prelimary experiment, the doses of this test were 0.6, 2, 6, 20, 60, and 200ug/plate. Blank control, solvent control (dimethylsulfoxide) and positive control had been included. Triplicate cultures were made per dose in the test.

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Dexon 2-aminofluorene 1,8-Dihydroxyanthraquinone 2,-aminoanthracene

Key result

Species / strain:

other: S. typhimurium TA97a

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:

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 100

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 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

Conclusions:

Under the conditions of this study, the results in both the first experiment and the validation experiment were negative. Thus the item E Stage 3 Intermediate is considered non-mutagenic in the bacterial reverse mutation assay using the histidine requiring tester strains of Salmonella typhimurium

Executive summary:

The study was performed to evaluate the ability of E Stage 3 Intermediate to induce reverse mutations in the genome of the histidine requiring test strains of Salmonella typhimurium with and without the metabolic activation system.

Five histidine requiring (his-) mutant tester strains of Salmonella typhimurium including TA97a, TA98, TA100, TA102 and TA1535 were treated with E Stage 3 Intermediate using the standard plate incorporation method and the pre-incubation method at six dose levels, in triplicate, in the presence and absence of co-factor supplemented S9 (S9mix). 

Based on the preliminary test results (initial toxicity test) previously performed, six dose levels in the first experiment were selected: 200, 60, 20, 6, 2 and 0.6μg/plate with and without metabolic evaluation. Dimethyl sulfoxide (DMSO) was used as a solvent. The validation experiment was then conducted with the same dose levels and solvent. 

In two experiments, the results of the viable count showed that the density of live bacteria in the cultures of each tester strain was within the acceptable range of 0.9~9 x 10⁹ colony forming units (CFU/ml). At the same time, all results of positive and solvent controls met the requiremetns of the test, so the sensitivity of the test and the efficacy of the S9 mix were validated. 

Test Item Precipitate: 

In the first experiment there was no precipitation at any dose level before and after incubation with and without metabolic activation system. In the validation experiment the same results were obtained as in the first experiment. 

Cytotoxicity

In the first experiment cytotoxicity was observed at 200μg/plate dose level of all tester strains in two treated conditions and 60μg/plate dose level of TA102 without metabolic activation. In the validation experiment the same results were obtained as in the first experiment.

Mutation

In the first experiment with and without metabolic activation, the mean number of revertant colonies at each dose level was less than two times that of the concurrent solvent control in TA1535. In the validation experiment the same results were obtained as in the first experiment. 

Under the conditions of this study, the results in both the first experiment and the validation experiment were negative. Thus the test item E Stage 3 Intermediate is considered to be non-mutagenic in the bacterial revrese mutation assay using histidine requiring tester strains of Salmonella typhimurium. 

 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25th September 2018 to 12th November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Qualifier:

according to guideline

Guideline:

other: PRC-MEP, The Guidelines for the Testing of Chemicals (Health Effect, 2nd Edition), 473 'In vitro Mammalian Chromosonal Aberration Test (2013)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

ATCC CRL-1935

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 prelimary experiment, the doses of this test were 1.54, 3.84, 9.6, 24, 60 and 150ug/plate. Blank control, solvent control (dimethylsulfoxide) and positive controls have been included. Triplicate cultures were made per dose in the test.

Vehicle / solvent:

Dimethylsulfoxide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Approximately 2 x 10 5 CHL cells were seeded in the cell culture plate and cultured overnight using 10% MEM. Duplicate cultures were included at each dose level and control. Before treatment the cells were washed with Hank's Balance Salt Solution (HBSS). Test solution and solvent were added to the corresponding cell mixtures. The finished test mixtures were treated for 3 hours with and without S9 and for 24 hours without S9. The cultures were observed by the naked eye for test-precipitation before and after treatment. After treatment the cells were washed with HBSS and cultured in 10% MEM.

At the end of cell treatment, the cells were recultured in 10% MM and harvested after approximately 24 hours. Before harvest, all cells were treated with Colchicine for approximately 2 hours at the final concentration of 1ug/ml. After harvest, the cells were washed in HBSS solution and treated with 150ul trypsin solution. The medium containing FBS was added to cultures and mixed repeatedly. The cells of each dose and control were counted using a haemocytometer under inverted microscope and the RICC was calculated to evaluate cytotoxicty.

Considering the cytotoxicty results and the dose-response relationship, the cells exposed to 24, 9.6 and 3.84ug/ml were chosen for chromosone preparation microscopic analysis.
Harvested cells were transferred to tubes to remove the medium by centrifugation at 1000rpm for 5 mins. The supernate was removed, 1ml of 75mM KCl hypotonic solution preheated to 37oC and mixed. The samples were incubated at 37oC for 40 mins, then centrifuged at 1000rpm for 5 mins. The supernate was then removed and 1.0ml of Carnoy's mixture added, mixed gently, fixed for 30min at room temperature, then centrifuged for 5 mins at 1000rpm. The supernate was removed, 1ml of Carnoy's mixture added and incubated at 2~8 oC overnight. The samples were centrifuged at 1000rpm for 5 mins, the supernate removed and 0.1ml of Carnoy's mixture added. The cell suspension was dropped onto a slide, and air dryed at room temperature.
The slides are then stained with Giemsa solution, washed with water and dried naturally.
The slides were then read microscopically and scored for chromosonal aberrations.

Evaluation criteria:

At least one of the test concentrations exhibits a statistically significant increase in the percentage of cells with structural chromosonal aberrations compared with the concurrent negative control.

The increase is dose-related or reproducible.

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The  RICC of the cells exposed for 3 hours in the absence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -29%, 29%, 60%, 82%, 100% and 92%. The  RICC of the cells exposed for 3 hours in the presence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -20%, 31%, 73%, 99%, 79% and 89%.The  RICC of the cells exposed for 24 hours in the absence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -20%, -4%, 55%, 111%, 103% and 116%.

Conclusions:

The results of this test in all treatment conditions were negative. Therefore, it is considered that the test item E Stage 3 Intermediate did not cause structural chromosonal aberrations in cultured mammalian cells under the conditions of this test. 

Executive summary:

This study was performed to evaluate E stage 3 Intermediate for its ability to cause structural chromosonal aberrations in cultured mammalian cells in three treatment conditions including exposure for three hours with and without metabolic activation and exposure for 24 hours without metablic activation. 

In this test, Chinese Hamster Lung (CHL) cell line was treated with E Stage 3 Intermdediate, in three treatment conditions at six dose levels. Positive and solvent controls were included in each condition concurrently. 

Based on the results of the preliminary test (initial toxicity test) previously performed, six dose rates of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml medium were included in each treament condition. DMSO was used as solvent. After exposure, all cells were harvested and counted. Then the relative increase in cell count (RICC) was calculated to evaluate the cytotoxicity of the test item. Based on cytotoxicity results, cells at the dose rate with an RICC near 50% (>50%) and two lower doses and cells of all controls in each treatment condition were selected for chromosone preparation. Then microscopic analysis was conducted for chromosonal aberrations.   

Test Item Precipitate: 

No precipitation was observed by the naked eye in the cell culture at any dose level in any of the three treatment conditions. 

Cytotoxicity

The  RICC of the cells exposed for 3 hours in the absence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -29%, 29%, 60%, 82%, 100% and 92%. The  RICC of the cells exposed for 3 hours in the presence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -20%, 31%, 73%, 99%, 79% and 89%. The  RICC of the cells exposed for 24 hours in the absence of S9 mix at the doses of 150, 60, 24, 9.6, 3.84 and 1.54 μg/ml were -20%, -4%, 55%, 111%, 103% and 116%.

The results of the microscopic analysis showed that there was no statistically significant difference (P>0.05) in the percentage of cells with structural chromosonal aberration was observed in any treated cells in each treament condition as compared with solvent control. At the same time, statistically significant differences (P< 0.01) were observed in the cells of all positive controls as compared with solvent controls. 

 

The results of this test in all treatment conditions were negative. Therefore, it is considered that the test item E Stage 3 Intermediate did not cause structural chromosonal aberrations in cultured mammalian cells under the conditions of this test. 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available