Chinomethionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2017 - 30 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source - The Sponsor; Osaka Soda Co. Ltd. Lot/batch number: 160722-01
- Expiration date of the lot/batch: 22nd July 2018
- Purity test date: 97%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70RH%)
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: The test item was insoluble at the concentration level of 100 mg/mL using Distilled water, but it was soluble with DMSO (solvent) at 100 mg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 100 mg/mL stock solution was prepared in DMSO after approximately 2 minutes ultrasonic water bath.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: 100 mg/mL
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) Dissolved in solvent (DMSO) as a liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver Homogenate S9 Fraction

Test concentrations with justification for top dose:

Initial Mutation Test (plate incorporation method) - examined concentrations for Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate (100, 31.62, 10, 3.162, 1, 0.3162, 0.1, 0.03162 and 0.01 mg/mL). For TA100 the examined concentrations were 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate (10, 3.162, 1, 0.3162, 0.1, 0.03162 and 0.01 mg/mL).

Confirmatory Mutation Test (pre-incubation method) - examined concentrations for Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate (100, 31.62, 10, 3.162, 1, 0.3162, 0.1, 0.03162, 0.01 and 0.003162 mg/mL). ForTA100 the examined concentrations were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate (10, 3.162, 1, 0.3162, 0.1, 0.03162, 0.01 and 0.003162 mg/mL).

Concentrations were selected on the basis of the Preliminary Compatibility Test and Preliminary Concentration Range Finding Test

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test item was soluble in 100 mg/mL of DMSO, with homogeneous suspension with slower sedimentation compared with other potential solvents e.g. DMF and Acetone

Untreated negative controls:

yes

Remarks:

Distilled Water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (NPD), 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
- Cell density at seeding (if applicable): Not specified

DURATION
- Preincubation period: Not specified
- Exposure duration: 48 hour +/- 1 hour
- Expression time (cells in growth medium): Not specified
- Selection time (if incubation with a selection agent): Not specified
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hour +/- 1 hour

SELECTION AGENT (mutation assays): Not specified

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not specified

NUMBER OF CELLS EVALUATED: Not specified

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: n/a
- Any supplementary information relevant to cytotoxicity: n/a

OTHER EXAMINATIONS:
- Determination of polyploidy: n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER: n/a

Rationale for test conditions:

Not specified

Evaluation criteria:

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- in all strains: the number of reversion was more than two times higher than the reversion rate of the negative (solvent) control.

Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without
metabolic activation.

Statistics:

The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

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:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: n/a
- Effects of osmolality: n/a
- Evaporation from medium: n/a
- Water solubility: n/a
- Precipitation: n/a
- Definition of acceptable cells for analysis: n/a
- Other confounding effects: n/a

RANGE-FINDING/SCREENING STUDIES: 100 mg/mL stock solution was prepared in DMSO, which was diluted in 6 steps by factors of 2, 2.5
and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains
(Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: -S9 Mix: TA98 (range 152-2336; mean 357.2; SD 113.8); TA100 (range 536-2120; mean 1229.3; SD 207.5); TA1535 (range 208-2440; mean 1169.8; SD 204.2); TA1537 (range 149-2104; mean 454.1; SD 169.7); E.coli (488-1708; mean 1034.3; SD 141.7).
+S9 mix: TA98 (312-4918; mean 2410.2; SD 317.7); TA100 (1192-5240; mean 2429.6; SD 291.6); TA1535 (101-2216; mean 235.1; SD 135.9); TA1537 (117-838; mean 221.3; SD 56.2); E-coli (125-2512; 257.4; SD 113.4)

- Negative (solvent/vehicle) historical control data: DMSO solvent control: -S9 Mix TA98 (6-55; mean 21.7; SD 5.7); TA100 (40-217; mean 98.9; SD 20.7); TA1535 (1-43; mean 12.0; SD 5.0); TA1537 (1-25; mean 7.1; SD 3.3); E-coli (7-81; mean 32.3; SD 9.6)
+S9 mix TA98 (11-67; mean 28.7; SD 7.0); TA100 (53-229; mean 109.5; SD 20.7); TA1535 (2-33; mean 11.3; SD 3.8); TA1537 (1-29; mean 8.7; SD 3.7); E-coli (9-85; mean 38.1; SD 9.7)


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: n/a
- Other observations when applicable: n/a

Remarks on result:

other: no mutagenic activity on the growth of the bacterial strains

Observations:

Reduced colony numbers was detected in the Confirmatory Mutation Test in Salmonella typhimurium TA100 strain with and without metabolic activation at 500, 158.1 μg/plate concentrations, in TA1535 strain with metabolic activation at 1581, 500 and 158.1 μg/plate concentrations, in TA1537 strain with and without metabolic activation at 1581, 500 and 158.1 μg/plate concentrations, and in Escherichia coli WP2 uvrA strain with metabolic activation at 5000 μg/plate concentration.

Precipitate/slight precipitate of the test item was detected in the Confirmatory Mutation Test with and without metabolic activation in Salmonella typhimurium TA98, TA1535, TA1537 strains on the plates at 5000 and 1581 μg/plate concentrations, in TA100 strain on the plates at 500 μg/plate concentration, and in Escherichia coli WP2 uvrA strain on the plates at 5000, 1581 and 500 μg/plate concentrations.

Slightly reduced background lawn was detected in the Confirmatory Mutation Test in Salmonella typhimurium TA98, TA1535, TA1537 strains with and without metabolic activation on the plates at 5000 μg/plate concentration.

Conclusions:

Under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item Chinomethionate (Batch Number: 160722-01) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Executive summary:

The test item Chinomethionate was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay with and without metabolic activation (S9) using Salmonella typhimurium TA98, TA100, TA1535 and TA1537, and Escherichia coli WP2 uvrA. The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and Confirmatory Mutation Test (Pre-incubation Method).

For the confirmatory mutation test, examined concentrations for Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA strains were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate (100, 31.62, 10, 3.162, 1, 0.3162, 0.1, 0.03162, 0.01 and 0.003162 mg/mL). For TA100 the examined concentrations were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate (10, 3.162, 1, 0.3162, 0.1, 0.03162, 0.01 and 0.003162 mg/mL). DMSO was used as the solvent as the test item was soluble at 100 mg/mL within it, and there was homogeneous suspension with slower sedimentation detected when DMSO was used compared to DMF and Acetone.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item Chinomethionate (Batch Number: 160722-01) has no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the results of these studies, Chinomethionate will not be classified as a genetic toxin.