thiacloprid (ISO);(Z)-3-(6-chloro-3-pyridylmethyl)-1,3-thiazolidin-2-ylidenecyanamide;{(2Z)-3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene}cyanamide

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:

6 Dec 1994 - 7 Apr 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

less than 2 million cells were seeded, data was not pooled for data analysis

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 1984

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

EC Number:

601-147-9

Cas Number:

111988-49-9

Molecular formula:

C10H9ClN4S

IUPAC Name:

3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells, provided by University of Ulm, Germany
- Suitability of cells: standard cell for this assay
- Normal cell cycle time (negative control): 10-14 h
For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: subculturing twice a week
- Cell cycle length, doubling time or proliferation index: 10-14 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: cells were cultured in hypoxanthine-free Eagle's Minimal Essential Medium (MEM), supplemented with nonessential amino acids, L-glutamine (2 mM), MEM-vitamins, NaHC03 , penicillin (100 units/mL), streptomycin (100 μg/mL) and heat-inactivated fetal calf serum (FCS, 10%). This medium is the culture medium. For treatment, the FCS was reduced to 2%. Cells were kept at 37 °C, 5% CO2.

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation system was cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with Aroclor 1254. S9 fraction was purchased commercially.

The protein concentration of the S9 preparation was 37.4 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 40% v/v in the S9 mix. The cofactors in the final S9 mix were concentrated as follows: 8 mM MgCI2 x 6 H2O, 33 mM KCI, 5 mM Glucose-6-phosphate, 1 mM NADP in buffer.

The final concentration of S9 in the culture was 5% (S9 mix) and 1.6% (S9 preparation).

The S9 was tested for its capability to activate the positive control 7,12-dimethylbenzanthracene (DMBA) during the test.

Test concentrations with justification for top dose:

The concentrations in the main experiment were based on a pre-test. Nine concentrations were tested ranging from 9 to 1000 µg/mL for 5 h +/- S9 mix. Without S9 mix, cytotoxicity was not seen up to the highest concentration. Precipitation occurred at 500 μg/mL and above.

Therefore, the following concentrations were chosen for the main test: 15.6, 31.3, 62.5, 125, 250, 500 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
- Percentage of solvent in the final culture medium: 1 % (v/v)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicates
- Number of independent experiments: 2 (- S9 mix), 3 (+ S9 mix)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 4x10^6 cells per 250 mL flask (exposure), 1.5 x 10^6 cells (expression), 3x10^5 (selection)
- Test substance added in medium (2% FCS)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment: 13 - 14 days

FOR GENE MUTATION:
- Expression time: 7 days (subcultured once)
- Selection time: 6-7 days
- Fixation time: 13-14 days
- Selective agent: 6-TG, final concentration 10 μg/mL, 8 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival to treatment (relative cloning efficiency), relative population growth, the ability of cells to form colonies at the time of mutant selection (absolute cloning efficiency)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Mutation frequency = (number of mutation colonies * 100/number of evaluated dishes x 3 x 10^5 x CE)
with CE = cloning efficiency

Rationale for test conditions:

The present study was conducted according to the recommendations of the OECD test guideline 476 version dated 1984, which was the standard at the time the study was conducted.

Evaluation criteria:

Acceptability
The assay is considered acceptable if it meets the following criteria
- the assay is performed twice
- at least 5 dishes per conditions are used to determine mutant frequency (better: 8)
- the numbers of mutant colonies per million cells found in the negative and/or solvent controls fall within the laboratory historical control data range
- the positive control substances produce a significant increase in mutant colonies (three times of
positive control)
- the average cloning efficiency of the negative and/or solvent controls must exceed 50%
- spontanous mutant frequency should be lower than 25x10^-6 cells
- absolute CE should be 10% or greater

An assay is considered positive if an increase in mutant frequencies is
- concentration-dependent (desirable over 3 concentrations, but at higher concentrations, reproducibiliy is sufficient to classify as mutagenic)
- significant (at least two to three times the mutant frequency of the solvent control)
- reproducible (seen in parallel cultures)

The results should be reproduced in the second experiment.

A test substance is considered equivocal if there is no concentration-dependency but one or more concentrations induced a reproducible, significant mutant frequency in all assays.

The assay is considered negative if none of the criteria described above are met (for a range of applied concentrations which extends to toxicity causing about 30% survival or less).

Statistics:

Mutation frequencies were submitted to weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. The weighted analysis of variance is followed by a Dunnett's test (nominal significance level of a = 0.05).
Mutation frequencies of 5 plates are too few to be considered for statistics. If the relative population growth was below 10%, the concentration is also discarded for evaluation.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated at 500 μg/mL and above. At 500 μg/mL (the highest soluble concentration), osmolality was not affected.

RANGE-FINDING/SCREENING STUDIES
A preliminary cytotoxicity test was conducted with a series of 9 concentrations ranging from 9 μg/mL to 1000 μg/mL, with and without S9 mix. Cytotoxicity was not affected up to the highest concentration tested but precipitation occurred at 500 µg/mL and above. Based on these results, 6 concentrations were retained for the main experiments ranging from 15.6 μg/mL to 500 μg/mL both, with and without S9 mix.

CYTOTOXICITY:
No toxicity occurred up to the highest concentration tested, both with and without S9 mix.

STUDY RESULTS
Without metabolic activation:
No relevant increase in mutation frequencies was observed. At 62.5 μg/mL, an increased mutant frequency compared to the vehicle controls was observed but this was not reproducible, not concentration-related and did not exceed the range, which is typical of vehicle control variation between trials. Therefore, the finding was considered incidental.

With metabolic activation:
No relevant increase in mutation frequencies was observed. A slight increase occurred at 125, 250 and 500 μg/mL in one culture compared to the vehicle controls but this was not reproduced in the parallel culture or the other trials. The joint statistical assessment of the 3 trials resulted in no statistically significant increase of the mutation frequency over the concurrent vehicle controls at any of the tested concentrations

Solvent, negative and positive controls gave the expected outcome.

HISTORICAL CONTROL DATA
Historical control data was provided by the testing facility. The data came from 17 and 16 experiments with and without metabolic activation, respectively. The experiments were conducted from February 1994 to April 1995.

Mutation frequency of negative controls
- S9-activation 6.1 ± 3.8 (range 0.5 to 15.0)
+ S9-activation 5.7 ± 5.9 (range 0.5 to 28.8)
Mutation frequency of vehicle controls
- S9-activation 5.6 ± 4.4 (range 1.0 to 22.6)
+ S9-activation 5.0 ± 5.5 (range 0.5 to 26.7)
Mutation frequency of positive controls
- S9-activation (EMS) 887.3 ± 193.9 (range 531.1 to 1291.5)
+ S9-activation (DMBA) 93.4 + 42.2 (range 21.8 to 240.2)

Applicant's summary and conclusion

Conclusions:

The present study was conducted according to the OECD guideline 476 dated 1984, under GLP conditions. Under the conditions of the assay, the test item did not induce any gene mutation at the HPRT locus in V79 cells, neither in the presence nor absence of metabolic activation.