clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 May - 16 Jun 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 2020

Deviations:

yes

Remarks:

2-AA was used as the sole indicator of the efficacy of the S9 mix

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

433-460-1

EC Name:

-

Cas Number:

210880-92-5

Molecular formula:

C6H8ClN5O2S

IUPAC Name:

(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Cytokinesis block (if used):

None

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Sprague Dawley rats which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254 five days before sacrifice.

The protein concentration of the S9 preparation was 28.4 mg/mL.

S9 fraction was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10 % v/v in the S9 mix. 70 mL cofactor solution are composed as follows: 162.6 mg MgCI2 x 6 H2O, 246.0 mg KCI, 179.1 Glucose-6-phosphate disodium salt, 315.0 mg NADP disodium salt in 100 mM phosphate buffer (pH 7.4).

The final concentration of S9 in the culture was 18.5% (S9 mix) and 1.8% (S9 fraction).

Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a
]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

- Preliminary experiment I (plate incorporation): 16, 50, 158, 500, 1581, 5000 µg/plate

No bacteriotoxic or mutagenic effects were found up to the highest concentration. Therefore, the concentrations were used in the second experiment as well. 5000 µg/plate is recommended as the highest test concentration by the guideline.

- Experiment II (preincubation): 16, 50, 158, 500, 1581, 5000 µg/tube (all strains)
- additional test (preincubation): 16, 32, 48, 64, 80, 96, 110 µg/tube (only TA 102 with S9 mix)
The additional test was neccessary because the revertant count for TA 102 with S9 at 50 µg/tube was ca. 100 colonies higher than control colony count.

Vehicle / solvent:

- Vehicle/solvent used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
- Justification for percentage of solvent in the final culture medium: not reported

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: Nitrofurantoin (NF), TA 100, 0.2 µg/plate, -S9 4-Nitro-1,2-phenylene diamine (4-NPDA), TA 1537, 10 µg/plate, and TA 98, 0.5 µg/plate, -S9 2-Aminoanthracene (2-AA), all strains, 3 µg/plate, +S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2 (+1 additional test)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar for plate incorporation test (experiment I) and in bacterial suspension for pre-incubation test (experiment II and additional test)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: clearing or diminution of the background lawn, reduction in revertant colonies, reduction in titer

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

The substance was considered mutagenic when a reproducible and dose-related increase in mutant count was observed in any strain with and/or without metabolic activation. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. If results are questionable, investigation should continue.

Statistics:

Mean values and standard deviation were calculated. According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was reported up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary plate incorporation experiment I, the concentration range of the test item was 16 - 5000 µg/plate. No bacterial mutagenicity could be evidenced for all tested S. typhimurium strains, both with and without S9 mix. No cytotoxicity was noticed at concentrations up to an including 5000 µg/plate

STUDY RESULTS
Bacteriotoxic effects were not observed up to and including 1581 µg/tube, titer reduction was seen occasionally at 5000 µg/tube. Since cytotoxicity was rather slight and only reported for some strains, assessment was not impaired.
No substantial increase in revertant colony numbers for any of the five strains investigated was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation. In experiment II, TA 102 at 50 µg/tube with metabolic activation had about 100 revertant colonies more than the controls. This was not dose-dependent and the result could not be confirmed in the additional experiment with concentrations between 16 and 110 µg/tube, so that the finding is considered incidental. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. For summarized results of both experiment I and experiment II, please refer to Attachment 1 in the attached background material.

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent and positive controls were within the range of the laboratory's historical control data. For details, please refer to Attachment 2 in the attached background material.

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 471 and compliant with GLP. Under the conditions of the assay, the test item was not mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.