imidacloprid (ISO); 1-(6-chloropyridin-3-ylmethyl)-N-nitroimidazolidin-2-ylidenamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan - 05 Feb 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

solubility and stability of test substance in vehicle not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

428-040-8

EC Name:

-

Cas Number:

138261-41-3

Molecular formula:

C9H10ClN5O2

IUPAC Name:

2-chloro-5-{[2-(nitroimino)imidazolidin-1-yl]methyl}pyridine

Method

Target gene:

his operon (for S. typhimurium strains)

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of Wistar rats which received triple treatments of 80 mg/kg bw phenobarbital/ß-naphthoflavone orally on consecutive days.

The protein concentration of the S9 preparation was 30.8 mg/mL in the pre-experiment/experiment I and 35.0 mg/mL in experiment II.

100 mL cofactor solution are composed as follows: 8 mM MgCI2, 33 mM KCI, 5 mM Glucose-6-phosphate, 4 mM NADP in 100 mM sodium phosphate buffer (pH 7.4). 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.

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:

Experiment I (pre-experiment): 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate (with and without metabolic activation)

Since no toxic effects were observed in experiment I, 5000 µg/plate was chosen as maximal concentration.

Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate (with and without metabolic activation)

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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine 4-NOPD: TA 1537 50 µg/plate and TA 98 10 µg/plate in DMSO; -S9 2-aminoanthracene 2-AA: TA 1535, TA 1537, TA 98, TA 100 2.5 µg/plate and TA 102 10 µg/plate in DMSO; + S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

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)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 1 h
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants and clearing or diminution of the background lawn

Rationale for test conditions:

according to OECD guideline

Evaluation criteria:

Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed
A dose dependent increase is considered biologically relevant if the threshold is exceeded at
more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Acceptability:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the
following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the
historical data
- the positive control substances should produce an increase above the threshold of twice
(strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony
count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels
showing no signs of toxic effects, evident as a reduction in the number of revertants below
the indication factor of 0.5.

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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: No precipitation of the test item occurred up to the highest investigated dose in both experiments.

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The preexperiment is reported as experiment I.

STUDY RESULTS
No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed in both experiments neither with nor without S9 mix. No substantial increase in revertant colony numbers of 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. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. For results of both experiment I and experiment II, please refer to the attached background material under "Overall remarks, attachments"

HISTORICAL CONTROL DATA
The number of revertant colonies observed for the solvent, untreated and positive controls were within the range of the laboratory's historical control data. For details, please refer to the attached background material under "Overall remarks, attachments".

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. typhimuirum strains TA 98, TA 100, TA 1535, TA 1537 and in TA 102 with and without metabolic activation.