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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 Jul - 30 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

Animals did not receive enriched environments, only males were used (justification was provided)

GLP compliance:

yes (incl. QA statement)

Remarks:

Certified by Food safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan

Type of assay:

mammalian erythrocyte micronucleus test

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

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) (SPF)

Details on species / strain selection:

commonly used for micronucleus tests, historical data is available

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc, Shiga, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 302.7-338.4 g
- Housing: plastic cages (W290xD340xH170 mm, Crea Japan Inc., Tokyo, Japan) with shavings (Whiteflake, Charles River Laboratories Japan, Inc.), in groups of maximum three animals
- Diet: Laboratory animal chows (CRF-1, Oriental Yeat Co., Ltd., Tokyo, Japan), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.0 - 24.1
- Humidity (%): 49.6 - 61.0
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.5% aqueous cremophor (Kolliphor EL) was used as a vehicle due its relative non-mutagenicicty and non-toxicity

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance was weighed to the approriate amount, ground with pestle and mortar and mixed with the cremophor solution. Constant stirring was achieved using a magnetic stirrer at room temperature. Stability of the test substance in the vehicle was proven for 2 h at room temperature for concentrations of 1 and 250 mg/mL. The dose volume was 10 mL/kg bw. The individual dose volumes were calculated based on body weights taken just before administration.

The positive control cyclophosphamide (CP) was dissolved in purified water (12 mg/mL) and administered once at 5 mL/kg bw (orally via gavage).

Duration of treatment / exposure:

2 days

Frequency of treatment:

Rats were dosed twice, once per day (24 h in between)
rats that received the positive control were only dosed once, together with the second dosing in animals receiving the test item and solvent control

Post exposure period:

24 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (for 0, 500 and 1000 mg/kg bw/day, and for the positive control), 7 (for 2000 mg/kg bw group)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: known mutagen
- Route of administration: orally via gavage
- Doses / concentrations: as described above, CP was solubilized at 12 mg/mL and administered at 5 mL/kg bw

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
No range finding study was conducted but doses were based on an alkaline comet assay in rats. The rats were of the same strain, age and were treated in the way described for this study (including dosing volume, route, frequency). No toxicity differences between the sexes were found and the rats tolerated all doses, up to the highest of 2000 mg/kg bw/day. As this is the limit dose recommended by the guideline, the doses in this experiment were chosen accordingly.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Groups of 5 or 7 male rats were dosed twice, approximately 24 h apart, with the vehicle alone (negative control) or the test item (500, 1000 or 2000 mg/kg bw/day). At the end of the observation period (approximately 24 h after the second test item administration), rats were euthanized by carbon dioxide inhalation.

OBSERVATIONS
Clinical signs and mortality were observed immediately, 3 h and 1 day after dosing. Body weights were recorded immediately before each dosing and 1 day after second dosing.

DETAILS OF SLIDE PREPARATION:
Bone marrow cells were washed out from the femurs with 1.0 mL of fetal bovine serum with 25 mM EDTA. After that, the cell suspension was centrifuged and the pellet was resuspended in residual serum and diluted with fetal bovine serum (including 25 mM EDTA). Small portions of this solution were smeared on a glass slide and the slides were fixed with methanol for 5 min within one day after preparation.

METHOD OF ANALYSIS:
The cells were analysed using acridine orange (40 µg/mL) using a fluorescent microscope (B excitation) using a 40-fold objective lens and 15-fold eye lens in a dry system. Erythrocytes with yellowish green fluorescence emitting particles (nuclei) and sharp outlines were identified as micronucleated erythrocytes. Due to the staining, polychromatic erythrocytes (PCEs) appeared bluish-grey and normochromatic erythrocytes (NCEs) appeared red. The incidences of micronucleated cells were scored in 4000 PCEs (per animal) and the incidence of PCEs in 1000 erythrocytes (including PCEs and NCEs) was examined.

Evaluation criteria:

Acceptability
The test was considered acceptable if
- incidence of PCEs (micronucleated) in the vehicle control group was within the acceptable negative control range from the historical control data
- the positive control induced a significant increase in micronucleated PCEs when compared with the concurrent vehicle control group.

Evaluation
An assay was considered positive if
- there was a statistically significant increase in the incidence of micronucleated PCEs when compared to the solvent or vehicle control
- the increase was dose-related
- the incidence of micronucleated PCEs in the dose group showing the increase exceeds the historical control range.

Statistics:

To statistically evaluate the incidences of PCEs, Kastenbaum-Bowman tables were used. Levels of significance were p=0.05 and 0.01. In case of an increased incidence, the dose-response relationship was tested with the Cochran-Armitage trend test (one-tailed, levels of significance were p=0.025 and 0.005).

The ratio of PCEs to total erythrocytes and animal body weight changes were evaluated using the t-test. The Student's t-test was used if p >= 0.05 and the F-test and the Welch's t-test was used if p<0.05 in the F-test (all two-tailed, levels of significance p=0.05 and 0.01).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Clinical signs: no clinical signs were observed in any of the treatment or control groups
- Body weight: Body weight and body weight gain was reduced in treatment groups before second dosing (500 and 1000 mg/kg bw/day) and one day after second dosing (all treatment groups)
- ratio of PCEs: the ratio of PCEs to total erythrocytes was decreased significantly (at all dose levels), a sign for bone marrow toxicity
- Induction of micronuclei: incidences of micronucleated PCEs were similar to vehicle controls for all dose levels and within the range of historical control data. Occassional incidences of slightly elevated increases were considered incidental and not of biological importance.
- Positive control: the positive control induced a statistically significant increase in the number of PCEs with micronuclei.

Historical control data
The historical control data was provided by the testing facility and can be found in Attachment 2 in the attached background material

Any other information on results incl. tables

The study is considered acceptable since 

1) the vehicle controls fell within the historical control range,
2) a sufficient number of animals were evaluable and
3) the positive control induced the expected result, confirming the sensitivity of the assay. 

ADME data had already been generated and decrease in % PCEs confirmed target organ exposure. 

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 474 and in compliance with GLP. According to the bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the limit dose of 2000 mg/kg bw/day in male rats.