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 vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Feb - 21 Mar 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 2016

Deviations:

yes

Remarks:

intraperitoneal injection not recommended as exposure route.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 27 - 43 g
- Assigned to test groups randomly: yes
- Housing: singly in type I cages with bedding of soft wood granules, type S 8/15
- Diet: Altromin 1324 Standard Diet (Altromin GmbH, lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1.5
- Humidity (%): 40 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

0.5 % aqueous Cremophor emulsion

Details on exposure:

Preparation of dosing solutions:
The test substance was suspended in 0.5 % aqueous Cremophor emulsion, using sonication for 30 minutes. The substance was injected intraperitoneally. The administered volume was 10 mL/kg bw. The same conditions were used for the positive control substance cyclophosphamide that was dissolved in deionized water.

Duration of treatment / exposure:

single injection

Frequency of treatment:

once

Post exposure period:

Test substance: 16, 24, 48 h
Control animals: 24 h
Positive control: 24 h

Doses / concentrations

Dose / conc.:

60 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): proven cytostatic agent and known clastogen
- Route of administration: injected intraperitoneally
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The doses were based on a pre-test in which groups of five animals received the test substance intraperitoneally at 10, 50, 75 and 100 mg/kg bw. For up to one hour, apathy, reduced motility, spasm, twitching, shivering and difficulty in breathing was observed in animals receiving 50 mg/kg bw or higher. In addition, 1 of 5 and 5 of 5 animals died in the 75 and 100 mg/kg bw groups, respectively. Therefore, the test substance was administered at a dose level of 60 mg/kg bw in the main test.

TREATMENT AND SAMPLING TIMES:
After the designated time, animals were sacrificed. At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). The bone marrow was flushed out with fetal calf serum. The serum-bone marrow suspension was centrifuged and most of the supernatant discarded. The pellet was resuspended in the remaining liquid to produce a homogeneous suspension.

DETAILS OF SLIDE PREPARATION:
One drop of the viscous suspension was placed on a well-cleaned slide and spread to allow proper evaluation of the smear. The slides were dried overnight and stained automatically with an Ames Hema- Tek Slide Stainer (Miles Company). The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.

METHOD OF ANALYSIS:
Micronuclei were analyzed with a light microscope at a magnification of about 1000. 1000 polychromatic erythrocytes were counted per animal. The number of normochromatic erythrocytes per 1000 polychromatic ones was noted. Additionally, the number of normochromatic erythrocytes showing micronuclei was assessed.

Evaluation criteria:

A test was considered positive if there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control at any of the time intervals investigated.

A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.

A test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.

Acceptability
A test was considered acceptable if the solvent and positive control fall withing the expected range, in accordance with the laboratory's experience and/or the available literature data

Statistics:

When the negative control mean was superceded by any group, the test group with the highest mean and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. Significance was set at p below 5%.

The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided Chi 2-test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality and clinical signs

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The doses were based on a pre-test in which groups of five animals received the test substance intraperitoneally at 10, 50, 75 and 100 mg/kg bw. For up to one hour, apathy, reduced motility, spasm, twitching, shivering and difficulty in breathing was observed in animals receiving 50 mg/kg bw or higher. In addition, 1 of 5 and 5 of 5 animals died in the 75 and 100 mg/kg bw groups, respectively.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: apathy, roughened fur, spasm and difficulty in breathing for up to 16 h after administration
- Mortality: 3/40 treated animals died during the test period
- Induction of micronuclei: no difference between control and treatment groups
- Ratio of PCE/NCE: no difference between control and treatment groups
- Positive control: the samples from the positive control had statistically significant increases in
the number of polychromatic erythrocytes compared with the concurrent control group which demonstrated that the test system was capable of detecting a known clastogen.

Summarized results can be found in Attachment 1.

- Historical control data: the negative and positive control are within the historical controls (refer to the attached background material 2).

Applicant's summary and conclusion

Conclusions:

The study was performed according to OECD guideline 474 dated 1983 and compliant with GLP. In the present bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following an intraperitoneal injection of 60 mg/kg bw of the test item, in both male and female mice.