8-Chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxylic acid

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: JMAFF 2-1-19-3 (2000)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

1

Reference substance name:

8-chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxylic acid

Cas Number:

353258-35-2

Molecular formula:

C9H4ClF3N2O2

IUPAC Name:

8-chloro-6-(trifluoromethyl)imidazo[1,2-a]pyridine-2-carboxylic acid

Specific details on test material used for the study:

Test Substance: IN-QEK31-011
Lot Number: SG0312574
Purity: 98.2%

Test animals

Species:

mouse

Strain:

other: Crl:CD1(ICR)

Details on species / strain selection:

Mice have been shown to exhibit micronuclei indicative of broken chromosomes (clastogenic effects) or spindle effects (aneugenic effects) in response to known mutagens and were therefore used in this assay. The Crl:CD1(ICR) mouse was selected based on extensive experience with this strain at test facility and its suitability for genetic toxicology studies.

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

0.1% Tween-80 in 0.5% aqueous methylcellulose

Frequency of treatment:

Single dose

Post exposure period:

Approximately 48 and 72 hours after dose administration, peripheral blood samples were collected for micronucleus evaluation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 600 mg/kg bw/day

Remarks:

not analyzed due to excessive mortality

Doses / concentrations 5

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

not analyzed due to excessive mortality

No. of animals per sex per dose:

Pre test: 3/sex/group
Main study: 5/sex/group (0, 500, 1000 mg/kg bw and positive control); 7/sex (2000 mg/kg bw)
Follow-up study: 5/sex (vehicle control); 7/sex/group (1300 and 1600 mg/kg bw)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CP), the positive control substance, was prepared in deionized water and administered once orally by gavage at a dose of 30 mg/kg and a dose volume of 10 mL/kg.

Examinations

Tissues and cell types examined:

Peripheral blood and micronucleated RET

Evaluation criteria:

Providing that all valid test criteria were fulfilled, a test substance was considered clearly negative if, in all experimental conditions examined:
• None of the treatment groups exhibited a statistically significant increase in the frequency of MN RETs compared with the concurrent negative control,
• There was no dose-related increase at any sampling time when evaluated by an appropriate trend test,
• All results (group means) were inside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits), and
• Bone marrow exposure to the test substance occurred (as evident by either a reduction in the RET frequency, measurement of the test substance levels in plasma or blood, or ADME data obtained in an independent study).
Providing that all valid test criteria were fulfilled, a test substance was considered clearly positive if:
• At least one of the treatment groups exhibited a statistically significant increase in the frequency of MN RETs compared with the concurrent negative control,
• This increase was dose-related at least at one sampling time when evaluated with an appropriate trend test, and
• Any of these results were outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
If only the highest dose was examined at a particular sampling time, a test substance was considered clearly positive if there was a statistically significant increase compared with the concurrent negative control and the results were outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits). When conducting a dose-response analysis, at least 3 treated dose groups should have been analyzed. Statistical tests should have used the animal as the experimental unit.

Statistics:

Male and female animals were evaluated separately. The frequency of RETs was evaluated for a decreasing response while the frequency of MN RETs was evaluated for an increasing response. For each sex and timepoint, an analysis of variance (ANOVA) and Dunnett’s test were performed on both the RET and MN RET frequencies. Where the data were found not consistent with the requirements for a Dunnett’s test, a Dunn’s non-parametric test was used. A t-test was used to compare positive and negative controls. Statistical significance was further evaluated for a dose response using the Williams’s test or the non-parametric Jonckheere-Terpstra test, where appropriate.
No statistical analysis was conducted on body weights or clinical signs

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

In the main study, signs of toxicity were observed in both male and female mice at 2000 mg/kg. Clinical observations included lethargy, eyelid ptosis, dehydration, labored breathing, ataxia, abnormal gate, discharge, prostration, moribundity, and death.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Clinical Observations and Mortality
No clinical signs of toxicity were observed in the range finding study conducted at 2000 mg/kg bw.
In the main micronucleus study clinical signs of toxicity were observed in both male and female mice at 2000 mg/kg bw. Clinical observations included lethargy (2/7 males and 1/7 females), eyelid ptosis (1/7 males), dehydration (2/7 males and 1/7 females), labored breathing (2/7 males and 1/7 females), ataxia (1/7 males), abnormal gate (1/7 males), discharge (1/7 females), prostration (1/7 females), moribundity (2/7 males and 1/7 females), and death (3/7 males and 1/7 females). The moribund animals were all removed from study on test day 2. The four animals in which death occurred were found dead on test day 3. Due to this excessive toxicity, all remaining mice in the 2000 mg/kg bw dose group were humanely sacrificed on test day 3 without blood collection.
In the follow-up micronucleus study no clinical signs of toxicity were observed; however, several mice were found dead. At 1600 mg/kg bw 3/7 male mice were found dead (two on test day 2 and one on test day 3) and 2/7 female mice were found dead (one on test days 2 and 3 each). At 1300 mg/kg bw 2/7 male mice were found dead on day test day 2.
It is believed that the reason moribundity/mortality were observed in both the main and follow-up studies but not the range finding study was the difference in the formulation procedure, where bead milling of the test substance formulation may have increased bioavailability.

Applicant's summary and conclusion

Conclusions:

Test substance was negative in this in vivo Mouse Micronucleus study

Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow by analyzing peripheral blood reticulocytes (RETs) in male and female Crl:CD1(ICR) mice according to the guidelines OECD 474, US EPA OPPTS 870.5395, EC B.12 and JMAFF. Based on range-finding results, doses of 500, 1000, and 2000 mg/kg body weight (bw) of the test substance were selected for the main micronucleus study. Concurrent control groups were administered 0.1% Tween-80 in 0.5% aqueous methylcellulose as the vehicle (negative) control, or 30 mg/kg bw of cyclophosphamide (positive control). However, in the main study 2000 mg/kg bw was found to be above the maximum tolerated dose (MTD), while no clinical signs of toxicity were observed at 500 or 1000 mg/kg bw. Therefore, a follow-up micronucleus study was conducted with animals administered a single dose of the test substance at 1300 or 1600 mg/kg bw, or the vehicle control.

All animals were given a single dose by oral gavage. The vehicle control, 500 and 1000 mg/kg bw dose groups, and the positive control groups contained 5 animals/sex. The 1300, 1600, and 2000 mg/kg bw group contained 7 animals/sex. Peripheral blood samples were collected via tail vein bleeding, and a micronucleus evaluation was conducted by flow cytometry. The samples were collected approximately 48 and 72 hours post-dosing for the test substance treated and vehicle control groups, and approximately 48 hours post-dosing for the positive control group. A total of 20,000 RETs were analyzed per blood sample for the induction of micronuclei and toxicity as indicated by the frequency of immature erythrocytes (% RETs) among total erythrocytes (i.e., RETs plus normochromatic erythrocytes, NCEs). All surviving animals were weighed and observed for mortality and clinical signs of toxicity at least daily.

The formulated test substance formed a suspension in all studies. However, based on the range finding study the formulations for the main and follow-up studies were bead milled to increase homogeneity at all dose levels. Aliquots of the vehicle control and each test substance concentration were taken to confirm homogeneity, dose concentrations, and stability. Homogeneity and target concentrations were verified, and the test substance was stable for the duration of the dosing period.

No clinical signs of toxicity were observed in the range finding study conducted at 2000 mg/kg bw.

In the main micronucleus study clinical signs of toxicity were observed in both male and female mice at 2000 mg/kg bw. Clinical observations included lethargy (2/7 males and 1/7 females), eyelid ptosis (1/7 males), dehydration (2/7 males and 1/7 females), labored breathing (2/7 males and 1/7 females), ataxia (1/7 males), abnormal gate (1/7 males), discharge (1/7 females), prostration (1/7 females), moribundity (2/7 males and 1/7 females), and death (3/7 males and 1/7 females). On test day 3 all the remaining mice in the 2000 mg/kg bw dose group were humanely sacrificed without blood collection.

In the follow-up micronucleus study no clinical signs of toxicity were observed; however, several mice were found dead. At 1600 mg/kg bw 3/7 male and 2/7 female mice died. At 1300 mg/kg bw 2/7 male mice died.

It is believed that the reason clinical observations and moribundity/mortality were observed in the main and follow-up study but not the range finding study was the difference in the formulation procedure, where bead milling of the test substance formulation may have increased bioavailability.

No abnormalities were detected in the vehicle or positive control groups.

Samples from the negative control, positive control, 500, 1000, and 1300 mg/kg bw dose group were analyzed at the 48-hour time point. At the 72-hour time point only samples from the negative control and 1300 mg/kg bw dose group were analyzed. Main study negative control, 500, and 1000 mg/kg bw dose groups were not analyzed at the 72-hour time point since no positive response was observed for the 1300 mg/kg bw dose group. The 1600 mg/kg bw dose group was not analyzed at either time point due to excessive mortality.

No statistically significant or biologically relevant increases in the micronucleated RET frequency were observed in any evaluated test substance treated group of male or female animals at either time point.

Statistically significant reductions in %RETs were observed with male mice in the 500 and 1000 mg/kg bw dose groups at the 48-hour time point and in the 1300 mg/kg bw dose group at the 72-hour time point. However, there was no reduction in %RETs observed in the 1300 mg/kg bw dose group at 48 hours and there were no statistically significant decreases in %RETs among the total erythrocytes observed with female mice at either time point at any dose group. Therefore, these reductions were not considered as sufficient evidence of target cell exposure.

Target cell exposure was evaluated in an independent study. The results of this study indicate that the systemic exposure in the male and female mice was evident based on the presence of quantifiable test substance in pooled plasma samples at 4 hours after a single oral gavage administration at a dose level of 500 mg/kg bw.

As expected, statistically significant increases in MN-RET frequency were found in CP-treated animals of both sexes. Statistically significant depressions in RET frequency were observed in both male and female animals administered CP, indicating that the 30 mg/kg bw dose level was sufficient to induce toxicity as well as induce MN-RETs.

All criteria for a valid study were met. Under the conditions of this study, test substance did not induce increases in micronucleated RETs in mouse peripheral blood. The test substance was concluded to be negative in this in vivo study.