1-[5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]ethanone

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2012

Report date:

2012

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: MAFF Japan Test Guidelines for Agricultural Chemicals 12-Nousan-8147: Guideline Number 2-1-19-3 (2000)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Purity: 99.9%

Test animals

Species:

mouse

Strain:

other: Crl:CD1(ICR)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult
- Weight at study initiation: male - 31.5 g; female - 24.8 g
- Fasting period before study: not reported
- Housing: At study start, male animals were housed individually and female animals were housed 2-3 per cage.
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.1% Tween-80 in 0.5% aqueous methylcellulose prepared with deionized water
- Amount of vehicle (if gavage or dermal): The test substance, vehicle, and positive controls were administered by single oral gavage at a dose volume of 10 mL/kg bw.

Details on exposure:

PREPARATION OF TEST SUBSTANCE FORMULATION:
-Preparation frequency: not reported
-Adjusted for purity: no

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

450 mg/kg bw/day

Remarks:

males and females

Doses / concentrations 2

Dose / conc.:

900 mg/kg bw/day

Remarks:

males and females

Doses / concentrations 3

Dose / conc.:

1 350 mg/kg bw/day

Remarks:

females

Doses / concentrations 4

Dose / conc.:

1 800 mg/kg bw/day

Remarks:

males

No. of animals per sex per dose:

Control: male 10, female 10
450 mg: male 10, female 10
900 mg: male 11, female 10
1350 mg: male 0, female 14
1800 mg: male 14, female 0
Positive control: male 5, female 5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive Control: yes, Cyclophosphamide
- Doses / concentrations: 40 mg/kg bw via gavage

Examinations

Tissues and cell types examined:

PCEs and NCEs from bone marrow taken from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based the on range-finder results, the dose levels selected for the main study were 450, 900, and 1800 mg/kg bw. However, based on the observations made during dosing of the mid dose female and high dose male animals, the high dose level was decreased for the female mice. Each group of male animals was administered a single dose of 450, 900, or 1800 mg/kg bw, or the vehicle or positive control substance for the main study. Each group of female animals was administered a single dose of 450, 900, or 1350 mg/kg bw, or the vehicle or positive control substance for the main study.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 24 and 48 hours after dose administration, treated animals were sacrificed and the bone marrow removed. Additional male and female animals were treated with the highest concentration to allow for unexpected mortality. Bone marrow was sampled at approximately 24 and 48 hours post-treatment from vehicle control, high-, intermediate-, and low-dose groups. Positive controls were sampled concurrently at the 24-hour interval, only.

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice, marrow from both femurs of each animal was aspirated into a syringe containing foetal bovine serum (FBS) and transferred to a centrifuge tube containing FBS. Erythrocytes were collected by centrifugation. Following centrifugation, most of the supernatant was removed. The pellet was suspended in FBS and a small drop was placed on a pre-cleaned microscope slide. Smears were made using a blood smearing instrument. At least 3 slides per animal were prepared. The slides were air-dried and fixed using absolute methanol. Bone marrow smears were stained in acridine orange, a DNA/RNA specific fluorochrome.

METHOD OF ANALYSIS:
Evaluations were conducted on 5 animals/sex/group. At the highest concentration, 5 males and 5 females were selected for evaluation based on survival until the scheduled sacrifice and then cage order. At least 2000 PCEs per animal were scored for the presence of micronuclei (round, bright yellow-green fluorescing bodies). In the event 2000 PCEs were unattainable for a given animal, the animal was excluded from the statistical evaluation of the scoring results. Fluorescent microscopy was used to examine the smears. Colour was used to distinguish PCEs (red-orange) from normochromatic erythrocytes (NCEs) (grey-green). Inclusions in PCEs which were improperly shaped or stained or were not in the focal plane of the cell were considered artefacts and not scored as micronuclei. Cells containing more than 1 micronucleus were scored as a single micronucleated PCE (MNPCE). The proportion of PCEs among 1000 total erythrocytes (expressed as the PCE/NCE ratio) was determined for each animal. The proportion of PCEs to total erythrocytes in the test substance-treated animals was not less than 20% of the control value.

Evaluation criteria:

Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result was not required to obtain a conclusive finding.
The test substance was judged negative if the following conditions were met:
• No statistically significant dose-related increases in the group mean MNPCEs above the concurrent vehicle control value occurred at any concentration of the test substance.
• The MNPCE values of the test substance-treated animals were within reasonable limits of the recent (past 3 years) laboratory historical control range.

The test substance was judged positive if the following conditions were met:
• The group mean MNPCEs was statistically significantly increased at one or more concentrations of the test substance compared to the concurrent vehicle control values.
• An accompanying statistically significant dose-response increase in MNPCEs was observed.

Statistics:

Data for the proportion of micronucleated PCEs among 2000 polychromatic erythrocytes and the proportion of PCEs among 1000 erythrocytes (MNPCE and PCE frequency, respectively) was transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation was appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the nontransformed proportion. Transformed data for PCE and MNPCE frequencies were analysed separately for normality of distribution and equal variance using the Shapiro-Wilk and Levene’s tests, respectively. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.

For those data that were normally distributed and had equal variance, parametric statistics (e.g., analysis of variance (ANOVA) and Dunnett’s test) were performed using the transformed data. For those data that were normally distributed but had unequal variance, a robust ANOVA and unequal-variance Dunnett test was done. For those data that were not normally distributed, nonparametric statistics (e.g., Kruskal-Wallis and Dunn’s tests) utilizing non-transformed data were performed. The individual animal was considered the experimental unit. All data analyses were one-tailed and conducted at a significance level of 5%.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality in range finding test (males only); clinical signs in definitive test (males and females)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 mg/kg bw and 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In the rangefinder experiment at 2000 mg/kg bw, 1/3 male mice was prostrate with laboured breathing and was later found dead, 1/3 male mice showed signs of ataxia, and 1/3 male mice showed no adverse clinical signs. In the second rangefinder experiment at 1500 mg/kg/bw, no abnormalities were detected in any of the male mice tested.
- Harvest times: The animals were observed for clinical signs of toxicity and mortality immediately after dosing and daily thereafter for 2 days (until approximately 48 hours post-dosing).

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint. A statistically significant decrease in PCEs among 1000 erythrocytes was observed with male mice in the top dose group of 1800 mg/kg/bw, at the 48 hour time point, indicating that the test substance reached the target cells. No other reductions in PCE frequency were detected at any other time point or at any other dose level for male or female mice administered the test substance.

Any other information on results incl. tables

Clinical signs:

In the main study, adverse clinical signs of toxicity were observed at all dose levels tested in male and female mice exposed to the test substance. At 1800 mg/kg bw, 10/14 male mice had clinical signs. By approximately 1 hour post dose 2/14 male mice were declared moribund; however, both animals began to recover showing lesser clinical signs by 3-5 hours post and no clinical signs by 24 hours post dose. Clinical observations included abnormal gait (1/14 males), ataxia (2/14 males), eyelid ptosis (1/14 males), decreased muscle tone (5/14 males), prostration (2/14 males), splayed limbs (4/14 males) and tremors (1/14 males).

At 1350 mg/kg bw, 13/14 female mice had clinical signs. Clinical observations included abnormal gait (3/14 females), ataxia (5/14 females), decreased muscle tone (12/14 females), prostration (5/14 females), splayed limbs (8/14 females), and tremors (1/14 females).

At 900 mg/kg bw, 1 male mouse was found dead shortly after dosing and was replaced on the study. Clinical signs were observed in 5/10 male mice and 9/10 female mice. Clinical signs include ataxia (1/10 males and 6/10 females), eyelid ptosis (1/10 females), decrease muscle tone (5/10 males and 6/10 females), low posture (2/10 females), leaning (1/10 males), splayed limbs (1/10 males and 6/10 females), and tremors (1/10 females).

At 450 mg/kg bw, 1/10 male mice and 2/10 female mice presented with decreased muscle tone. No abnormalities were detected in the vehicle or positive control groups.

All adverse clinical observations were observed on test day 0. No test substance related abnormalities were detected with any animals at either the 24 or 48 hour observation time point.

Micronucleus Evaluation for Male Mice
	Group 1 0 mg/kg	Group 2 450 mg/kg	Group 3 900 mg/kg	Group 4 1800 mg/kg	Group 5 40 mg/kg CP
PCEs/1000 Erythrocytes
24-Hour	536 38 (5)	563 14 (5)	563 39 (5)	545 16 (5)	566 39 (5)
48-Hour	565 17 (5)	a	a	537* 27 (5)	b
PCE/NCE Ratio
24-Hour	1.165 0.178 (5)	1.291 0.076 (5)	1.304 0.186 (5)	1.200 0.077 (5)	1.318 0.214 (5)
48-Hour	1.301 0.091 (5)	^a	^a	1.164* 0.121 (5)	^b
MNPCE/2000 PCEs
24-Hour	0.8 1.3 (5)	1.4 0.9 (5)	1.6 0.9 (5)	1.2 0.4 (5)	30.6@* 5.2 (5)
48-Hour	1.2 1.3 (5)	^a	^a	2.0 0.7 (5)	^b
Data arranged as:           Mean                                      Standard Deviation (Number of values included in calculation)   PCE/NCE Ratio = mean of the PCE/NCE for the individual animals   a Group not evaluated at this time point. b Group not included at this time point. * Statistically significant difference from control at p <0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p <0.05 by Dunn’s test. ~ Due to lack of control group values or variability among group means, statistical analyses were unable to be performed.

 

Micronucleus Evaluation for Female Mice
	Group 1 0 mg/kg	Group 2 450 mg/kg	Group 3 900 mg/kg	Group 4 1350 mg/kg	Group 5 40 mg/kg CP
PCEs/1000 Erythrocytes
24-Hour	523 70 (5)	558 17 (5)	565 24 (5)	543 39 (5)	522 44 (5)
48-Hour	548 9 (5)	a	a	571 14 (5)	b
PCE/NCE Ratio
24-Hour	1.127 0.271 (5)	1.267 0.086 (5)	1.303 0.132 (5)	1.199 0.184 (5)	1.107 0.193 (5)
48-Hour	1.214 0.043 (5)	^a	^a	1.333 0.080 (5)	^b
MNPCE/2000 PCEs
24-Hour	1.4 0.9 (5)	1.0 1.2 (5)	2.4 2.3 (5)	12.6 1.1 (5)	29.4@* 6.5 (5)
48-Hour	1.6 1.1 (5)	^a	^a	0.8 0.4 (5)	^b
Data arranged as:           Mean                                       Standard Deviation (Number of values included in calculation)   PCE/NCE Ratio = mean of the PCE/NCE for the individual animals   a Group not evaluated at this time point. b Group not included at this time point. * Statistically significant difference from control at p <0.05 by Dunnett/Tamhane-Dunnett test. @ Statistically significant difference from control at p <0.05 by Dunn’s test. ~ Due to lack of control group values or variability among group means, statistical analyses were unable to be performed.

Applicant's summary and conclusion

Conclusions:

The test substance was concluded to be negative in this in vivo study.

Executive summary:

An oral micronucleus study was conducted in mice to determine whether the test substance induces an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow.  In this study, groups of male and female Crl:CD1(ICR) mice were given doses of 0, 450, 900, 1350 (female only) and 1800 (male only) mg/kg body weight (bw) of the test substance.  Bone marrow smears were prepared approximately 24 and 48 hours after dosing.  Two thousand PCEs per animal were evaluated for micronuclei and 1000 total erythrocytes per animal were evaluated for bone marrow toxicity.

In the main study, adverse clinical signs of toxicity were observed at all dose levels tested in male and female mice exposed to the test substance. At 1800 mg/kg bw, 10/14 male mice had clinical signs. By approximately 1 hour post dose 2/14 male mice were declared moribund; however, both animals began to recover showing lesser clinical signs by 3-5 hours post and no clinical signs by 24 hours post dose. Clinical observations included abnormal gait (1/14 males), ataxia (2/14 males), eyelid ptosis (1/14 males), decreased muscle tone (5/14 males), prostration (2/14 males), splayed limbs (4/14 males) and tremors (1/14 males). At 1350 mg/kg bw, 13/14 female mice had clinical signs. Clinical observations included abnormal gait (3/14 females), ataxia (5/14 females), decreased muscle tone (12/14 females), prostration (5/14 females), splayed limbs (8/14 females), and tremors (1/14 females). At 900 mg/kg bw, 1 male mouse was found dead shortly after dosing and was replaced on the study. Clinical signs were observed in 5/10 male mice and 9/10 female mice. Clinical signs include ataxia (1/10 males and 6/10 females), eyelid ptosis (1/10 females), decrease muscle tone (5/10 males and 6/10 females), low posture (2/10 females), leaning (1/10 males), splayed limbs (1/10 males and 6/10 females), and tremors (1/10 females). At 450 mg/kg bw, 1/10 male mice and 2/10 female mice presented with decreased muscle tone. No abnormalities were detected in the vehicle or positive control groups. All adverse clinical observations were observed on test day 0. No test substance related abnormalities were detected with any animals at either the 24 or 48 hour observation time point.

No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint. A statistically significant decrease in PCEs among 1000 erythrocytes was observed with male mice in the top dose group of 1800 mg/kg/bw, at the 48 hour time point, indicating that the test substance reached the target cells. No other reductions in PCE frequency were detected at any other time point or at any other dose level for male or female mice administered the test substance.  Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.