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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 January 2017 - 05 April 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:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Methyl 3-phenyloxirane-2-carboxylate
EC Number:
253-370-2
EC Name:
Methyl 3-phenyloxirane-2-carboxylate
Cas Number:
37161-74-3
Molecular formula:
C10H10O3

Test animals

Species:
mouse
Strain:
other: Hsd:ICR (CD-1)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS, Inc., Frederick, MD
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 30.6 - 36.1g; females: 24.3 - 26.7g
- Assigned to test groups randomly: yes
- Fasting period before study: No data.
- Housing: Animals of the same sex were housed up to three per Micro-Barrier cage.
- Diet: Free access to a certified laboratory rodent chow (Envigo 2018C Teklad Global 18% Protein Rodent Diet).
- Water: Free access to tap water.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 February 2017 To: 05 April 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: Corn oil was the vehicle of choice based on the solubility of the test substance and compatibility with the test system.
- Concentration of test material in vehicle: The test substance formed a solution in corn oil at a concentration of approximately 200 mg/mL, the maximum concentration tested.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
24 hours/dose
Frequency of treatment:
dosed on four consecutive days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide and methylmethanesulfonate
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg/day / 2 mg/mL (cyclophosphamide), dosed on study days 1 and 2; 40 mg/kg/day / 4 mg/mL (methylmethanesulfonate), dosed on study day 4

Examinations

Tissues and cell types examined:
The frequency of micronucleated reticulocytes in peripheral blood was analyzed after flow cytometry calibration using Malaria infected biostandard and CD71-negative control standards provided in the Litron kit. Up to 20,000 RETs per animal, when possible, were analyzed.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In the dose range finding test, clinical signs of toxicity were observed at all dose levels in male mice and at 1000 and 2000 mg/kg/bw day in female mice. Mortality was observed in all animals at 2000 mg/kg/bw day. Due to the clinical observations and mortality observed, both sexes were used in the definitive assay.

Peripheral Blood Collection for Flow Cytometry:
Approximately 3 to 4 hours after the last dose, peripheral blood (>120 μL) was collected for the detection of micronuclei using flow cytometry. MicroFlow Anticoagulant, supplied by Litron Labs (stored at 2-8 ºC) was used as Anticoagulant.

Collection Location
Retro-orbital Sinus:
Animals were anesthetized prior to blood collection by exposure to 70% CO2/30% O2. Each sample was collected into a blank micro-centrifuge tube; then, 100 μL was transferred into a pre-labeled tube with anticoagulant, pipetted up and down several times to mix and placed on wet ice until all of the samples were collected. Blood samples in anticoagulant were kept on wet ice for less than two hours until fixation was complete. Approximately 180 μL of the blood mixture with anticoagulant was transferred into 2 sets of tubes containing fixative (freezing cold methanol). After fixation, samples were stored at -80 ± 5°C for 15 days prior to flow cytometric analysis.

METHOD OF ANALYSIS:
Detection of Micronucleated Reticulocytes with Flow Cytometry:
Peripheral blood samples were washed with ice cold buffer solution to remove the fixative. The cells were then pelleted by centrifugation, and the supernatant was poured off leaving a small amount of supernatant with the pellet. The cells were re-suspended and 20 μL of suspension was added to 80 μL of staining solution containing RNase, FITC-conjugated anti-CD 71 antibodies (mouse antibody) and PE-conjugated anti-CD 61 antibodies (platelet antibody). The samples were incubated at 2-8ºC for 30 minutes, re-suspended, and then incubated at room temperature for an additional 30 minutes. 1.5 mL of DNA staining solution (propidium iodide) was added; then, the samples were placed on wet ice for 5 minutes prior to flow cytometric analysis.

Calculation of Flow Cytometric Analysis
The proportion of reticulocytes to total number of cells scored (% RETs) was determined for each animal and treatment group. This calculation was carried out as indicated below:
% RET = (UL + UR) X 100 / (UL + UR + LL + LR)
UL: The number of events in the upper-left quadrant
UR: The number of events in the upper-right quadrant
LL: The number of events in the lower-left quadrant
LR: The number of events in the lower-right quadrant

The % RETs served as a parameter of the test substance cytotoxicity in peripheral blood. A decrease in this ratio in the test substance groups, as compared to the vehicle (negative) control, would indicate a toxic effect of the test substance, while an increase would represent a sign of recovery from earlier toxic insult. The animal with a <5% %RET of the vehicle control was considered excessively cytotoxic and was excluded from evaluation.
The quantization of the MnRETs in peripheral blood was expressed as percentage of MnRETs per total number of cells evaluated. The % MnRETs was presented for each animal and the mean ± standard deviation was calculated and presented for each treatment group as follows:
% MnRET = (UR) X 100 / (UL + UR)
Evaluation criteria:
A test substance was considered to have induced a positive response if:
a) at least one of the test substance doses exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
b) when multiple doses were examined at a particular sampling time, the increase was dose-related (p ≤ 0.01), and
c) results of the group mean or of the individual animals in at least one group were outside the 95% control limit of the historical negative control data.
A test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met and there was evidence that the peripheral blood was exposed to the test substance (unless intravenous administration was used).
Statistics:
Statistical analysis was performed on the micronucleus frequency (%MnRET) and RET% using the animal as the unit. The mean and standard deviation of %MnRET and RET% were presented for each treatment group.
The use of parametric or non-parametric statistical methods in the evaluation of data was based on the variation between groups. The group variances for micronucleus frequency for the vehicle and test substance groups at the respective sampling time were compared using Levene’s test (significant level of p ≤ 0.05). Since the variation between groups was found not to be significant, a parametric one-way ANOVA was performed followed by a Dunnett’s post-hoc analysis to compare each dose group to the concurrent vehicle control.
A linear regression analysis was conducted to assess dose responsiveness in the test substance-treated groups (p ≤ 0.01).
A pair-wise comparison (Student’s T-test, p ≤ 0.05) was used to compare the positive control groups to the concurrent vehicle control groups.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the dose range finding test, clinical signs of toxicity were observed at all dose levels in male mice and at 1000 and 2000 mg/kg/bw day in female mice. Mortality was observed in all animals at 2000 mg/kg/bw day. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study.
The following clinical signs were observed:
Dose Level (mg/kg/bw day) Males Females
500 Piloerection Normal
1000 Piloerection Piloerection
2000 Piloerection, ataxia and Piloerection and ataxia
irregular breathing

RESULTS OF DEFINITIVE STUDY
Mortality occurred in 1/6 female mice at 1000 mg/kg/bw day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study.
The following clinical signs were observed:
Dose Level (mg/kg/bw day) Males Females
Vehicle Normal Normal
250 Piloerection Piloerection
500 Piloerection Piloerection
1000 Piloerection Piloerection
Positive control Normal Normal

The scoring results and a statistical analysis of data indicated the following:
• No appreciable reductions in %RETs were observed in the test substance-treated groups compared to the vehicle control groups, indicating the test substance did not induce cytotoxicity.
• Group variances for mean of means of the micronucleus frequency in the vehicle and test substance groups were compared using Levene’s test. The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.
• No statistically significant increases in the incidence of MnRETs were observed in the test substance-treated groups relative to the vehicle control groups (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).
• The positive control, CP, induced a statistically significant increase in the incidence of MnRETs (Student’s t-test, p ≤ 0.05).
• The number of MnRETs in the vehicle control groups did not exceed the historical control range

Applicant's summary and conclusion

Conclusions:
A micronucleus study with the substance was performed according to OECD 474 guideline and GLP principles, in male and female mice. It is concluded that the substance is not mutagenic in the mouse micronucleus assay.
Executive summary:

Method: In an in vivo micronucleus study (as part of the Comet assay), 6 male and 6 female mice per dose were exposed to 250, 500 and 1000 mg/kg bw day of the substance for four consecutive days, performed according to OECD 474 guideline and GLP principles. The maximum tolerated dose for the definitive Micronucleus assay was set at 1000 mg/kg/bw day based on mortality observed in all animals at 2000 mg/kg/bw day in a dose range finding assay.

Results on toxicity: Mortality occurred in 1/6 female mice at 1000 mg/kg/bw day in the definitive assay. No mortality occurred in male mice. No considerable reductions in mean group body weights were observed in the test substance-treated groups during the course of the study. Clinical signs were observed at all dose levels and included piloerection.

Results on genotoxicity: No appreciable reductions could also be increase in %RETs (reticulocytes) were observed in the test substance-treated groups compared to the vehicle control groups, indicating the test substance did not induce cytotoxicity in the red blood cells. In view of the systemic effects seen (mortality) the substance (and its metabolites) have reached the systemic circulation. No statistically significant increases in the incidence of MnRETs (reticulocytes containing micronuclei) were observed in the test substance-treated groups relative to the vehicle control groups (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).

Conclusion: The substance is negative in the in vivo micronucleus assay.

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