Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16/07/2015 - 19/10/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas
Specific details on test material used for the study:
Lot No.: 150625
Storage conditions: Room temperature (actual range: 19.0°C to 20.1°C, permissible range: 10°C to 30°C) and air tight condition

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
Air
Details on exposure:
Inhalation exposure (nose-only inhalation exposure) was selected as dosing route to assess the safety of the test substance when it will be exposed to human by inhalation. Fig. 1 shows the inhalation exposure system used in this study. The rats held in the restraint tube (Muenster Ltd.) were subjected to the nose-only inhalation exposure using a flow-past nose-only inhalation exposure chamber (hereinafter abbreviated as “chamber”, Muenster Ltd.) according to the method widely used in similar study. The chamber is constructed from stackable tiers, which has 16 exposure ports per tier. A single-tier chamber was used in this study. The flow rate of air-supply to the chamber was set at 1 L/min per exposure port, 16 L/min in total. The flow rate of the chamber exhaust was set at 14 L/min, which was approximately 10% lower than the air-supply, because the inner pressure of the chamber has to be positive to ensure a reliable exposure. The positive control was orally dosed using a 5 mL-disposable syringe with a gastric tube for rats. The dose volume was set at 10 mL/kg.
Duration of treatment / exposure:
4 hours
Frequency of treatment:
Twice
Post exposure period:
24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
910 920 mg/m³ air (nominal)
Dose / conc.:
150 000 ppm (nominal)
Dose / conc.:
303 640 mg/m³ air (nominal)
Dose / conc.:
50 000 ppm (nominal)
Dose / conc.:
91 092 mg/m³ air (nominal)
Dose / conc.:
15 000 ppm (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Positive control(s):
Cyclophosphamide

Examinations

Tissues and cell types examined:
Bone marrow cell
Details of tissue and slide preparation:
Tissue Preparation
(1) Rats were euthanized under anesthesia with sodium thiopental (Ravonal®, Mitsubishi Tanabe Pharma Corporation, Lot No. V003). The abdominal cavity was opened, and the abdominal aorta was cut for exsanguination. The right femur was removed from the rat.
(2) The bone marrow cells were collected by washing the cavity with 10 v/v% buffered formalin (abbreviation: formalin) of the quantity of 0.5 to 1 mL to obtain the cell suspensions.
(3) The bone marrow cell suspension was left to rest for 5 minutes to obtain supernatant.
(4) The supernatant was further mixed with an about 1 mL of formalin and centrifuged at approximately 170 × g for 5 minutes. The resultant supernatant was discarded.
(5) The precipitate was suspended again in a small amount of formalin to prepare the cell suspension and stored at room temperature.
(6) The bone marrow cell suspension of each animal was stained with the same volume of acridine orange solution (500 μg/mL) just before the microscopic observation and was spread on a clean glass slide.
Slide Preparation
(1) The slide specimens prepared as described above were observed under a fluorescent microscope with a B-excitation filter in a blind manner at a magnification of 600 ×.
(2) One thousand erythrocytes (including both immature erythrocytes [IMEs] and mature erythrocytes [MEs]) per animal were examined to determine the ratio of IMEs among the total erythrocytes. A total of 4000 IMEs were examined for the number of micronucleated IMEs (MNIMEs).
(3) Types of erythrocytes (IMEs or MEs) were identified in accordance with the methods of Hayashi et al. Small bodies with yellowish green fluorescence in the cytoplasm were recognized as micronuclei.
Evaluation criteria:
Because the MNIMEs (%) satisfied the following criteria, this study was judged to be conducted appropriately. (1) The individual values in the negative control group were within the laboratory historical control data (mean ± 2SD). (2) The individual data in the positive control group were within the range of the maximum and minimum values of the laboratory historical positive control, and the total number of the MNIMEs in the positive control group was statistically higher than that in the negative control group.
The ability of the test substance to induce MNIMEs was judged positive if the test substance significantly increased the number of MNIMEs as compared to the negative control with a dose-dependency, and the value in the test substance group deviated from the laboratory historical control range (mean ± 2SD).
Statistics:
The frequency (number) of MNIMEs, animal body weight and percentage of IMEs were statistically analyzed in this study.
The method of Kastenbaum and Bowman was applied to compare the number of MNIMEs in each treatment group including the positive control group with that in the negative control group (significance levels: 5% and 1%). Since no statistically significant differences were detected in any of the test substance groups, Cochran-Armitage test was not applied in this study.
Ratio of IMEs was statistically analyzed using EXSUS system (CAC EXICARE Corporation, Ver. 7.7.1). The significance levels were set at 5% for Bartlett test, 5% (with two-tailed) for Williams test, 5% for F test, 5% and 1% (with two-tailed) for the other tests.
(1) All the data except for the positive control group were tested by Bartlett’s test for homogeneity of variance among the groups. Williams’ test was applied to determine the statistical differences between the test substance groups and the negative control group since the variance was homogeneous by Bartlett’s test.
(2) Since there were no significant differences by Williams’ test, Dunnett test was applied to compare the mean value in each test substance group with that in the negative control group.
(3) The data from the positive control groups was compared to that from the negative control group by F test for homogeneity of variance between the two groups. Since the variance was heterogeneous, Aspin-Welch t-test was performed to compare the mean values of the two groups.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

An in vivo bone marrow micronucleus assay was conducted to assess the potential of HCFO-1224yd(Z) to induce chromosomal aberrations using 5 male rats per group dosed at 0 (negative control), 91,092, 303,640 and 910,920 mg/m3 (15,000, 50,000 and 150,000 ppm respectively) by nose-only inhalation exposure for 4 hours daily with two-day regimen.

Actual exposure concentrations were 99,618, 315,385 and 925,877 mg/m3 (16,404, 51,934 and 152,463 ppm respectively) as the mean concentrations. It was not considered that there were environmental changes affected the study results during the exposure.

One animal in the 303,640 mg/m3 (50,000 ppm) group was found dead just after the exposure on day 1. The cause of death was not clear; however, it was considered unrelated to the exposure of the test substance, because there were neither abnormal clinical signs nor irregular body weight changes in other animals including even at the higher dose level during the experimental period.

The frequencies of MNIMEs obtained from all test substance groups were comparable to that of the negative control group. There were no statistically significant increases in the values in any test substance groups even if there were four analyzable animals in the middle dose group being shorter than the other groups. There were no adverse effects in the study results to evaluate the micronucleus inducibility.

The historical control data at the test facility are shown in Appendix. The individual incidences of MNIMEs ranged from 0.075% to 0.175% in the negative control group. These values were within the laboratory historical control data (Mean ± 2SD = 0.149% ± 0.142%). In the positive control group, the individual values of MNIMEs were within the maximum and minimum individual range of the historical positive control. Additionally, a significant increase in the frequency of MNIMEs was observed in the positive control group compared to the negative control group. These control results satisfied the acceptance criteria of this study and supported the validity of this study.

It is considered that a chemical treated to a mammalian by inhalation exposure directly contact with alveolus cells and circulatory blood around the cells, so that the chemical is estimated to be systemically exposed to the treated animal.

Based on the results described above, HCFO-1224yd(Z) was judged negative in the rat bone marrow micronucleus assay and did not have a genotoxic potential to induce chromosome aberration in vivo under the conditions employed in this study.

Applicant's summary and conclusion

Conclusions:
Based on the results described above, HCFO-1224yd(Z) was judged negative in the rat bone marrow micronucleus assay and did not have a genotoxic potential to induce chromosome aberration in vivo under the conditions employed in this study.
Executive summary:

Based on the results described above, HCFO-1224yd(Z) was judged negative in the rat bone marrow micronucleus assay and did not have a genotoxic potential to induce chromosome aberration in vivo under the conditions employed in this study.