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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:
Study initiation: 15 March 2011; Submission of final report: 18 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Korea
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Chemical name : 1,3,2-Dioxathiolane, 2,2-dioxide
Product name: ESA
Lot No. : Not available
Received date : 18 November, 2010
Appearance : Lemon yellow solid
Purity : 99.1 %
Storage condition : Recommended temperature: 2-8ºC
Stability : Stable in recommended storage condition

Test animals

Species:
mouse
Strain:
ICR
Details on species / strain selection:
Reason for animal selection: ICR mice has been widely used in mammalian erythrocyte micronucleus test. It has been known for suitable experimental animal for general toxicity. Moreover, sufficient fundamental data have been accumulated in toxicological study using ICR mice. Such data will be useful for interpretation and evaluation of test results.
Sex:
male
Details on test animals and environmental conditions:
Species and strains: SPF (Specific Pathogenic Free), CrljOri:CD1 (ICR) mice
Supplier: Orient Bio Co., Ltd.
Number of animals at the time of arrival:
Preliminary dose-range finding study: 33 male mice
Main study: 33 male mice
Age at the time of arrival: 7 weeks old
Period of quarantine and acclimation:
Preliminary dose-range finding study : 7 days after receipt
Main study I: 9 days after receipt
Main study II: (Confirmation test) : 7 days after receipt
(The only healthy animals were selected for this study by observation of general symptoms during the acclimation period.)
Age of test animal at administration: 8 weeks old
Grouping Method
Animals were weighted one day before the administration, and grouped by using the graded body weight randomly.
Identification of individual animals
Individual animals was identified by tail marking using oil marker and individual cages was identified by card labeling. The record sheet was posted at the entrance of animal room.
Weighing method of test animals
Body weight of test animals was measured at the time of animal acquisition, grouping, administration of test substance and autopsy. Body weight was measured one by one in accordance with individual animal number using electrical balance calibrated by calibration standard weight and recorded in data sheet.
Disposal of remaining animals: Animals were euthanized on the last autopsy.

Range of acceptable temperature and humidity
Preliminary dose-range finding study I
: 21.6 ± 0.9 ºC of temperature, 44.5 ± 2.6 % of relative humidity
Preliminary dose-range finding study II
: 21.4 ± 1.0 ºC of temperature, 45.0 ± 7.5 % of relative humidity
Main study I
: 21.6 ± 1.2 ºC of temperature, 54.4 ± 8.6 % of relative humidity
Main study II (Confirmation test)
: 21.7 ± 0.6 ºC of temperature, 53.1 ± 1.4 % of relative humidity

Ventilation frequency : 10~15 times/hr.

Lighting cycle : 12 hours of lighting duration (lighting up at 8 a.m.~lighting out at 8 p.m.)

Luminous intensity
Preliminary dose-range finding study I : 278 Lux
Preliminary dose-range finding study II: 278 Lux
Main study I : 275 Lux
Main study II (Confirmation test) : 275 Lux

Breeding cage (size) : During the quarantine, acclimation, administration and observation period, all animals were housed in polycarbonate cage (170W x 235L x 125H mm).

Animal number per cage
Preliminary dose-range finding study : All animals were housed 5 animals per cage during quarantine and acclimation period and 3 animals per cage during administration and observation period.

Main study and Confirmation test : All animals were housed 5 animals per cage during quarantine and acclimation period and 3 animals per cage during administration and observation period.

Feed
Type: Solid feed for laboratory animals
Source: Polas International
Produced by: Harlan
Salvage method: Free ingestion by feeding.
Identification of pollutants: Data from feed producers were used.

Water
Type : Tap water purified by reverse osmosis filtering system
Feeding method : ad libitum using polycarbonate water bottles
Contamination analysis : Water certification from nationally certified inspection organization was referred to examine contamination.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
distilled water.
Details on exposure:
Preparation and dosage of test substance
The test substance was weighed on the day of administration in accordance with the concentration of the preparation, and heated at about 40 ºC in hot water to dissolve in distilled water. The administration level of prepared test substance was determined to 10 ml/kg based on body weight measured at the administration day. The stability test and the homogeneity test were not performed because the test solution was prepared fresh on administration day.
Duration of treatment / exposure:
single injection
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
16 mg/kg bw/day (nominal)
Remarks:
Main tests I and II
Dose / conc.:
32 mg/kg bw/day (nominal)
Remarks:
Main tests I and II
Dose / conc.:
63 mg/kg bw/day (nominal)
Remarks:
Main tests I and II
No. of animals per sex per dose:
Preliminary tests: 3 male mice per group
Main tests: 6 males mice per group
Control animals:
yes, concurrent vehicle
other: yes, concurrent positive control
Positive control(s):
Mitomycin C (MMC) 2.0 mg/kg
Positive control substances were selected according to OECD guidelines for the Testing of Chemicals No. 474.

Examinations

Tissues and cell types examined:
Erythrocytes in bone marrow from thigh bone.
Details of tissue and slide preparation:
Extraction of bone marrow and preparation of slides
Three slides of bone marrow were prepared per animal. In case of preliminary dose-range finding study, the thigh bone of the test animal was collected in 24, 48 hours after administration. In case of main test and confirmation test, the thigh bone of the test animal was collected in 24 hours after administration. The thigh bone of the test animal was collected with care to avoid blood contamination. The bone marrow was collected in a centrifuge tube by flushing the thigh bone inside with in-activated FBS (Hyclone, Lot No. AWA93840). The extracted bone marrow was centrifuged at 1000 rpm for 5 minutes and then resuspended with small aliquots of FBS after discarding the supernatant. The bone marrow was smeared on a slide glass and then dried at room temperature and fixed in methanol for 5 minutes.

Staining of specimen
For the scoring of the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio, the slides were stained in 4 % Giemsa staining solution. For the scoring of the micronucleated polychromatic erythrocytes (MNPCE) from PCEs, the slides were fixed and then stained with acridine orange (40 µg/ml) and covered with cover glasses.

Slide analysis
Observation method
The observation of slides was performed with blind method and PCE to NCE ratio was examined by optical microscope at over 400× magnification. The observation of MNPCEs was performed by fluorescence microscope equipped with FITC filter.

Criteria
The PCE/NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring 200 erythrocytes per animal. The micronuclei frequency (expressed as percent micronucleated cells) was determined by analyzing the number of MNPCEs from 2000 PCEs per animal. In case of observation by fluorescence microscope, PCE was counted cells which appeared as red-fluorescence color without nucleus by acridine orange staining. NCE stained with acridine orange was appeared as only shadow entity without fluorescence. In case of observation by optical microscope, PCE stained with Giemsa staining solution was counted cells appeared as purple or blue color. NCE stained with Giemsa staining solution was counted cells appeared as pink color. According to criteria in micronucleus judgement, the largest size was defined as 1/2 size of erythrocyte diameter and the smallest size was defined as up to the limit of identification. Shape included circle, donut, semi-circle. Color was defined as same color as nucleus of cell.

Observation of general symptoms
It was conducted once at the administration date and autopsy date for observation of dead and abnormal sign of test animals.

Measurement of body weight
The body weight of the animals was measured before the administration and autopsy
date.
Evaluation criteria:
In case PCE/(PCE+NCE) ratio (Mean±SD) is more than 0.1, test is judged to be reasonable. It is determined as a positive result if there is dose-related increase or
statistically significant increase in frequency of MNPCE (MNPCE/2000PCEs) in at least single dose group.
Statistics:
Frequency of MNPCE (MNPCE/2000PCEs), PCE/(PCE+NCE) ratio
1) Comparison between negative control and treated group: ANOVA test
2) In case of P<0.05 at 1), verification of capacity correlation : Linear logistic regression
3) Comparison between negative control and positive control: ANOVA test

Weight
1) Weight of each individual at the time of autopsy: ANOVA test
2) Statistically significant case in 1) : Dunnett T3 or Duncan's multiple range test

The result of statistical evaluation is regarded as significant when the P value is less than 0.05. statistical analysis was performed with SPSS 12.0 program.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Micronuclei frequency and cytotoxicity
The frequencies of MNPCEs in 2000 PCEs per animal were 0.23 %, 0.14 %, 0.13 %, 0.20 % and 9.59 % in order of negative control group, 16, 32, 63 mg/kg administration group and positive control group. There was no statistically increase in frequency of PCEs having micronuclei in administration group compared to negative control group. There was clear statistically significant increase in positive control group compared with negative control group in frequency of MNPCE (p<0.01).
The PCE ratios of 200 erythrocytes (PCE+NCE), as an index of cytotoxicity, were 0.48, 0.52, 0.49, 0.54 and 0.36 in order of negative control, 16, 32, 63 mg/kg group and positive control. There was no significant decrease in PCE/(PCE+NCE) ratio compared to negative control group.

Clinical sign
As a result of observation, no special abnormality was observed compared to negative control group.

Body weight
There was no statistically change in body weight at any group compared to negative control group.

Applicant's summary and conclusion

Conclusions:
It was concluded that, under the condition of this study, test substance ESA did not induce micronucleus in mice bone marrow.
Executive summary:

The test substance, ESA, was evaluated for its potential to induce micronucleus in bone marrow of intraperitoneal administrated mice.

Animals were about 8 weeks old at administration of test substance. The test substance was intraperitoneally administered to the mice twice with a 24-hour interval, three dose levels (16 ㎎/㎏, 32 ㎎/㎏, 63 ㎎/㎏) and then frequencies of MNPCEs and cytotoxicity (PCE ratio in the erythrocytes) were evaluated by collecting bone marrow at 18~24 hours after administration of test substance. As a results of counting the frequency of MNPCEs of 2000 PCEs, there was no statistically significant increase at any dose groups compared to negative control group. Also, there was statistically significant increase in positive control group compared to negative control group (p<0.01). The PCEs ratio of 200 erythrocytes (PCE/(PCE+NCE)), as a index of cytotoxicity, did not significantly decrease in PCE/(PCE+NCE) ratio compared to negative control group.

It was concluded that, under the condition of this study, test substance ESA did not induce micronucleus in mice bone marrow.