1,2,3-tris(2,3-epoxypropoxy)propane

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1,2,3-tris(2,3-epoxypropoxy)propane

EC Number:

236-211-1

EC Name:

1,2,3-tris(2,3-epoxypropoxy)propane

Cas Number:

13236-02-7

Molecular formula:

C12H20O6

IUPAC Name:

1,2,3-tris(2,3-epoxypropoxy)propane

Constituent 2

Reference substance name:

N/A

Cas Number:

178667-42-0

Molecular formula:

C12H24O8

IUPAC Name:

N/A

Test material form:

liquid

Test animals

Species:

mouse

Strain:

ICR

Details on species / strain selection:

The ICR mice were recommended by the testing guideline and had been widely used in this type of study. A large amount of historical control data have been accumulated to aid the interpretation and evaluation of the test results.

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Dimethyl sulfoxide(DMSO)

Duration of treatment / exposure:

2 days

Frequency of treatment:

The negative control substance and test substance were administered twice, divided dose/day at an interval of 2 hours, and administrated two days at an interval of 24 hours. The positive control substance will be administered intraperitoneally once on the day of the second administration day of the test substance.

Post exposure period:

3.3.2.1. Clinical signs observation
On the day of administration, two observations were carried out including within 30 minutes after administration. On the day of autopsy, one observation was carried out before bone marrow collection.
3.3.2.2. Body weight
Body weight was measured at receipt of animals, on the day of test group composition, the day of administration (before administration), and the day of bone marrow collection.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

(male)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

(male/female)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

(male/female)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

(female)

No. of animals per sex per dose:

Dose range finding test : 3 each per dose (male/female)
Main test : 6 each per dose (male/female)

Control animals:

yes

Positive control(s):

Cyclophosphamide(CPA)

Examinations

Tissues and cell types examined:

3.4. Collection and specimen production of bone marrow cells
Bone marrow cells were collected within 24 hours after the second administration of the test substance. Both ends of the femur were cut with scissors and extracted, and bone marrow cells were harvested by perfusion with fetal bovine serum(FBS). The harvested bone marrow cells were centrifuged at 1,000 rpm for 5 minutes. After removing the supernatant, the bone marrow cells were spread on a slide glass and dried sufficiently at room temperature. Two specimens were prepared for each animal. After drying, specimens were fixed for about 8 minutes with methanol.
3.5. Staining of Specimen
The specimens fixed with methanol were stained with 5 % Giemsa staining solution for approximately 20 minutes. The stained specimens were washed with buffer phosphate-buffered saline(PBS). For light discoloration of the specimens stained with Giemsa solution, specimens were treated in 0.004% citric acid solution for 12 seconds, and then thoroughly washed with distilled water so that no citric acid solution remained.
3.6. Reading of Specimen
According to the rule, blind method was applied. To obtain the ratio of polychromatic erythrocytes among all erythrocytes[PCE/(PCE+NCE)], each animal was counted so that the sum of polychromatic erythrocytes(PCE) and normochromic erythrocytes(NCE) becomes 500. To obtain the ratio(MNPCE/4000PCE) of micronucleated polychromatic erythrocytes (MNPCE), at least 4,000 polychromatic erythrocytes per animal were counted and calculated.

Evaluation criteria:

If the test substance meets the following criteria, it was judged as positive.
- When there is a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes in one or more test substance group compared to the negative control group.
- When this increase is dose-dependent on the frequency of micronucleated polychromatic erythrocytes.
- When these results are outside the 95 % confidence range of the historical negative control data.

Statistics:

Statistical processing was performed using the SPSS program for the frequency of micronucleated polychromatic erythrocytes, the frequency of polychromatic erythrocytes among total red blood cells, and changes in body weight. According to the result of statistical processing. It was determined that there is statistical significance when p<0.05. ANOVA(One-way analysis of variation) was performed to compare the means between groups, Dunnett test was performed if equivariability was satisfied through Levene’s test, and Dunnetts T3 test was performed if eqsssuivariability was not satisfied.
Linear regression confirmed the significance of dose-dependent of frequency of micronucleated polychromatic erythrocytes, and the frequency of polychromatic erythrocytes among total red blood cells.
Student t-test was performed for frequency of polychromatic erythrocytes among total red blood cells and frequency of micronucleated polychromatic erythrocytes in the negative and positive control groups to confirm their statistical significance.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

no effects

Additional information on results:

4.3.1. Clinical signs
In all test substance groups, no clinical signs and death were observed following administration of the test substance.
4.3.2. Body weight
No significant body weight change was observed in all dose groups compared with the negative control group.
4.3.3. Frequency of polychromatic erythrocyte in total erythrocyte
In males, the frequency ratio of polychromatic erythrocytes was statistically significantly decreased in the 1,000 mg/kg dose group compared to the negative control group(p<0.05). But, dose-dependent no increase was observed, and there was inside the range of historical negative control data.
In females, the frequency ratio of polychromatic erythrocytes was statistically significantly decreased in at 2,000 mg/kg dose group compared to the negative control group(p<0.05), and dose-dependent decrease was observed(p<0.05), and there was an outside the range of historical negative control data.
In addition, the frequency ratio of polychromatic erythrocytes in the positive control group was statistically significantly decreased compared to the negative control group(p<0.05).
4.3.4. Frequency of micronucleus
In males, the frequencies of micronucleated polychromatic erythrocyte observed in 4,000 polychromatic erythrocytes per group were 0.06±0.03 % in the negative control group, 0.92±0.27 % in the 250 mg/kg B.W. dose group, 0.95±0.24 % in the 500 mg/kg B.W. dose group, 1.28±0.39% in the 1,000 mg/kg B.W. dose group, and 1.31±0.16 % in the positive control group.
The frequency of micronucleated polychromatic erythrocytes in polychromatic erythrocytes was statistically significantly increased above 250 mg/kg B.W. dose groups compared to the negative control group(p<0.05) and dose-dependent increase was observed(p<0.05), and there was outside the range of historical negative control data.
Also, the frequency of micronucleated polychromatic erythrocytes in polychromatic erythrocytes was significantly increased in the positive control group when compared to the negative control group(p<0.05).
In females, the frequencies of micronucleated polychromatic erythrocyte observed in 4,000 polychromatic erythrocytes per group were 0.05±0.03 % in the negative control group, 1.30±0.60 % in the 500 mg/kg B.W. dose group, 1.55±0.30 % in the 1,000 mg/kg B.W. dose group, 1.88±0.61% in the 2,000 mg/kg B.W. dose group, and 1.35±0.19 % in the positive control group.
The frequency of micronucleated polychromatic erythrocytes in polychromatic erythrocytes was statistically significantly increased above 500 mg/kg B.W. dose groups compared to the negative control group(p<0.05) and dose-dependent increase was observed(p<0.05), and there was outside the range of historical negative control data.
Also, the frequency of micronucleated polychromatic erythrocytes in polychromatic erythrocytes was significantly increased in the positive control group when compared to the negative control group(p<0.05).

Applicant's summary and conclusion

Conclusions:

All validity criteria of this test were fulfilled.
As a result of counting micronucleated polychromatic erythrocyte for 4,000 polychromatic erythrocyte per group, there was a statistically significantly increased in above the lowest dose group of males and females compared to the negative control group, and dose-dependent increase was observed, and there was an outside range of historical negative control data(p<0.05).
In conclusion, the test substance, KF EPIOL-PE311(GPGE), does induce micronucleus in polychromatic erythrocytes under the present test conditions.