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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

The Chinese Hamster Lung(CHL/IU) cell line was used. Modal chromosome number of CHL/IU cell line is 25 and doubling time is about 15 hours. The CHL/IU cell line has a high detection sensitivity, is commonly used in in vitro chromosomal aberration test and recommended in the regulatory guidelines.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

2.6. Metabolic activation system
2.6.1. S9 fraction
Species : Rat
Sex : Male
Strain : Sprague-Dawley
Supplier : Molecular Toxicology, Inc.
Storage condition : Frozen(-20 °C below)
Product No. : 11-01L
Lot No. : 4504
Inductive material : Aroclor 1254-induced rat liver S-9
2.6.2. S9 cofactor
Name : Cofactor Ⅰ
Manufacturer : Genogen Co., Ltd.
Lot No. : 220330-I
Storage condition : Frozen(-70±20 °C)
2.6.3. Concentration of S9 Mix
Composition of S9 Mix mL (Final concentration)
S9 fraction 1 (10 % v/v S9)
Cofactor I 0.4 mol/L MgCl2 0.2 (8 μmol/mL)
1.65 mol/L KCl 0.2 (33 μmol/mL)
1.0 mol/L glucose-6-phosphate 0.05 (5 μmol/mL)
0.1 mol/L NADPH 0.4 (4 μmol/mL)
0.1 mol/L NADH 0.4 (4 μmol/mL)
0.2 mol/L sodium phosphate buffer(0.2 M SPB, pH7.4) 5.0 (100 μmol/mL)
Purified water 2.75 -
Total volume 10


S9 Mix was prepared by mixing the S9 fraction and cofactor, but mixing so that the concentration of S9 fraction is 10 %, and then was used while maintaining the refrigerated state(-1~10 °C). S9 Mix was treated with 0.5 mL/5 mL total volume/T-25 flask and used at a concentration of 1%(v/v) in the final medium. Enzyme activity was confirmed by chromosomal aberration of B[a]P.

Test concentrations with justification for top dose:

Based on results in the concentration range finding study, highest concentration indicating 45±5 % RICC was selected as the highest concentration in all treatment series in the main study. It was serially diluted to consist of three concentration levels by the common ratio of 2. The prepared test substance was dissolved in the vehicle. The negative and positive control group were added. The positive control was treated at 5 μg/mL of B[a]P in the 6-hour treatment series in the presence of metabolic activation system. And, the positive control was treated at 0.2 μg/mL of 4NQO in the 6-hour treatment series and 24-hour treatment series in the absence of metabolic activation system. After preparing specimen from all treatment series, metaphase cells were checked to determine whether test substance induce genotoxicity. Concentrations in the main study are presented in the table below.


Series S9 Mix Concentration(μg/mL)
6+S + 27.5, 55, 110
6-S - 3.8, 7.5, 15
24-S - 2.5, 5, 10

Vehicle / solvent:

Sterile distilled water(SDW)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Evaluation criteria:

If the test substance meets all the following criteria, it was judged as positive, and if it does not meet all criteria, it was judged as negative.
- When at least one treatment group of test substance shows a statistically significant increase in the frequency of the metaphase cells with structural or numerical chromosomal aberration compared to the negative control group.
- When increases of frequency of metaphase cells with structural or numerical chromosomal aberration in the treatment group of test substance is concentration-related
- When at least one treatment group of test substance is outside the 95 % confidence range of the historical control data of the negative control group.

Statistics:

Statistical analysis was performed by a chi-squared test using the SPSS program for the frequency of the metaphase cells(excluding gaps) with structural and numerical chromosomal aberration in the negative control group, treatment group of test substance and positive control group.
The result was judged to be significant when p<0.05

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

All validity criteria of this test were fulfilled.
In the all treatment series, there was a statistically significant increase of metaphase cells with structural chromosomal aberration when compared to the negative control group(p<0.05), and concentration-related increase was observed(p<0.05). Also, frequency of metaphase cells with structural chromosomal aberration was outside the 95 % confidence range of the historical negative control data.
In conclusion, the test substance, KF EPIOL-PE311(GPGE), was considered as inducing chromosomal aberration in the chinese hamster lung cells under the present study conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

metabolic active system(S9 Mix, +S9) or not(-S9)

Test concentrations with justification for top dose:

Dose range finding study was the highest dose of the test substance was selected as 5000 μg/plate dose level, the highest dose was sequentially diluted by a common ratio of 3 to produce total 7(6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 μg/plate) dose levels.

Vehicle / solvent:

Dimethyl sulfoxide(DMSO)

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene(2-AA), N-Methyl-N’-nitro-Nnitrosoguanidine (MNNG)

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Growth inhibition and precipitation were not observed in all strains in the presence and absence of a metabolic activation.

Conclusions:

In conclusion, the test substance, 1,2,3-tris(2,3-epoxypropoxy)propane was considered as have reverse mutagenic potential of bacterial strain under the present study conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records

Reference

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)

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

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

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.

Dose / conc.:

250 mg/kg bw (total dose)

Remarks:

(male)

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

(male/female)

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

(male/female)

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)

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.

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).

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)