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

Genetic toxicity: in vitro

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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

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

Method

Species / strain

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)

Controls

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

Results and discussion

Test results

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

Applicant's summary and conclusion

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.