Fatty acids, C8-18 and C18-unsatd., zinc salts

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 Chromosomal 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:

Zinc dioctanoate

EC Number:

209-156-6

EC Name:

Zinc dioctanoate

Cas Number:

557-09-5

Molecular formula:

C16H30O4Zn

IUPAC Name:

zinc(II) octanoate

Constituent 2

Reference substance name:

Zinc decanoate

EC Number:

235-909-3

EC Name:

Zinc decanoate

Molecular formula:

C10H20O2.1/2Zn

IUPAC Name:

zinc(II) decanoate

Constituent 3

Reference substance name:

Zinc dilaurate

EC Number:

219-518-5

EC Name:

Zinc dilaurate

Cas Number:

2452-01-9

Molecular formula:

C12H24O2.1/2Zn

IUPAC Name:

zinc(II) dodecanoate

Constituent 4

Reference substance name:

Zinc dimyristate

EC Number:

240-369-7

EC Name:

Zinc dimyristate

Cas Number:

16260-27-8

Molecular formula:

C28H54O4Zn

IUPAC Name:

zinc(II) tetradecanoate

Constituent 5

Reference substance name:

Zinc dipalmitate

EC Number:

225-652-5

EC Name:

Zinc dipalmitate

Cas Number:

4991-47-3

Molecular formula:

C16H32O2.1/2Zn

IUPAC Name:

zinc(II) hexadecanoate

Constituent 6

Reference substance name:

Zinc distearate

EC Number:

209-151-9

EC Name:

Zinc distearate

Cas Number:

557-05-1

Molecular formula:

C18H36O2.1/2Zn

IUPAC Name:

zinc(II) octadecanoate

Constituent 7

Reference substance name:

Zinc dioleate

EC Number:

209-154-5

EC Name:

Zinc dioleate

Cas Number:

557-07-3

Molecular formula:

C18H34O2.1/2Zn

IUPAC Name:

zinc(II) 9-octadecenoate

Constituent 8

Reference substance name:

Zinc dilinoleate

EC Number:

235-870-2

EC Name:

Zinc dilinoleate

Cas Number:

13014-44-3

Molecular formula:

C36H62O4Zn

IUPAC Name:

zinc(II) 9,12-octadecdienoate

Test material form:

solid: bulk

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

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:

2.6.1. 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, the lowest concentration at which precipitation of the test substance was observed until the end of treatment in 6-hour treatment series in the presence of metabolic activation system was set as the highest concentration. In case of 6-hour treatment series and 24-hour treatment series in the absence of metabolic activation system, highest concentration indicating 45±5 % RICC was selected. 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 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 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.

Vehicle / solvent:

Tetrahydrofuran (THF)

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

Test results 1

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Test results 2

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

As a result of the main study, precipitation of test substance was observed at 250 μg/mL in 6-hour treatment series in presence of metabolic activation system at the start and end of the treatment.


In the 6-hour treatment series in the presence of metabolic activation system, the frequencies of metaphase(excluding gap) cells with structural chromosomal aberration were observed to be 0.67, 0.33, 0.33, and 0.00 % respectively for 0, 62.5, 125, and 250 μg/mL treated group. There was no statistically significant increase of metaphase cells with structural chromosomal aberration at any treatment groups when compared to the negative control group, and concentration-related increase was not observed. Also, the frequency of metaphase cells with structural chromosomal aberration was inside the 95 % confidence range of the historical negative control data. The frequencies of metaphase cells with numerical chromosomal aberration were observed to be 0.33, 0.33, 0.33, and 0.00 % respectively for 0, 62.5, 125, and 250 μg/mL treated group. There was no statistically significant increase of metaphase cells with numerical chromosomal aberration at any treatment groups when compared to the negative control group, and concentration-related increase was not observed. Also, the frequency of metaphase cells with numerical chromosomal aberration was inside the 95 % confidence range of the historical negative control data.
In the positive control group(B[a]P 5 μg/mL), the frequency of metaphase(excluding gap) cells with structural chromosomal aberration was 11.00 %, and there was a statistically significant increase when compared to the negative control group(p<0.05). The frequency of metaphase cells with numerical chromosomal aberration was 0.00 %, and there was no statistically significant increase when compared to the negative control group.


In the 6-hour treatment series in the absence of metabolic activation system, the frequencies of metaphase(excluding gap) cells with structural chromosomal aberration were observed to be 0.00, 0.33, 0.33, and 8.67 % respectively for 0, 16.9, 33.8, and 67.5 μg/mL treated group. There was a statistically significant increase of metaphase cells with structural chromosomal aberration at 67.5 μg/mL treated group when compared to the negative control group(p<0.05), and concentrationrelated increase was observed(p<0.05). Also, the frequency of metaphase cells with structural chromosomal aberration was outside the 95 % confidence range of the historical negative control data. The frequencies of metaphase cells with numerical chromosomal aberration were observed to be 0.33, 0.00, 0.00, and 0.00 % respectively for 0, 16.9, 33.8, and 67.5 μg/mL treated group. There was no statistically significant increase of metaphase cells with numerical chromosomal aberration at any treatment groups when compared to the negative control group, and concentration-related
increase was not observed. Also, the frequency of metaphase cells with numerical chromosomal aberration was inside the 95 % confidence range of the historical negative control data.
In the positive control group(4NQO 0.2 μg/mL), the frequency of metaphase(excluding gap) cells with structural chromosomal aberration was 4.67 %, and there was a statistically significant increase when compared to the negative control group(p<0.05). The frequency of metaphase cells with numerical chromosomal aberration was 0.00 %, and there was no statistically significant increase when compared to the negative control group.


In the 24-hour treatment series in the absence of metabolic activation system, the frequencies of metaphase(excluding gap) cells with structural chromosomal aberration were observed to be 0.00, 0.00, 0.00, and 5.33 % respectively for 0, 16.3, 32.5, and 65 μg/mL treated group. There was a statistically significant increase of metaphase cells with structural chromosomal aberration at 65 μg/mL treated group when compared to the negative control group(p<0.05), and concentrationrelated increase was observed(p<0.05). Also, the frequency of metaphase cells with structural chromosomal aberration was outside the 95 % confidence range of the historical negative control data. The frequencies of metaphase cells with numerical chromosomal aberration were observed to
be 0.33, 0.00, 0.00, and 0.00 % respectively for 0, 16.3, 32.5, and 65 μg/mL treated group. There was no statistically significant increase of metaphase cells with numerical chromosomal aberration at any treatment groups when compared to the negative control group, and concentration-related increase was not observed. Also, the frequency of metaphase cells with numerical chromosomal aberration was inside the 95 % confidence range of the historical negative control data.
In the positive control group(4NQO 0.2 μg/mL), the frequency of metaphase(excluding gap) cells with structural chromosomal aberration was 5.33 %, and there was a statistically significant increase when compared to the negative control group(p<0.05). The frequency of metaphase cells with numerical chromosomal aberration was 0.00 %, and there was no statistically significant increase when compared to the negative control group.

Applicant's summary and conclusion

Conclusions:

All validity criteria of this test were fulfilled.
In the 6-hour treatment series and 24-hour treatment series in the absence of metabolic activation system, 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, Fatty acids, C8-18 and C18-unsatd., zinc salts, was considered as inducing chromosomal aberration in the chinese hamster lung cells under the present study conditions.