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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

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 Chromosomal Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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)

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 fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

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.

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.

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:

CD-1

Details on species / strain selection:

2.5.2. Reason for test system selection
The ICR mice are widely used in In vivo micronucleus study, and a large amount of historical data have been accumulated, facilitating the interpretation and evaluation of the test results easy.
2.5.3. Quarantine and acclimation
At receipt, all animals were recorded clinical signs and weight. After receipt, clinical signs were to be observed once a day during the acclimation period of quarantine 3 days and acclimation 4 days under the environment of CentralBio's animal room 20. At the end of the acclimation, measured weight and checked clinical signs and weight changes. And then, Only healthy individuals were used in the study.
2.5.4. Animal and cage identification
Individually identification of animals was carried out by marking each individual number on tail using an oil-based red pen during the acclimation period and blue pen during test period. Cages were identified by attaching an individual identification card.
2.5.5. Group separation
After acclimation, healthy animals were grouped using a randomization method based on body weight. At the time of group separation, uniformity was checked based on average body weight and standard deviation.
2.5.6. Remnant animal
The remnant animals were euthanized using CO2 gas on the day of necropsy.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain : Mouse SPF, Hsd:ICR(CD-1®)
Breeder/Supplier : KOATECH Co., Ltd. (181-21 Jeonwi-ro, Jinwei-myeon, Pyeongtaek-si, Gyeonggi-do, Republic of Korea, 17711)


Step Age (Week) Sex Number of animals Range of body weight

Animals at acquisition
Dose range finding test I 7 Male 17 28.1~33.4 g
7 Female 17 23.0~27.7 g
Dose range finding test II 7 Male 17 29.9~33.5 g
7 Female 17 24.2~26.3 g
Main test 7 Male 33 28.6~32.9 g

At administration
Dose range finding test I 8 Male 15 1st,2nd administration (day1): 28.5~33.1 g
3nd,4nd administration (day2): 28.9~34.4 g
8 Female 15 1st,2nd administration (day1): 23.0~28.8 g
3nd,4nd administration (day2): 23.1~28.8 g
Dose range finding test II 8 Male 15 1st,2nd administration (day1): 33.2~35.6 g
3nd,4nd administration (day2): 30.7~36.2 g
8 Female 15 1st,2nd administration (day1): 24.3~28.3 g
3nd,4nd administration (day2): 24.2~28.3 g
Main test 8 Male 30 1st,2nd administration (day1): 31.7~35.1 g
3nd,4nd administration (day2): 28.8~35.2 g

Route of administration:

oral: gavage

Vehicle:

Dimethyl sulfoxide (DMSO)

Frequency of treatment:

The negative control substance and test substance were administrated twice divided doses/day at an interval of 2-hour, and administrated two days at an interval of 24-hour, and positive control substance was administrated intraperitoneally once on the day of the second administration of the test substance.

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

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.

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 in the frequency of micronucleated polychromatic erythrocytes.
- When these results are outside the range of the historical negative control data.
- If the micronucleus test result does not meet all criteria, it is judged as negative.

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 Dunnett's 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:

negative

Toxicity:

not specified

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
There were no statistically significant differences in the frequency ratio of polychromatic erythrocytes in all test substance groups compared to the negative control group.
The frequency ratio of polychromatic erythrocytes in the positive control group was significantly decreased compared to the negative control group(p<0.05).
4.3.4. Frequency of micronucleus
The frequencies of micronucleated polychromatic erythrocytes observed in 4,000 polychromatic erythrocytes per group were 0.08±0.03 % in the negative control group, 0.09±0.05 % in the 500 mg/kg B.W. administration group, 0.08±0.04 % in the 1,000 mg/kg B.W. administration group, 0.08±0.04 % in the 2,000 mg/kg B.W. administration group, and 1.96±0.35 % in the positive control group.
The frequency of micronucleated polychromatic erythrocytes in polychromatic erythrocytes was no statistically significant increase in all test substance groups compared to the negative control group, and no dose-dependent increase was observed.

Conclusions:

All validity criteria of this test were fulfilled.
As a result of counting micronucleated polychromatic erythrocytes for 4,000 polychromatic erythrocytes per group, there was no statistically significant increase in all test substance groups compared to the negative control group, and dose-dependent no increase was observed. Also, the frequency of polychromatic erythrocytes with micronucleus was included in the historical negative control data.
In conclusion, the test substance, Fatty acids, C8-18 and C18-unsatd., zinc salts do not induce micronucleus in polychromatic erythrocytes under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)