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

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:

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

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.