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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

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 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

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

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Species and strain:CBA/JCrHsd mice
Microbiological grade:Specific Pathogen Free(SPF)
Sex:Female
Breeder/Supplier:KOATECH_Korea(181-21 Jinwi-ro, Jinwi-myeon, Pyeongtaek-si, Gyeonggi-do, Republic of Korea, 17711)

Step Age(Week) Sex Number of animals
Animals at acquisition (Pre-screen test) 8 Female 6
At administration (Pre-screen test) 9 Female 5
Animals at acquisition (Main test) 8 Female 25
At administration (Main test) 9 Female 24

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

The concentrations of 10.0 %(w/v), 5.0 %(w/v) and 2.5 %(w/v) were selected in the main test based on the result of the pre-screen test. In addition, additional negative and positive controls were set up to administer Acetone:Olive oil (4:1, v/v), MEK and Eugenol 25.0 %(w/v) respectively to compare with the test substance treatment groups.

No. of animals per dose:

Main test: 4

Details on study design:

Test substance Group Dose Sex Number of animals Animal number
Acetone:Olive Oil 4:1(v/v) G1 – Female 4 1~4
MEK G2 – Female 4 5~8
Eugenol G3 25.0 %(w/v) Female 4 9~12
Fatty acids, C8-18 and C18-unsatd., zinc salts
G4 2.5 %(w/v) Female 4 13~16
G5 5.0 %(w/v) Female 4 17~20
G6 10.0 %(w/v) Female 4 21~24


3.2.3. Experimental schedule
3.2.3.1. Treatment of substance: Day 1~Day 3
The test substance, vehicle, and positive control were treated over the entire dorsal surface of the ear once a day up to Day 3 using a micropipette at 25 μL/ear to each corresponding group.
3.2.3.2. No treatment: Day 4
No Treatment
3.2.3.3. BrdU solution injection: Day 5
Intraperitoneal(IP) injection 0.5 mL(5 mg/mouse) of BrdU at a concentration of 10 mg/mL solution approximately 24 hours prior to the extraction of the auricular lymph node.
3.2.3.4. Auricular lymph node extraction: Day 6
Approximately 24 hours after BrdU injection, all animals were euthanized with CO2 gas. The auricular lymph nodes were excised from each mouse and gently disassembled using 70 micro-mesh(SPL, Cell Strainer) and distributed with PBS(1×) for a total volume of 15 mL.
3.2.3.5. Cellular proliferation measurement: Day 6
BrdU intensity was measured by ELISA using a commercial kit. Briefly, 100 μL of the lymph node cells(LNC) suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and then the substrate solution was added and allowed to produce chromogen. Absorbance at 370 nm(Emission wavelength, em) with a reference wavelength of 492 nm(Reference wavelength, ref) was measured.

Positive control substance(s):

eugenol (CAS No 97-53-0)

Statistics:

The SPSS Statistical Program(IBM, Ver. 25) was used to conduct statistical analysis of the body weights and the ear thickness. After testing the normality of the variance(Anderson Darling or other appropriate method), statistics was processed as One-way ANOVA test. Then, homogeneity of variances was verified with Levene’s test. In case of homogeneity, the Dunnett test was conducted as a post-hoc test and in case of heteroscedasticity, the Dunnett T3 was conducted as a post-hoc test to confirm the significance with the control group. P-values<0.05 was considered statistically significant.

Results and discussion

Positive control results:

The Stimulation index(SI) of the positive control group, Eugenol 25.0 %(w/v), was calculated as 2.414 and the result was confirmed as the SI of the positive control met the reference of "SI≥1.6".

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.702

Test group / Remarks:

2.5 %(w/v)

Results 2

Key result

Parameter:

SI

Value:

ca. 1.926

Test group / Remarks:

5.0 %(w/v)

Results 3

Key result

Parameter:

SI

Value:

ca. 2.545

Test group / Remarks:

10.0 %(w/v)

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

This study was conducted to assess the skin sensitization potential of the test substance, Fatty acids, C8-18 and C18-unsatd., zinc salts, by measuring the proliferation of lymphocytes in the auricular lymph nodes through 5-Bromo-2'-Deoxyuridine content analysis following applications to female CBA/J mice.
The results are as follows.
The Stimulation index(SI) of the positive control group, Eugenol 25.0 %(w/v), was calculated as 2.414 and the result was confirmed as the SI of the positive control met the reference of "SI≥1.6".
The SI index of the test substance for 2.5 %(w/v), 5.0 %(w/v) and 10.0 %(w/v) treated group were determined as 1.702, 1.926 and 2.545 respectively, and all test substance groups were confirmed to be positive as the results.
In conclusion, the test substance, Fatty acids, C8-18 and C18-unsatd., zinc salts, was considered to cause skin sensitization at 2.5 %(w/v), 5.0 %(w/v) and 10.0 %(w/v).