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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2018- November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An in vivo study was conducted because an experimental study according to OECD 442E was not applicable, and the results obtained from OECD 442D and OECD 442C studies were not conclusive for classification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Name: Oxazolin (A 45 A)
Chemical name (name used in the study): 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol
Batch No.: UR010-076
Molecular Formula: C23H43NO2
Molecular Weight: 365.59 g/mol

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca Ola Hsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90. Hungary
- Females: yes, nulliparous and non-pregnant
- Hygienic level at arrival: SPF
- Age at study initiation: 12 weeks old (at start of both Dose Range Finding tests-DRFs)
- Weight at study initiation: 19.4 – 21.5 g
- Diet (e.g. ad libitum): ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice
- Water (e.g. ad libitum): ad libitum
- Acclimation period (Main Test): 7 days
- Acclimation period (DRF): 28 days prior to the first DRF, 21 days prior to the second DRF
- Indication of any skin lesions:
- Animal health: Only healthy animals (and not showing any sign of skin lesion) were used.

ENVIRONMENTAL CONDITIONS

Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. polypropylene/polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The test item (formulated in AOO) was tested at four test concentrations (10 %, 5 %, 2.5 % and 1 % w/v).
No. of animals per dose:
4 animals per dose (in total 24 animals*)
* Including control animals shared with the concurrent LLNAs. Number of shared animals: 8.
Details on study design:
The pooled treatment group approach was used in this test.
The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and two consecutive Dose Range Finding tests (DRFs).
The liquid test item was adequately miscible in Acetone : Olive oil 4:1 (v/v) mixture (AOO) or N,N-Dimethylformamide (DMF) (standard vehicles which are most preferred in the LLNA). Formulations prepared with both vehicles were evaluated in the DRFs to find the most adequate vehicle and the highest applicable concentration. Symptoms of adverse effects were observed in both vehicles at high test concentrations (irritation in AOO, irritation and systemic toxicity in DMF) in the DRFs. Based on the results AOO was more adequate vehicle than DMF and the highest applicable non-toxic, non-irritant concentration was 10 % (w/v) in AOO. According to this the test item was examined in the main test as 10 %, 5 %, 2.5 % and 1 % (w/v) formulations in AOO.
Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and a negative control group dosed with the vehicle of both the positive control and the test item (AOO) were employed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Significance of the dose-response was evaluated by linear regression using the calculated SI values. All calculations were made by Microsoft Excel Software.

Results and discussion

Positive control results:
- Visually larger lymph nodes compared to the vehicle control (AOO)
- No mortality, cutaneous reactions or signs of toxicity
- calculated SI value: 20.9.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
10%
Cellular proliferation data / Observations:
Visually larger lymph nodes compared to the vehicle control (AOO) were observed in the positive control group and in the 10 % (w/v) dose group. Appearance of the lymph nodes was normal in the negative control group (AOO) and in the other test item treated groups (5 %, 2.5 % and 1 %, w/v). Significant lymphoproliferative response (SI ≥ 3) was observed for the test item at all tested concentrations.
The observed stimulation index values were 20.4, 12.0, 4.6 and 4.1 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. Significant dose-response correlation was observed (p = 0.01, r = 0.99).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol tested at the maximum feasible (non-toxic, non-irritant) concentration of 10% (w/v) and also at concentrations of 5 %, 2.5 % or 1 % (w/v) as formulations in a suitable vehicle (Acetone : Olive oil 4:1 (v/v) mixture, AOO) was shown to have skin sensitization potential in the Local Lymph Node Assay in mice.
Executive summary:

The aim of this study was to determine the skin sensitization potential of 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol following dermal exposure in the Local Lymph Node Assay in mice. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and two consecutive Dose Range Finding tests (DRFs).

The liquid test item was adequately miscible in Acetone : Olive oil 4:1 (v/v) mixture (AOO) or N,N-Dimethylformamide (DMF). Formulations prepared with both vehicles were evaluated in the DRFs to find the most adequate vehicle and the highest applicable concentration. Symptoms of adverse effects were observed in both vehicles at high test concentrations (irritation in AOO, irritation and systemic toxicity in DMF) in the DRFs. Based on the results AOO was more adequate vehicle than DMF and the highest applicable non-toxic, non-irritant concentration was 10 % (w/v) in AOO. According to this the test item was examined in the main test as 10 %, 5 %, 2.5 % and 1 % (w/v) formulations in AOO.

Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and a negative control group dosed with the vehicle of both the positive control and the test item (AOO) were employed.

The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 20.9). Thus, confirming the sensitivity and validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.

Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the control was noted for 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol at the applied test concentrations. Significant dose-response correlation was observed. The observed stimulation index values were 20.4, 12.0, 4.6 and 4.1 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Significant dose-response correlation was observed (p = 0.01, r = 0.99, evaluated by linear regression using the SI values).

According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation (indicated by an SI ≥ 3) at the maximum feasible concentration of 10 % (w/v) and also at lower concentrations as well as the significant dose-response correlation are considered as evidence that 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is a skin sensitizer.