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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24-06-2020 to 08-07-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V. Inc, Postbus 6174, 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Housing:
- Diet: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water: ad libitum tap water
- Acclimation period: 5 days minimum
- Indication of any skin lesions/Microbiological status of animals: Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°
- Humidity (%): 45-65
- Air changes (per hr): 8 minimum
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: To:
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25, 50%
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Fully miscible up to 100% in vehicle
- Irritation: Two mice treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days.
- Systemic toxicity: No, but irritation screened animals showed slight weight loss at 100% concentration
- Ear thickness measurements: Yes, 100% caused excessive thickening at 100% concentration.
- Erythema scores: 100% caused irritation beyond acceptable levels and eschar formation..

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index and that a dose response relationship is observed.

TREATMENT PREPARATION AND ADMINISTRATION:Each test group treated by topical application to the dorsal surface of each ear with test item (25 µL/ear/day spread over entire surface (~8 mm dia) for 3 consecutive days. A further group of mice were treated with vehicle alone. Five days after the first application (day 6) 250 µL of phosphate buffered saline containing 20 µCi of 3H-methyl thymidine (equivalent to 79.8 µCi/mL 3HTdR) were injected into tail vein. Mice sacrificed 5 hours later and lymph nodes harvested.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity.
Positive control results:
EC3 = 12.5%. Historic control range 5.7 - 17.6%.
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
The EC3 value could not be calculated, since all S.I.´s up to the maximum tolerable concentration of 50% were below the threshold value of 3.
Parameter:
SI
Value:
1.12
Test group / Remarks:
10% concentration
Parameter:
SI
Value:
1.31
Test group / Remarks:
25% concentration
Parameter:
SI
Value:
2.67
Test group / Remarks:
50% concentration
Remarks on result:
other: maximum tolerated concentration that does not cause irritation
Cellular proliferation data / Observations:
CLINICAL OBSERVATIONS: Top dose group showed slight to well defined ear erythema in all animals by day 3/4 which completely reversed by the day of sacrifice (day 6). No effects seen in any other dose group

BODY WEIGHTS: No adverse effects noted.
Interpretation of results:
GHS criteria not met
Conclusions:
Not a skin sensitiser
Executive summary:

In a guideline mouse LLNA study, the test substance was not found to meet the criteria to be considered as a skin sensitiser when tested up to the maximum tolerated concentration. The study was performed using test item concentrations of 10, 25, and 50%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with 50% test item concentration showed a very slight to well defined erythema of the ear skin (Score 1 to 2) and one animal showed scaly ears although this reversed after a few days of non-treatment.  The animals treated with 10 and 25% test item concentration did not show any signs of local irritation. In this study Stimulation Indices (S.I.) of 1.12, 1.31, and 2.67 were determined with the test item at concentrations of 10, 25, and 50% in acetone/olive oil (4+1 v/v), respectively. As an SI of 3 was not reached, the substance is not considered as a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Justification for classification or non-classification

Substance does not meet the criteria for classification under the GHS system. At the maximum tolerated concentration, the stimulation index (EC3) remained below 3.