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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Mouse (healthy females only), strain: CBA/J Rj with appropriate range of bodyweight at study start.
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 Le Genest-St-Isle, France
- Hygienic level at arrival: SPF
- Age at treatment start (1st induction): Nine weeks.
- Weight at treatment start (1st induction): Minimum 20.0 g, maximum 22.9 g.
- Housing: Group caging (4 animals/cage) in Type II polypropylene/polycarbonate cages
- Bedding material: Wood-based, Lignocel Hygienic Animal Bedding,
J. Rettenmaier & Söhne GmbH + Co. KG, Rosenberg, Germany
- Cage enrichment: Glass tunnel tubes
- Diet (ad libitum): Ssniff SM R/M-Z+H, autoclavable complete breeding and maintenance diet for rats and mice,
Ssniff Spezialdiäten GmbH, Soest, Germany.
- Water (ad libitum): Tap water from municipal supply
- Acclimation period: 13 days before treatment start under laboratory conditions.

Water was regularly analysed for contaminants, detailed information on diet and bedding material were provided by the suppliers. Water, diet and bedding material used in the present study were not considered to adversely affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS

Controlled environment, environmental conditions were set at:
- Ventilation, air changes per hour: 15-20
- Temperature (°C): 22 ± 3°C
- Relative Humidity (%): 30 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
There was no mentioning of any deviations from these ranges, which compromised the integrity or validity of the study.



Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Induction administrations on Days 1, 2 and 3 at the following concentrations of WS400102 in vehicle (% w/v):
- Pre-screen Test (2 females/dose level): 25, 50
- Main Study (4 females/dose level): 0 (vehicle control), 5, 10, 25

Induction administration at the following concentration of Hexyl cinnamic aldehyde (positive control) in vehicle (% w/v):
- Main Study (4 females/dose level): 25
No. of animals per dose:
Pre-screen Test: 2 female animals per dose level
Main Study: 4 female animals per dose level
Positive Control: 4 female animals (1 dose group)
Details on study design:
TEST SUBSTANCE SOLUBILITY
A vehicle trial has demonstrated that WS400102 is soluble in 4:1 v/v acetone:olive oil at 100% w/v suitable for dose administration.

TREATMENT PREPARATION AND ADMINISTRATION
- Pre-screen Test
In view of increases in ear thickness of > 30% after topical treatment with WS400102 in both animals at 50% w/v, 25% w/v was selected as high dose level for the main study. In animals dosed at 25% w/v, the increase in ear thickness was considerably less than 25% in both animals (ca. 10%). Mortality, systemic toxicity or erythema skin reactions were not evident during the pre-screen test.

- Main Study
On three consecutive days, groups of 4 female mice were treated by topical application to the dorsal surface of both ears with 25 μL/ear/day at the test or positive control material concentrations listed above in the field "LLNA – Concentration". All formulations were freshly prepared on each day of administration using acetone:olive oil (v/v 4:1) as the vehicle. Negative control animals received the vehicle alone.

OBSERVATIONS, MEASUREMENTS AND ENDPOINTS (POOLED TREATMENT GROUP APPROACH) DURING THE MAIN STUDY
Each animal was checked twice a day (before and after treatment) on Days 1 to 3 and once daily on Days 4 to 6 for signs of ill health or toxicity. At the same intervals, the ears were examined for signs of irritation. In addition, individual bodyweights were recorded on Days 1 (prior to treatment) and 6 (three days after the third induction administration). On Day 6, all animals were injected into the tail vein Tritiated (^3H)-methyl Thymidine (^3HTdR) diluted in sterile phosphate buffered saline at a nominal dose of 20 µCi per mouse, in order to measure lymphocyte proliferation by radioactive labelling. Five hours (± 30 minutes) afterwards the draining (auricular) lymph nodes were excised and pooled for each experimental group. After sample processing and precipitating macromolecules (DNA) of the lymph node cells in 5% trichloracetic acid (TCA), radioactivity measurements were performed on Day 7. Radioactivity was expressed as the number of radioactive disintegrations per minute (DPM). The ratio of the proliferation (reflected by the magnitude of measured DPM/node) in treated groups to that in the vehicle control group, termed the stimulation index (SI) or test/control ratio, was subsequently calculated for each group. Background ^3HTdR levels were also measured in two 1 mL aliquots of 5% TCA and accounted for in the study results.

Criteria Used to Consider a Positive Response:
The test material is regarded as a sensitizer if at least one concentration of the test material produces a stimulation index (SI) ≥ 3.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data were not statistically analysed.
Positive control results:
A stimulation index (SI) of 4.6 was attained in a concomittant positive control assay with the same strain of mice (CBA/J Rj) in response to 25% w/v hexyl cinnamic aldehyde in acetone:olive oil (4:1 v/v), thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory. In addition, the disintegrations per lymph node value attained was within the historical reference range. Mortality, cutaneous reactions or signs of toxicity were not evident in the postive control group. Lymph nodes of the positive control group were larger than normal (vehicle control) which is consistent with the sensitization response attained in this group.
Key result
Parameter:
SI
Value:
0.4
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.3
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
0.5
Test group / Remarks:
25%

During the pre-screen test, treatment with 50% w/v test material dilution elicited increases in ear thickness of > 30% in both animals, whereas increases of only approximately 10% were elicited by 25% w/v. Mortality, systemic toxicity or erythema skin reactions were not evident and ear punch weights were unaffected during the pre-screen test.

During the main study, deaths, signs of ill health or toxicity or signs of local irritation over the treated area were not evident. Larger than normal (vehicle control) lymph nodes observed in the 25 (w/v) % test material group and the positive control group were not considered to represent a sensitization response in the test material treated group, because the disintegration per lymph node (DPN) values of all test material treated groups were well within the historical reference range of vehicle controls and because of the absence of any increase in stimulation indices in the test material treated groups in the present study.

Marginal to slight bodyweight loss was seen in a number of animals from all groups in this study, but this was not attributable to treatment with the test material.

Interpretation of results:
other: not sensitising
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the low stimulation indices attained in the local lymph node assay, WS400102 is considered not to be a skin sensitiser and does not warrant any classification regarding skin sensitisation according to European classification rules [REGULATION (EC) 1272/2008].