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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
of 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
of 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

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): 8 to 9 weeks.
- Weight at treatment start (1st induction): Minimum 20.0 g, maximum 21.7 g.
- Housing: Group caging (4 animals/cage) in Type II polypropylene/polycarbonate cages
- Bedding material: Woodbased, 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: 6 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.



Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Induction administrations on Days 1, 2 and 3 at the following concentrations of WS400402 in vehicle (% w/v):
- Pre-screen Test: 25, 50
- Main Study: 0 (vehicle control), 10, 25, 50

Induction administration at the following concentration of Hexyl cinnamic aldehyde (positive control) in vehicle (% w/v):
- Main Study : 25
No. of animals per dose:
Pre-screen Test: 2 female animals per dose level
Main Study, Vehicle Control: 8 female animals (1 group)
Main Study, Test Groups: 4 female animals per dose level
Positive Control: 4 female animals (1 dose group)
Details on study design:
TEST SUBSTANCE SOLUBILITY
WS400402 is a viscous liquid at room temperature, which is not applicable to the animals undiluted. A vehicle trial has demonstrated that WS400402 is soluble in 4:1 v/v acetone:olive oil at 50% w/v suitable for dose administration.

TREATMENT PREPARATION AND ADMINISTRATION
- Pre-screen Test
Administration of WS400402 at 25 or 50% w/v in the selected vehicle did not produce deaths, signs of ill health or systemic toxicity, relevant increases in ear thickness or excessive local irritation over the treated area. The only findings were erythema grade 1 (very slight in degree) confined to Day 4 in all animals. Based on this information 50% w/v of WS400402 was selected as high dose level for the main study.

- 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 substance concentrations listed above in the field "LLNA – Concentration". A group of 8 control females received only the vehicle, acetone:olive oil (v/v 4:1). All formulations were freshly prepared on each day of administration.

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 substance produces a stimulation index (SI) ≥ 3.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data were not statistically analysed.

Results and discussion

Positive control results:
A stimulation index (SI) of 10.6 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.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
41.1
Test group / Remarks:
50% test substance concentration
Key result
Parameter:
SI
Value:
45.8
Test group / Remarks:
25% test substance concentration
Key result
Parameter:
SI
Value:
9.7
Test group / Remarks:
10% test substance concentration

Any other information on results incl. tables

The lymph nodes were visibly larger in animals of the test material treated groups and the positive control group than in vehicle control animals. This is consistent with the sensitization response attained in the treated groups and positive control group.

Premature deaths, signs of ill health or systemic toxicity, or excessive local irritation over the treated area were not evident in the pre-screen or main tests. The only irritation findings recorded were erythema grade 1 (very slight in degree) confined to Day 4 in all animals treated with 25 or 50% w/v test material dilutions in the pre-screen test, and seen on Days 4, 5 and 6 in all animals treated with 50% w/v test material dilution in the main test. Ear punch weight and ear thickness were also unaffected by treatment during the pre-screen test.

Marginal bodyweight loss was seen in a few pre-screen and main test animals in this study, but this was not attributable to treatment with the test material.

Applicant's summary and conclusion

Interpretation of results:
other: sensitising according to Regulation 1272/2008