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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
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
of 2002
Deviations:
yes
Remarks:
Contrary to OECD 429 of 2002, but in accordance with OECD 429 of 2010, scintillation vials were filled with 10 mL of scintillation fluid for 3H-counting. This did not compromise the validity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
of 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS (including 1 animal for preliminary investigation + 16 main study animals).
- Mouse (healthy females only), strain: CBA/Ca with appropriate range of bodyweight at study start.
- Source: Harlan UK.
- Age at treatment start (1st induction): Eight to twelve weeks.
- Weight at treatment start (1st induction): Minimum 17.2 g, maximum 23.8 g
- Housing: Individual housing in polycarbonate cages inside a barriered rodent facility.
- Bedding material: Woodflake bedding.
- Cage enrichment: Nestlets and plastic shelter
- Diet (ad libitum): Standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) containing no added antibiotic,
chemotherapeutic or prophylactic agent.
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before treatment start under laboratory conditions.

Analysis of the batch of diet used and water did not provide evidence of contamination that might have prejudiced the study.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Air changes per hour in the animal room: ca. 15
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 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:
dimethylformamide
Concentration:
Induction on Days 1, 2 and 3 at the following concentrations of WS400152 in vehicle (w/v):
- Preliminary Range-finding Test: 50% (1 female).
- Main Study: 0% (vehicle control, 4 females), 10% (4 females), 25% (4 females), 50% (4 females).
No. of animals per dose:
Preliminary Range-finding Test: 1 female animal
Main Study: 4 female animals per dose
Details on study design:
TEST SUBSTANCE SOLUBILITY
A vehicle trial has demonstrated that WS400152 was unsuitable for dosing as supplied, due to its high viscosity, and that it was imiscible with the vehicle, acetone:olive oil (4:1 v/v). At 50% w/v in dimethylformamide (DMF), WS400152 formed a dark brown thick cloudy suspension which was suitable for dose administration in the LLNA.

TREATMENT PREPARATION AND ADMINISTRATION
- Preliminary Range-finding Test
Administration of WS400152 at 50% w/v to one animal on three consecutive days did not produce death, signs of ill health, toxicity or local irritation over the treated area. Greasy and wet fur along with slight hair loss was noted post Dose on Day 1, slight hairloss being still present at termination on Day 4. Based on this information 50% w/v 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 entire dorsal surface of both ears with 25 μL/ear/day at the following concentrations (w/v) of test substance in the vehicle:

Group 1 (Vehicle Control): 0%,
Group 2 (Low Dose): 10%,
Group 3 (Mid Dose): 25%,
Group 4 (High Dose): 50%

The test substance preparations were prepared on each day of administration and dosed within 4 hours of preparation.

OBSERVATIONS, MEASUREMENTS AND ENDPOINTS (POOLED TREATMENT GROUP APPROACH) DURING THE MAIN STUDY

All animals were checked daily for signs of ill health or toxicity. The ears were also examined daily for signs of irritation. In addition, 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 3H-methyl thymidine diluted in phosphate buffered saline at a nominal dose of 20 µCi per mouse, in order to measure lymphocyte proliferation by radioactive labelling. Five hours afterwards the draining (auricular) lymph nodes were excised and pooled for each experimental group. After precipitating the DNA of the lymph node cells, 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.

Criteria Used to Consider a Positive Response:

The test substance 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.
Positive control results:
A stimulation index (SI) of 7.8 was attained in an about contemporaneous positive control assay with the same strain of mice (CBA/Ca) in response to 25% v/v hexyl cinnamic aldehyde in dimethylformamide (DMF), thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory.
Key result
Parameter:
SI
Value:
1.9
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
5.7
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
7.8
Test group / Remarks:
50%

There were no deaths and no signs of ill health or systemic toxicity during the main study. However, unforeseen dermal irritation (reddening and dry skin abrasions) was seen at 25% w/v having completely resolved in all affected animals by Day 5. In addition, hair loss, slight to moderate in degree, was noted at all treated dose levels having not resolved by termination of the animals on Day 6. Wet fur was noted for all control and test animals post-dose from Day 1 having resolved by Day 6. Dose residue was still present at termination on Day 6 on all animals of the 25 and 50% w/v groups.

Bodyweight loss, marginal in degree, was recorded for one main study animal of the 25 and one of the 50% w/v dose group. All other main study animals gained bodyweight during the main study.

Interpretation of results:
other: sensitising
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a local lymph node assay with mice, the stimulation index (SI) threshold of ≥ 3.0, indicating a positive sensitisation response, was attained in the mid and high dose groups and there was a dose related increase in SI over all groups. The EC3 was calculated to be 14%. Therefore, according to EU classification rules the test substance was classified as “Category 1; subcategory 1B” [Warning: May cause an allergic skin reaction; other (than strong) skin sensitiser] [REGULATION (EC) 1272/2008].

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