Fatty acids, tall-oil, compds. with oleylamine

Skin sensitisation

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

of 2010

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS (including 10 animals for preliminary investigation + 20 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): 8 to 12 weeks.
- Weight at treatment start (1st induction): Minimum 16.4 g, maximum 22.0 g
- Housing: 2 animals per cage in polycarbonate cages inside a barriered rodent facility.
- Bedding material: Autoclaved 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.

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 WS400101 in vehicle (% w/v):
- Pre-screen Test (2 females/dose level): 1, 10, 25, 50, 100
- Main Study (4 females/dose level): 1, 2.5, 5

Induction administration at the following concentration of Hexyl cinnamic aldehyde (positive control) in vehicle (% v/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 WS400101 is miscible with 4:1 v/v acetone:olive oil at 100% w/v forming a pale yellow/orange liquid suitable for dose administration.

TREATMENT PREPARATION AND ADMINISTRATION
- Pre-screen Test
In view of edema formation or increases in ear thickness > 25% after topical treatment with WS400101 at 10% w/v and higher concentrations, 5% w/v was selected as high dose level for the main study. In animals dosed at 1% w/v the increase in ear thickness was less than 25%.

- 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 test or positive control substance concentrations listed above in the field LLNA – Concentration. All formulations were prepared on each day of administrationusing acetone:olive oil (v/v 4:1) as the vehicle 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.

Results and discussion

Positive control results:

A stimulation index (SI) of 7.0 was attained in a concomittant positive control assay with the same strain of mice (CBA/Ca) in response to 25% v/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 vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

1.0%

Remarks on result:

other: see Remark

Remarks:

Stimulation Index (SI) values for the experimental groups treated with 1, 2.5, and 5 % w/v test substance dilutions were 1.2, 4.2 and 10.4, respectively. From the stimulation indices attained at the two lower doses an EC3* of 1.9% w/v was calculated by linear interpolation. *EC3 = estimated concentration needed to produce a stimulation index of 3. Reference: Ryan CA, Chaney JG, Gerberick GF, Kern PS, Dearman RJ, Kimber I & Basketter DA 2007, Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutan. Ocul. Toxicol. 26(2): 135-145.

Results 2

Key result

Parameter:

SI

Value:

4.2

Test group / Remarks:

2.5%

Results 3

Key result

Parameter:

SI

Value:

10.4

Test group / Remarks:

5.0%

Results 4

Key result

Parameter:

EC3

Test group / Remarks:

would correspond to 1.9%

Remarks on result:

other: calculated by linear interpolation

Any other information on results incl. tables

There were no deaths, no signs of ill health or toxicity and no signs of local irritation over the treated area during the main study. Greasy fur was noted for all control and test animals following each dosing occasion. In the main study, this finding had resolved in all vehicle control and test animals by Day 4 and in the positive control animals by Day 6. This finding was not attributable to the test substance itself but was considered to be related to unoccluded dermal administration of a liquid formulation/vehicle.

Bodyweight loss, marginal in degree, was recorded in one main study animal of the 2.5% w/v and, slight to marked in degree, in animals treated at 25% w/v and higher test substance concentrations in the pre-screen test. All other animals of the main study and pre-screen test gained bodyweight.

During the pre-screen test, increases in ear thickening by more than 25% were recorded in all animals treated with WS400101 at 10% w/v and higher concentrations, whereas in animals treated at 1% w/v an increase in ear thickness was not evident or less than 25%. No edema were observed up to the highest concentration of 100%.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria