Fatty acids, C18-unsatd., trimers, 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:

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:

2010

Report date:

2010

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 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.

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 1 animal 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): Eight to twelve weeks.
- Weight at treatment start (1st induction): Minimum 17.5 g, maximum 22.1 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.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Induction on Days 1, 2 and 3 at the following concentrations of WS400151 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
Positive Control: 4 female animals (1 dose group)

Details on study design:

TEST SUBSTANCE SOLUBILITY
A vehicle trial has demonstrated that WS400151 was unsuitable for dosing as supplied, due to its high viscosity. At 50% w/v in the vehicles, acetone:olive oil (4:1 v/v), dimethylformamide (DMF) or methyl ethyl ketone it formed a pale brown emulsion, which separated out very quickly. All of these emulsions were unsuitable for dosing. At 50% w/v in propylene glycol WS400151 formed a pale brown emulsion suitable for dose administration in the LLNA.

TREATMENT PREPARATION AND ADMINISTRATION
- Preliminary Range-finding Test
Administration of WS400151 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 fur was noted post Dose and was 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%
Concomitant with the Main Study a positive control group of 4 female mice received the following concentration in the vehicle, propyplene glycol (v/v):
Group 5 (Positive Control): 25%

The test formulations 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.

Results and discussion

Positive control results:

A stimulation index (SI) of 12.5 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 propylene glycol, 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

Parameter:

SI

Remarks on result:

other: Stimulation Index (SI) values for the experimental groups treated with 10, 25 and 50% w/v test substance dilutions were 8.6, 19.3 and 15.4, respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Overall DPM/lymph node values for the experimental groups treated with 10, 25 and 50% w/v test substance dilutions were 2617.81, 5873.68 and 4683.76 DPM/node, respectively. For the vehicle control group, 304.11 DPM/node were recorded.

Any other information on results incl. tables

There were no deaths and no signs of ill health, toxicity or local irritation over the treated area during the study. Greasy fur was noted for all control and test animals post Dose from Day 1 having completely resolved in all animals of the vehicle control and low dose test substance group by Day 4 and in the positive control group by Day 6. In the mid and high dose test substance groups this sign was evident during the whole study period post dose from Day 1.

Bodyweight loss was recorded for one animal of the mid dose and all animals of the high dose test substance groups. All other main study animals gained bodyweight during the main study.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU