Fatty acids, C14-18 and C16-18-unsatd., maleated, reaction products with oleylamine

Endpoint summary

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:

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.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2010

Deviations:

no

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 2 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 17.5 g, maximum 21.0 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.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Induction administrations on Days 1, 2 and 3 at the following concentrations of WS400107 in vehicle (% w/v):
- Pre-screen Test (2 females): undiluted, i.e. as supplied
- Main Study (4 females/dose level): 25, 50, as supplied

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 (1 dose group)
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 WS400107 is miscible with the vehicle, acetone:olive oil (4:1 v/v) forming a thick orange liquid suitable for dose administration.

TREATMENT PREPARATION AND ADMINISTRATION
- Pre-screen Test
Administration of undiluted WS400107, i.e. as supplied, did not produce deaths, signs of ill health, toxicity, relevant increases in ear thickness or local irritation over the treated area. Based on this information, WS400107 'as supplied' 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 test or positive control substance concentrations listed above in the field LLNA – Concentration. All formulations were prepared on each day of administration using acetone:olive oil (4:1 v/v) as the vehicle and dosed within 4 hours of preparation. Negative control animals received the vehicle alone.

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 cellson Day 7, radioactivity measurements were performed on Day 8. 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 12.3 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.

Parameter:

SI

Remarks on result:

other: Stimulation Index (SI) values for the experimental groups treated with WS400107 at 25 and 50 % w/v and 'as supplied' were 3.5, 3.3 and 5.1, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Overall DPM/lymph node values for the experimental groups treated with WS400107 at 25 and 50 % w/v and 'as supplied' were 1364.48, 1255.91 and 1963.56 DPM/node, respectively. For the vehicle control group, 385.18 DPM/node were recorded.

There were no deaths, no signs of ill health or toxicity and no signs of local irritation over the treated area during the pre-screen and main tests. Ear thickness was also unaffected during the pre-screen test.

Greasy fur on the head was noted for all control and test animals following each dosing occasion. This finding had resolved in all vehicle control animals by Day 4 and in the 25 and 50% w/v test animals and the positive control animals by Day 5 in the main study,

but was still evident at animal sacrifice on Day 6 in 'as supplied' animals both in the pre-screen test and the main study. 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.

Toxicologically relevant adverse effects on bodyweight were not evident during the pre-screen test or main study.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 all treated groups. Therefore, according to EU classification rules the test substance was classified as "irritant (Xi)" and "May cause sensitisation by skin contact (R43)” [DIRECTIVE 67/548/EEC] and as “Category 1 (Warning: May cause an allergic skin reaction) [REGULATION (EC) 1272/2008].