Addition reaction products of conjugated sunflower-oil fatty acids and tall-oil fatty acids with maleic anhydride

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:

2012

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
- 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 22.0 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 WS400104 in vehicle (% w/v):
- Pre-screen Test: 25, 50
- Main Study: 0 (vehicle control), 5, 10, 25, 50

Induction administration on Days 1, 2 and 3 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
WS400104 is a wax-like liquid at room temperature, which is not applicable to the animals undiluted. A vehicle trial has demonstrated that WS400104 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 WS400104 at 25 or 50% w/v in the selected vehicle did not produce deaths or signs of systemic toxicity and did not affect bodyweight. Rigid ears were recorded in all animals from Day 2 to 6, erythema grade 1 (very slight in degree) were seen from Day 2 to 5 in all animals treated with 50% w/v test material dilution and alopecia was detected on Day 6 only in the animals treated with 25 % w/v test material dilution. At both test concentrations, increases in ear thickness >25% were evident on Days 3 and 6, and ear punch weights were considerably higher than the upper limit of their normal range. In view of the test material being wax-like at 20°C, possible interference of bedding material sticking to the ear surface with these measurements and the absence of visible signs of excessive local irritation, the ear thickness and ear punch weight results may not have reflected excessive local skin irritation. Based on this information a total of four test concentrations were selected for the main study, whereby 50% w/v of WS400104 was considered to be a suitable high dose level.

- 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)

Results

Results 1

Key result

Parameter:

SI

Value:

13.7

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

28.1

Test group / Remarks:

10%

Results 3

Key result

Parameter:

SI

Value:

62.9

Test group / Remarks:

25%

Results 4

Key result

Parameter:

SI

Value:

71.9

Test group / Remarks:

50%

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.

 

At all test concentrations, premature deaths or signs of systemic toxicity were not evident. All control animals and animals treated with 5 or 10% w/v test material dilution were free from clinical signs or local irritation during the entire study. At 25 and 50% w/v, all main test animals showed rigid ears with test item precipitate on them from Day 2 to Day 6, erythema grade 1 (very slight in degree) and alopecia, the latter increasing in incidence of affected animals as the study progressed. The erythema were only transient having disappeared in all animals treated with 25% w/v by Day 5 and in animals treated with 50% w/v by Day 3 or 4. Rigid ears, erythema grade 1 and alopecia were also seen in the pre-screen test [detailed in the above field “Details on study design (LLNA)].

 

Bodyweight was unaffected by treatment with the test material, both in the pre-screen and in the main test.

Applicant's summary and conclusion

Interpretation of results:

sensitising