[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

supporting study

Study period:

27 September 2016 - 25 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 2008, including amendments

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

ω-hydroxy fatty acid methyl esters, C16 (saturated and unsaturated) alkyl and fatty acid methyl esters, C16 (unsaturated) alkyl

EC Number:

943-164-7

Molecular formula:

A generic molecular formula cannot be given for this UVCB substance.

IUPAC Name:

ω-hydroxy fatty acid methyl esters, C16 (saturated and unsaturated) alkyl and fatty acid methyl esters, C16 (unsaturated) alkyl

Test material form:

solid

Details on test material:

Identification: (w-hydroxy) fatty acid methyl esters and (w-hydroxy) fatty acids
Appearance: Brown solid (at 2-8°C)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 20.4 - 23.3 g
- Housing: Animals were group housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 5%, 10%, and 25%

No. of animals per dose:

5

Details on study design:

WEIGHT OF EVIDENCE ANALYSIS: In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

RANGE FINDING TESTS: Two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines. The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per conce ntration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT: Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed. The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch.

STUDY ADMINISTRATION:
Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.
Observations:
- Mortality/Viability: Twice daily.
- Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
- Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.
- Necropsy: No necropsy for gross macroscopic examination was performed according to the study plan.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis not performed

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Laboratories Den Bosch B.V. is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

5%

Results 2

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

10%

Results 3

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test item concentrations 5% and 10% were 1321 and 2176 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1144 DPM.

Results 4

Key result

Parameter:

EC3

Value:

> 10

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION: A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is
the ratio of the DPM/animal compared to the DPM/vehicle control group mean. The SI values calculated for the test item concentrations 5% and 10% were 1.2 and 1.9, respectively.

EC3 CALCULATION: It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 10%.

CLINICAL OBSERVATIONS: At 25%, signs of systemic toxicity (hunched posture and/or piloerection) were noted for the majority of animals between Days 2 and 5. These signs could have affected the sensitization response of the animals and therefore this group was excluded from interpretation. Sufficient dose groups were available to warrant the study integrity. No further clinical signs of systemic toxicity were observed in the remaining animals of the main study. No mortality occurred.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

SKIN REACTIONS/IRRITATION: Very slight erythema and/or scaliness were noted for the majority of vehicle and test item animals dosed at 5 and 10%. This erythema was considered not to have an effect on the activity of the lymph nodes.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Not sensitising according to Regulation (EC) No. 1272/2008

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, the substance was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 10% v/v.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 test guideline and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 5%, 10% and 25% v/v. Reliable negative and positive controls were included. At 25%, signs of systemic toxicity (hunched posture and/or piloerection) were noted for the majority of animals between Days 2 and 5. These signs could have affected the sensitization response of the animals and therefore this group was excluded from interpretation. Sufficient dose groups were available to warrant the study integrity. No further clinical signs of systemic toxicity were observed in the remaining animals of the main study. No mortality occurred. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Very slight erythema and/or scaliness were noted for the majority of vehicle and test item animals dosed at 5% and 10%. This erythema was considered not to have an effect on the activity of the lymph nodes. Mean DPM/animal values for the experimental groups treated with test item concentrations 5% and 10% were 1321 and 2176 DPM, respectively. The mean DPM/animal value for the vehicle control group was 1144 DPM. The SI values calculated for the test item concentrations 5 and 10% were 1.2 and 1.9, respectively. As the SI appeared not to be ≥ 3 when tested up to 10% v/v, the substance is considered to NOT be a skin sensitiser and does not meet the criteria for classification as skin sensitisation according to Regulation (EC) No. 1272/2008.