Reaction mass of Fatty acids, tallow, Guerbet reaction Products, Ca salts and Fatty acids, tallow, Guerbet reaction Products, Na salts and Fatty acids, tallow, Guerbet reaction Products

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 October, 2015 - 09 November, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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 most recent 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:

73138-58-6, 73138-59-7, 73138-60-0

IUPAC Name:

73138-58-6, 73138-59-7, 73138-60-0

Details on test material:

- Name of test material (as cited in study report): LICOWAX R 21 S FL
- Physical state: Slightly yellow flakes
- Analytical purity: 99.6 % (w/w)

Fatty acids, tallow,
Guerbet reaction Products 73138-58-6 21% (w/w)
Fatty acids, tallow,
Guerbet reaction Products, Ca salts 73138-59-7 48% (w/w)
Fatty acids, tallow,
Guerbet reaction Products, Na salts 73138-60-0 31% (w/w)

- Lot/batch No.: DEF2084336
- Expiration date of the lot/batch: 2018-11-13
- Storage condition of test material: Room temperature, protected from light in the tightly closed original container
- Solubility : ≤ 0.01 g/L (20 °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: 18.4 - 23.2 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:

propylene glycol

Concentration:

0, 10, 25 and 50%

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 (undiluted for liquids, 50% for solids).
The test system, procedures and techniques were identical as those used in the main study except that the application method may have been different (see tables) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages. Ear thickness measurements were conducted using a digital thickness gauge 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 AND ADMINISTRATION:
Test substance preparation: The test item was ground to a powder using a mortar and pestle prior to weighing. 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.
Rationale for vehicle: 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 WIL Research Europe B.V..

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. In addition, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.

Positive control substance(s):

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

Statistics:

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 WIl Research Europe is an appropriate model for testing for contact hypersensitivity (see attached background material).

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

50% test item concentration

Remarks on result:

other: The SI values calculated for the item concentrations 10, 25 and 50% were 1.1, 1.4 and 1.1, respectively.

Any other information on results incl. tables

Results Pre-screen test:

No signs of systemic toxicity were observed in any of the pre-screen animals. Very slight erythema was noted for all animals on Day 4. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test item concentration selected for the main study was a 50% concentration.

Other results - main study:

No irritation was observed in any of the animals. White staining of test item remnants on the dorsal surface of the ears of one animal treated at 10% (Day 2) and all animals treated at 25% (Days 2-3) and 50% (Days 1-3) did not hamper scoring for erythema.

 

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

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.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, LICOWAX R 21 S FL was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to and including 50% 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 10%, 25% and 50% v/v. Reliable positive and negative controls were included.

No irritation was observed in any of the animals. White staining of test item remnants on the dorsal surface of the ears of one animal treated at 10% (Day 2) and all animals treated at 25% (Days 2-3) and 50% (Days 1-3) did not hamper scoring for erythema. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 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.

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 724, 908 and 722 DPM, respectively. The mean DPM/animal value for the vehicle control group was 653 DPM. The SI values calculated for the item concentrations 10, 25 and 50% were 1.1, 1.4 and 1.1, respectively.

As the SI appeared not to be ≥ 3 when tested up to and including 50% v/v, LICOWAX R 21 S FL was considered not to be a skin sensitiser

and does not have to be classified according to Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (including all amendments).