Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 30, 2015 to October 12, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

EC Number:

943-172-0

IUPAC Name:

Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium salt

Test material form:

semi-solid (amorphous): gel

Remarks:

Paste

Details on test material:

- Name of test material (as cited in study report): Vegetable or animal oil, hydrogenated, reaction products with diethanolamine, maleic anhydride and sodium sulphite
- Lot No.: CH 192435/01
- Purity: Not applicable (UVCB)
- Organic Carbon: 30.2%
- Hydrotox No.: 15/5241
- Origin: DACC
- Stability: Stable within the durability and with appropriate storage conditions
- Solubility in water: ~1 g/L
- Appearance: Light beige liquid/paste
- Storage conditions: Ambient temperature, dark, dry
- Expiration date: July 15, 2015
- Safety directions: H315, H319. Use protective clothing: gloves and protection goggles.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Age at study initiation: 7 to 8 weeks old
- Weight at study initiation: approximately 25 g
- Housing: 1/cage during the study; up to 5 during acclimatisation in polysulphone solid bottomed cages with nesting material
- Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy), ad libitum
- Water: drinking water supplied to each cage via a water bottle, ad libitum
- Acclimation period: at least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±2°C
- Humidity: 55±15%
- Air changes: Approximately 15 to 20 air changes per h
- Photoperiod: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 h

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary test: 2.5, 5, 10, 25 and 50% w/w (maximum feasible concentration).
Main test: 10, 25 and 50% w/w.

No. of animals per dose:

Preliminary test: 1 animal per dose group
Main test: 7 animals per dose group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: up to 50%
- Irritation: The evaluation of visible reactions showed no erythema at any of the concentrations investigated (2.5, 5, 10, 25 and 50% w/w).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA-BrdU
- Criteria used to consider a positive response: The test substance is considered to positive when the SI for any single treatment dose group is ≥ 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION:
During the study, the test substance was mixed in acetone/olive oil 4:1 v/v as vehicle. Concentrations were calculated considering the test substance as supplied. No correction factor was applied.

Main assay:
Animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle, test substance or control formulations. A dose volume of 25 μL/ear/day of each selected concentration and controls was applied to the dorsal surface of each ear (50 μL/animal/day), using a micropipette. On Day 5, The animals were treated intraperitoneally with 0.5 mL/animal of a solution of BrdU at a concentration of 10 mg/mL in physiological saline (0.9% NaCl), using a plastic graded syringe. The negative control was acetone:olive oil 4:1 v/v.

In vivo observations:
Animals were observed twice daily for mortality and morbidity throughout the study. Animals were observed for clinical signs before dosing commenced and daily up to sacrifice (approximately 1 h after dosing on Days 2, 3 and 5). The animals were weighed at allocation (Day 1) and on sacrifice (Day 6). The animals were killed on Day 6, approximately 24 h after BrdU injection, by carbon dioxide. Shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected in a solution of 2% BSA-PBS [2% bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation at lymph node. BrdU was measured by ELISA using a commercial kit.

Positive control substance(s):

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

Statistics:

Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.

Results and discussion

Positive control results:

SI value for the positive control group was 4.80. The assay is considered satisfactory as the Stimulation Index (SI) of the positive control group is higher than 2.0.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 8.57

Test group / Remarks:

Test substance 10%

Results 2

Key result

Parameter:

SI

Value:

ca. 12.56

Test group / Remarks:

Test substance 25%

Results 3

Key result

Parameter:

SI

Value:

ca. 12.97

Test group / Remarks:

Test substance 50%

Any other information on results incl. tables

Preliminary test:

No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations. The evaluation of visible reactions showed no erythema at any of the concentrations investigated (2.5, 5, 10, 25, 50% w/w).

The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated that no increase was found at any dose level investigated. Based on the results described above, the highest concentration selected for the main assay was 50% w/w.

Main assay - In vivo phase:

Mortality and clinical observation

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application.

Body weight

No treatment related effects were observed on body weights.

Evaluation of cell proliferation: Dose-related increases in cell proliferation of draining lymph nodes, significant at statistical analysis, were observed in all treated groups when compared to controls. The calculated Stimulation Indices (SI) were 8.57, 12.56 and 12.97, respectively at the low, mid- and high dose levels. In the group treated with the positive control substance, a Stimulation Index of 4.80 was calculated. As it was greater than 2, the study was regarded as valid.

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (skin sensitising) based on CLP criteria

Conclusions:

The test substance is considered to be a skin sensitiser.

Executive summary:

A study was conducted to assess the skin sensitising potential of the test substance in mouse (local lymph node assay) according to OECD Guideline 442b, in compliance with GLP. Based on the results of a preliminary test, animals were treated at concentrations of 10, 25 and 50% (w/w) on three consecutive days by application on the ears (50 μL/animal/day). Negative control animals were treated with vehicle alone (acetone/olive oil 4:1 v/v), while positive control animals received 25% (w/w) alpha-hexylcinnamaldehyde. No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No treatment-related effects were observed on body weight. The assay was considered satisfactory as the stimulation index (SI) of the positive control group was higher than 2.0. The stimulation index values calculated for the test substance were 8.57, 12.56 and 12.97 at concentrations of 10, 25 and 50% (w/w), respectively. No EC3 value could be extrapolated from these values. The results obtained in this study indicate that the test substance may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the stimulation index was greater than 1.6. Based on the results of the study, the test substance was considered to be a skin sensitiser (Longobardi C, 2015b).