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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Release date certificate 3 November 2015
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: white crystalline powder
Test item storage: In refrigerator (2-8°C)
Specific details on test material used for the study:
- Trial formulation preparation (for optimal vehicle selection) had a non-GLP status but was carried out in the quality assured environment of WIL Research Europe’s GLP testing facility.
- The characterization of the test item was conducted in a GMP environment.
- Analysis of test item in vehicle for concentration, stability, homogeneity was not performed, however, to limit the impact, the test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 4 hours after preparation of the formulation.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 19.4 - 23.6 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): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES: From: 6 January 2016 to 25 January 2016

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
Vehicle was selected on the basis of maximizing the solubility using test item data provided by the Sponsor, trial preparation results performed at WIL Research Europe and the vehicles specified in the test guideline.
Concentration:
0, 10, 25, 50% w/w Diona-R
No. of animals per dose:
5
Details on study design:
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). Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted prior to dosing on Days 1 and 3, and on Day 6.


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 propylene glycol.

TREATMENT PREPARATION AND ADMINISTRATION:
There was no information available regarding the solubility or stability in vehicle.
Test substance preparation: The test item was ground to a powder using a pestle and mortar 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..


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 the following numerical scoring system. In addition, a description of all other (local) effects was recorded.

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.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
50%
Cellular proliferation data / Observations:
Pre-screen Test
No irritation and no signs of systemic toxicity were observed in any of the pre-screen animals. White staining of the dorsal surface of the ears by test item remnants was noted for all animals (Days 1-3). The staining did not hamper the scoring of the ears. 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.

Skin Reactions / Irritation
No irritation was observed in any of the animals. The scaliness, noted for five animals, was considered not to have a toxicologically significant effect on the activity of the nodes. White staining of test item remnants on the dorsal surface of the ears of the experimental animals between Days 1 and 4, did not hamper scoring for erythema.

Systemic Toxicity
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.

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area
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.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 734, 768 and 723 DPM, respectively. The mean DPM/animal value for the vehicle control group was 714 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.0, 1.1 and 1.0, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, Diona-R was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50% w/w.
Executive summary:

An LLNA skin sensitisation study was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 10%, 25% and 50% w/w. No irritation was observed in any of the animals. The scaliness, noted for five animals, was considered not to have a toxicologically significant effect on the activity of the nodes. 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. 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. Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 734, 768 and 723 DPM, respectively. The mean DPM/animal value for the vehicle control group was 714 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.0, 1.1 and 1.0, respectively. As the SI appeared not to be ≥ 3 when tested up to 50% w/w, Diona-R was considered not to be a skin sensitiser.