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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
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:
Appearance: off-white powder
Test item storage: at room temperature

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: 18.9 - 23.1 g
- Housing: Animals were group housed in labeled Makrolon cages. Paper and shelters were supplied as cage-enrichment.
- 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

IN-LIFE DATES: From: 13 January 2016 to 25 January 2016

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10, 25% \\\(zie report: alleen ts conc vermelden en niet de conc van pos control substance)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN 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 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 (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

TREATMENT PREPARATION AND ADMINISTRATION:
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 immediately 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 study plan.

- 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.
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:
1.1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
25%
Cellular proliferation data / Observations:
Pre-screen Test
No irritation was 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 on Days 2 and 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. Hunched posture and piloerection were noted for both animals treated at 50% on Days 3 and 4. Additionally, hunched posture was seen on Day 5. The 25% was well tolerated in the animals. Based on these results, the highest test item concentration selected for the main study was a 25% concentration.

Skin Reactions / Irritation:
No irritation was observed in any of the animals. Scaliness was noted for two animals treated at 5% and all animals treated at 10% and 25% on Day 6.
White staining of the dorsal surface of the ears by test item remnants was noted but did not hamper the scoring of the skin reactions.

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 5, 10 and 25% were 723, 975 and 755 DPM, respectively. The mean DPM/animal value for the vehicle control group was 661 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.1, 1.5 and 1.1, 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, Norethyl was considered not to be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 25% 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 5%, 10% and 25% w/w. No irritation was observed in any of the animals. Scaliness was noted for two animals treated at 5% and all animals treated at 10% and 25% on Day 6. 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 5, 10 and 25% were 723, 975 and 755 DPM, respectively. The mean DPM/animal value for the vehicle control group was 661 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 1.1, 1.5 and 1.1, respectively. As the SI appeared not to be ≥ 3 when tested up to 25% w/w, Norethyl was considered not to be a skin sensitiser.

Based on these results, Norethyl would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).