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Description of key information

Skin sensitisation (OECDTG429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 March, 2016 - 20 April, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
Deviations:
no
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)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Female CBA/J strain mice were supplied by Janvier, Le Genest-Saint-Isle, France. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acc limatisation period of at least five days the animals were selected at random and given a tail mark with marker pen. At the start of the study the animals were in the weight range of 19.8 to 23.5 g, and were eight to ten weeks old.
Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding mater ial. Paper and shelters were supplied as cage-enrichment. Free access to tap water and food (pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)) was allowed throughout t he study.
The temperature and relative humidity were controlled to remain within target ranges of 18 to 24°C and 40 to 70 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least 10 changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Undiluted test item or the test item at concentrations of 10%, 25 % or 50 % v/v in vehicle.
No. of animals per dose:
Groups of five mice were treated
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.

Preliminary Screening Test:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 (see section 7.8) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 50% and 100% 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. 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 Test:
Test Item Administration:
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50 %, 25 % or 10 % v/v in acetone/olive oil 4:1. The vehicle was selected on the basis of maximizing the solubility using the test substance data provided by the Sponsor and trial preparation results performed at WIL Research Europe. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide. There was no information available regarding the solubility or stability in vehicle. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 μl of the positive control item, α Hexylcinnamaldehyde, technical grade, at a concentration of 5, 10 and 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear. The positive control group served as common control with another study according to the summary of positive control data for the local lymph node assay.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

Terminal Procedures - Day 6
Termination: After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Preparation of Single Cell Suspension: Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter: 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Determination of 3HTdR Incorporation: Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract back ground and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability Twice daily.

Clinical Observations: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6 (prior to termination).

Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the numerical scoring system (see any other information on materials and methods incl. tables). In addition, a description of all other (local) effects was recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The positive control item, α-Hexylcinnamaldehyde, technical grade gave a Stimulation Index of 4.4 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.
Parameter:
SI
Remarks on result:
other: The SI values calculated for the test item concentrations 10, 25 and 50% were 1.3, 1.9 and 5.3, respectively.
Key result
Parameter:
EC3
Remarks:
%
Value:
33.1
Parameter:
other: NOEC %
Value:
25
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 10, 25 and 50% were 1.3, 1.9 and 5.3, respectively.

EC3 CALCULATION
These results indicate that the test item elicits a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 33.1% was calculated.

CLINICAL OBSERVATIONS/BODY WEIGHTS:
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.

Pre-screen test:

Very slight to well-defined erythema was noted for the pre-screen animals. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. The animals treated at 100% showed piloerection and erythema between the ears. No signs of systemic toxicity were noted for the animals treated at 50%. Based on the tolerability of the test item, the highest test item concentration selected for the main study was a 50% concentration.

Interpretation of results:
other: Category 1B
Remarks:
based on CLP criteria
Conclusions:
The SI values calculated for the substance concentrations 10, 25 and 50% were 1.3, 1.9 and 5.3, respectively. These results show that the test substance could elicit a SI ≥ 3. An EC3 has been derived resulted in an EC3 of 33.1%. A NOEC of 25% is derived. Based on these results the substance should be classified as skin sensitizer (Category 1B) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin sensitisation potential of the substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 10, 25 and 50% the substance showed SI values of 1.3, 1.9 and 5.3, respectively. Reliable negative and positive controls were included. All auricular lymph nodes of the experimental animals treated at 10% and 25% and control groups were considered normal in size. Most auricular lymph nodes of the animals treated at 50% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. These results show that the test substance could elicit a SI ≥ 3. An EC3 has been derived resulted in an EC3 of 33.1%. A NOEC of 25% is derived. The substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008 and GHS.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitisation potential of the substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. At 10, 25 and 50% the substance showed SI values of 1.3, 1.9 and 5.3, respectively. Reliable negative and positive controls were included. All auricular lymph nodes of the experimental animals treated at 10% and 25% and control groups were considered normal in size. Most auricular lymph nodes of the animals treated at 50% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. These results show that the test substance could elicit a SI ≥ 3. An EC3 has been derived resulted in an EC3 of 33.1%. A NOEC of 25% is derived.

Justification for classification or non-classification

Based on the results of the key study, Cedryl acetate is considered to be a sensitiser and needs to be classified as such (Skin Sens. 1B / H317) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).