Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 19 October 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The maximum tested concentration was 25% because at higher concentrations systemic toxicity was seen.

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
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
liquid

In vivo test system

Test animals

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. The animals were nulliparous and non pregnant and the acclimatisation period was at least five days. At the start of the study the animals were in the weight range of 19.1 to 24.6 g, and were approximately ten weeks old.
Animals were group housed in labeled Makrolon (MIII type) cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and 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 the study.
The temperature and relative humidity were controlled to remain within target ranges of 18 to 24°C and 40 to 70 %, respectively. Animal rooms were set with at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
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.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The test item was tested at concentrations of 5%, 10% or 25% w/w 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 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Initially, two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-11 weeks (at initiation of treatment) 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 (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 Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 5 %, 10 % or 25 % w/w in acetone/olive oil (4:1 v/v). 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 Charles River Den Bosch. 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, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). There was no information available regarding the solubility or stability in vehicle. 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. Correction of the purity/composition of the test item is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed. 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. Based on the tolerability of the test item, the highest test item concentration selected for the main study was a 25% concentration. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at this selected concentration. The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. 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 the positive control item, α Hexylcinnamaldehyde, techical grade, at a concentration of 5, 10 and 25 % in acetone/olive oil 4:1 was applied to the dorsal surface of each ear. The positive control group served as common control with other studies according to the summary of positive control data for the local lymph node assay. This reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used.

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 background 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).

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

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.

Results and discussion

Positive control results:
The positive control item, α-Hexylcinnamaldehyde, techical grade, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % in acetone/olive oil (4:1 v/v).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks:
%
Value:
> 25
Parameter:
other: NOEC %
Value:
> 25
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 5, 10 and 25% were 0.9, 1.0 and 1.5, respectively.
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The SI values calculated for the test item concentrations 5, 10 and 25% were 0.9, 1.0 and 1.5, respectively.

EC3 CALCULATION
There was no indication that the test item elicits a SI ≥ 3 when tested up to 25%. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 25%.

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.

Skin Reactions / Irritation:
Very slight erythema was noted for the animals treated at 25% between Days 2 and 4. Scaliness was noted for the animals treated at 10% and 25% between Days 2 and 6.

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.

Any other information on results incl. tables

Pre-screen Test:

At 100%, hunched posture and/or piloerection were noted between Days 1 and 4. Very slight to well-defined erythema was noted between Days 1 and 3 and scaliness was noted between Days 2 and 6. At 50%, hunched posture was noted on Day 3. Very slight to well-defined erythema was noted between Days 1 and 3 and scaliness was noted between Days 2 and 6. At 25% and 10%, no signs of systemic toxicity were noted. Very slight erythema was noted for one animal treated at 25% on Day 2 only. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all pre-screen animals.

White test item remnants were present on the dorsal surface of the ears of both animals treated at 10% (between Days 2 and 6), which did not hamper scoring of the skin reactions.

Based on the tolerability of the test item, the highest test item concentration selected for the main study was a 25% concentration.

Applicant's summary and conclusion

Interpretation of results:
other: Not a skin sensitiser.
Remarks:
According to EU CLP Regulation (EC) No 1272/2008 and its amendments.
Conclusions:
The SI values calculated for the substance concentrations 5, 10 and 25% were 0.9, 1.0 and 1.5, respectively. These results show that the test substance does not elicit a SI ≥ 3 when tested up to 25%. An EC3 has been derived resulted in an EC3 of >25%. A NOEC of >25% is derived. The test substance was considered not to be a skin sensitiser under the conditions of the test.
Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: “Local Lymph Node Assay" method and GLP. Test item concentrations selected for the LLNA study were based on the results of a pre-screen test using 100, 50, 25 and 10% test substance. In the pre-screen test systemic effects such as hunched posture and piloerection were noted after treatment with concentrations of 50 and 100% test substance. In addition, well-defined erythema and scaliness was observed but ear thickness increase was below 25%. Based on these results the highest concentration in the main study was lowered to 25% at which also very slight irritation was seen. In addition 10% and 5% concentrations were selected for the main study. In the main study, very slight erythema was noted for the animals treated at 25% between Days 2 and 4. Scaliness was noted for animals treated at 10% and 25% between Days 2 and 6. At 5, 10 and 25% the substance showed SI values of 0.9, 1.0 and 1.5, respectively. Reliable negative and positive controls were included. There was no indication that the test item elicits a SI ≥ 3 when tested up to and including 25%, the highest well tolerated concentration, and therefore the substance was not considered to be a skin sensitizer. An EC3 has been derived resulted in an EC3 of >25%. A NOEC of >25% is derived.