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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 August 2012 to 5 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A reliability 1 is assigned because it is a GLP-compliant guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
WIL Research Europe B.V. Hambakenwetering 7 5231 DD ‘s-Hertogenbosch The Netherlands
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
p-menth-1-en-8-yl acetate
EC Number:
201-265-7
EC Name:
p-menth-1-en-8-yl acetate
Cas Number:
80-26-2
Molecular formula:
C12H20O2
IUPAC Name:
1-methyl-1-(4-methylcyclohex-3-en-1-yl)ethyl acetate
Test material form:
other: liquid
Details on test material:
- Identification (Trade Name): Terpinyl Acetate Alpha (mono-constituent)
- Molecular weight: 196.286
- Description: Clear colourless liquid
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: 20-24 g
- Housing: labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA) 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.
- 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 before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50, 100% ( w/w) 25 μL/ear
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TEST:
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. Two test substance concentrations were tested; a 50% and 100% concentration. The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). 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 prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.
MAIN STUDY
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.
- Induction - Days 1, 2 and 3: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
- Excision of the nodes - Day 6: 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). After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with 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.
- Tissue processing for radioactivity - Day 6: 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 stored in the refrigerator until the next day.
- Radioactivity measurements - Day 7: 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. Radioactive 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, 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. Furthermore, a description of all other (local) effects was recorded.
- Interpretation: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 1.7 and 4.7 respectively. An EC3 value of 16.5% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.9, 16.0, 11.9, 16.9, 14.4 and 12.8%. Based on these results it was concluded 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
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 1.3, 2.2 and 2.4 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 808, 1350 and 1486 DPM respectively. The mean DPM/animal value for the vehicle control group was 627 DPM.

Any other information on results incl. tables

The slight irritation of the ears as shown by the animals treated at 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No irritation of the ears was observed in any of the animals treated with vehicle or at test substance concentration of 25% or 50%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. The body weight loss noted for some animals across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent. Most auricular lymph nodes of the animals at 50% and 100% were considered enlarged in size. The auricular lymph nodes of the animals at 25% were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The SI values calculated for the substance concentrations 25, 50 and 100% were 1.3, 2.2 and 2.4 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 100%, Terpinyl-Acetate-Alpha was considered not to be a skin sensitiser.
Executive summary:

In a GLP compliant sensitivity study performend according to OECD guideline 429 the contact hypersensitivity to alpha-Terpinyl Acetate in the Mouse (Local Lymph Node Assay) was assessed. Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.3, 2.2 and 2.4 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 100%, alpha-Terpinyl Acetate was considered not to be a skin sensitiser.