Registration Dossier

Administrative data

Description of key information

In Vitro OECD 429 Local Lymp Node Assay (LLNA) showed no indication of sensitisation.

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:
14 August 2013 to 12 December 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: E00031-633
- Retest date of the lot/batch: 30 January 2014
- Purity test date: 99%

RADIOLABELLING INFORMATION (if applicable)
This chemical was received from Perkin Elmer Inc., Boston, USA, on 20 February 2013. The chemical was stored refrigerated (2 to 8°C) in the dark in the radiochemistry laboratory at Charles River, Edinburgh. The supplier stated that purity value of the chemical was >97%.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at ambient temperature
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.6 g to 21.2 g
- Housing: Housed in pairs in solid-bottomed cages with a stainless steel grid top and integrated food hopper.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 8 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 °C to 23 °C
- Humidity (%): 42% to 63%
- Air changes (per hr): minimum of 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark (light hours 0700 h to 1900 h)
- IN-LIFE DATES: From: 28 August 2013 To: 16 October 2013
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 µL at 0%, 25%, 50% and 100% formulation concentration
No. of animals per dose:
4 animals per dose
Details on study design:
Preliminary tests were conducted as no data on the toxicity or irritancy of the test item was available. Treatment was administered on 3 consecutive days (Days 1 to 3). On each day of treatment the animals received an open application of 25 µL of undiluted test item onto the dorsum of each ear. Doses were administered using an appropriate micropipette.
There was no further treatment after the 3rd application.
Animals were checked for viability early in the morning and again as late as possible on each day. All animals were examined for reaction to treatment. The animals were observed frequently on each day of dosing (predose, immediately post dose and approximately 1 and 2 h after dosing) and once daily thereafter.The body weight of each individual animal was recorded on Day 1 (before the first dose) and on Day 6.
On Day 1 (pre-dose) and on Day 3 (approximately 48 h after the first dose) and Day 6 the thickness of both ears of each mouse was measured using digital callipers. In addition, both ears were observed for erythema and the scores recorded.
Animals were euthanised by cervical dislocation on Day 6 and were then discarded.

The dose concentrations of EXP0700332 indicated below were selected for the main study as suitable non-toxic dosages. Treatment was administered on 3 consecutive days (Days 1 to 3). On each day of treatment the animals received an open application of 25 µL of the appropriate formulation onto the dorsum of each ear. Formulations were administered using an appropriate micropipette. There was no treatment on Days 4 and 5.

On Day 6 each animal received an intravenous injection (250 µL) of phosphate buffered saline (PBS) containing 18.5 µCi of [methyl-3H] thymidine into the lateral tail vein.
Approximately 5 h after intravenous administration, all animals were euthanised by exposure to a rising concentration of carbon dioxide and the major blood vessels were severed to exsanguinate. Each pair of draining auricular lymph nodes was collected from each animal and the animal was then discarded. A single cell suspension of lymph node cells from each paired sample was prepared by gentle disaggregation through a 200 µm mesh stainless steel gauze. The mesh was rinsed with PBS (approximately 1 mL) and discarded. The lymph node cells were then washed (centrifuged at approximately 1300 g for 10 min at 4°C). The supernatant was drawn off, approximately 1 mL PBS was added and the cells were washed (centrifuged) for a second time. The supernatant was discarded and the DNA was precipitated with approximately 1 mL 5% trichloroacetic acid at 2 to 8°C for approximately 21 h. The resulting pellet underwent a further centrifugation and was re-suspended in 200 µL ‘Solvable’, (Perkin Elmer Inc., Waltham, USA) an aqueous-based solubiliser, and the suspension transferred to a vial containing 10 mL scintillation fluid (Aquasafe 500 plus liquid, Zinsser Analytic, Maidenhead, UK). Incorporation of tritiated thymidine was measured by ß-scintillation counting and was expressed as disintegrations per minute (DPM).

Excessive local skin irritation is indicated by an erythema score of 3 and/or an increase in ear thickness of 25% on any day of measurement.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
A group of mice was treated with a formulation of hexylcinnamicaldehyde that was prepared at a concentration of 25% in April 2013. This result was considered to provide evidence that the test methods employed at these laboratories are valid.
Key result
Parameter:
SI
Value:
ca. 1
Test group / Remarks:
Group 2
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
ca. 1.7
Test group / Remarks:
Group 3
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
ca. 1.8
Test group / Remarks:
Group 4
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Key result
Parameter:
SI
Value:
ca. 1
Test group / Remarks:
Group 1
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Results were corrected for background radiation and expressed as the Stimulation Index (SI). This was obtained by dividing the mean DPM obtained from each group by the mean DPM for the vehicle control group. The SI for the vehicle control group, therefore, is one. A positive response is indicated by an SI =3, together with consideration of dose-response and, where appropriate, statistical significance.

EC3 CALCULATION
The estimated concentration for the test item required to produce a 3-fold increase in draining lymph node cell proliferation (EC3) could not be determined because no group produced a 3-fold increase.

CLINICAL OBSERVATIONS:
There were no systemic signs and no signs of local irritation in any animal during the observation period. Clinical signs were restricted to wetness to the head, which was observed frequently in all animals, including controls. This was considered to be merely vehicle or test formulation residues, and not an adverse clinical sign.

BODY WEIGHTS
Body weights were considered to be unaffected by treatment with EXP0700332 and were acceptable for mice of this age and strain.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study, since treatment with EXP0700332 at concentrations of up to 100% did not achieve a stimulation index of =3, it was considered that the test item does
not have the potential to cause skin sensitisation.
Applying the criteria for classification according to the EU labelling regulations Commission Directive 2001/59/EC, no symbol and no risk phrase are required for EXP0700332.
Executive summary:

The objective of this study was to determine the delayed contact hypersensitivity potential of

EXP0700332.

The study was performed using female CBA/Ca mice. A formulation trial showed that acetone:olive oil in a ratio of 4:1, v/v (AOO) was a suitable vehicle for EXP0700332 and that a concentration of 50% was practicable. A preliminary test in two animals was conducted. Each mouse received an open application of 25 µL of undiluted test item onto the dorsum of each ear on 3 consecutive days. There were no signs of either systemic toxicity or local irritation and there was no effect on body weight in either animal.

The animals received 25 µL of the appropriate formulation onto the dorsum of each ear on 3 consecutive days. There were no signs of either systemic toxicity or local irritation and there was no effect on body weight in any animal. Three days after the final application, each animal received an intravenous injection of [methyl-3H] thymidine into the lateral tail vein. Approximately 5 hours later the draining lymph nodes were collected in order that incorporation of tritiated thymidine could be assessed by scintillation counting.

The stimulation indices (SI) for Groups 2, 3 and 4, when compared with Group 1, were 1.0, 1.7 and 1.8, respectively.

Under the conditions of the study, since treatment with EXP0700332 at concentrations of up to 100% did not achieve a stimulation index of 3, it was considered that the test item does not have the potential to cause skin sensitisation.

Applying the criteria for classification according to the Regulation (EC) 1272/2008, no symbol and no risk phrase are required for EXP0700332.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test item is not classed as a skin sensitizer based on the Local Lymph Node Assay results which were conducted in accordance to the OECD guideline (429) and GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Based on the LLNA study conducted in accordance with the OECD 429 guideline, the test material does not meet the criteria for classification as a skin sensitizer under the CLP Regulation.

The respiratory sensitization potential of the Substance has not been tested experimentally, however based on the lack of dermal sensitization potential it is concluded that no classification for respiratory sensitization is required.

Justification for classification or non-classification

Based on the negative result in the Local Lymph Node Assay, no classification for skin sensitisation is required.

The respiratory sensitisation potential of the substance has not been tested experimentally, but based on the lack of dermal sensitisation potential, it is concluded that based on current knowledge no classification for respiratory sensitisation is required.