Cineole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 07 January and 24 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Constituent 2

Reference substance name:

Eucalyptol

IUPAC Name:

Eucalyptol

Details on test material:

- Name of test material (as cited in study report): Eucalyptol
- Physical state: clear colourless liquid
- Analytical purity: 99.6%
- Lot/batch No.: AS00113945
- Expiration date of the lot/batch: 06 May 2014
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet, ad libitum
- Water (e.g. ad libitum): Mains tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Positive control substance(s):

yes

Remarks:

α-Hexylcinnamaldehyde; 85% purity

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25 % and 50 % v/v in acetone/olive oil 4:1, and 100 % v/v

No. of animals per dose:

Five per dose

Details on study design:

Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 to 3 and Days 1 to 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 μl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
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 at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR:80μCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. 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 Day 6 (prior to termination).
Ear thickness: The ear thickness measurements of each mouse were recorded on Day 1 (pre-dose) and Days 3 and 6.

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by β-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: A concentration of the test item at 100 % v/v gave a stimulation index of 5.08. and was considered positive for sensitisation. Concetrations of 25 and 50% v/v gave scores of 1.43 and 2.08 respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean dpm/animal (Standard deviation): Vehicle acetone/olive oil 4:1: 1819.65 (± 460.90) Test item 25 % v/v in acetone/olive oil 4:1: 2596.31 (± 502.30) Test item 50 % v/v in acetone/olive oil 4:1: 3692.15 (± 890.17) Test item 100 % v/v in acetone/olive oil 4:1: *9235.45 (± 3525.55) Positive control item 25 % v/v in acetone/olive oil 4: ** 13343.09 (±4295.79) The results of the statistical analysis of the data indicated that there was a significant difference between the control group and the test groups with animals treated with the undiluted test item and the positive item. * = Significantly different from control group P < 0.01 ** = Significantly different from control group P < 0.001

Any other information on results incl. tables

Preliminary Screening Test

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment group	Concentration	Stimulation Index	Result
Test item	25 % v/v in acetone/olive oil 4:1	1.43	Negative
Test item	50 % v/v in acetone/olive oil 4:1	2.03	Negative
Test item	100 % v/v	5.08	Positive
Positive control	25 % v/v in acetone/olive oil 4:1	7.33	Positive

Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Ear Thickness Measurements and Ear Thickness Changes, Local Skin Irritation

There was no increase in ear thickness (>25%) in any of the test or control animals on Days 3 and 6. No signs of irritation were seen in any of the animals throughout the test.

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 65.90%.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance was assessed for skin sensitisation according to OECD guideline 429. The test item was considered to be a sensitiser under the conditions of the test.
The positive control, α-Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

 Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008 Methods.

Following a preliminary screening test in which no clinical signs of toxicity were noted with the undiluted test item, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde at a concentration of 25% v/v in acetone/olive oil 4:1. Results. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Treatment group	Concentration	Stimulation Index	Result
Test item	25 % v/v in acetone/olive oil 4:1	1.43	Negative
Test item	50 % v/v in acetone/olive oil 4:1	2.03	Negative
Test item	100 % v/v	5.08	Positive
Positive control	25 % v/v in acetone/olive oil 4:1	7.33	Positive

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 65.90%. Conclusion. The test item was considered to be a sensitiser under the conditions of the test. α-Hexylcinnamaldehyde gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1.