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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 March - 04 April 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the OECD Guideline No 429 and in compliance with GLP without any deviations.
Cross-reference
Reason / purpose for cross-reference:
read-across source

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
p-mentha-1,4(8)-diene
EC Number:
209-578-0
EC Name:
p-mentha-1,4(8)-diene
Cas Number:
586-62-9
Molecular formula:
C10H16
IUPAC Name:
4-isopropylidene-1-methylcyclohexene
impurity 1
Chemical structure
Reference substance name:
p-mentha-1,4-diene
EC Number:
202-794-6
EC Name:
p-mentha-1,4-diene
Cas Number:
99-85-4
Molecular formula:
C10H16
IUPAC Name:
1-isopropyl-4-methylcyclohexa-1,4-diene
impurity 2
Chemical structure
Reference substance name:
Dipentene
EC Number:
205-341-0
EC Name:
Dipentene
Cas Number:
138-86-3
Molecular formula:
C10H16
IUPAC Name:
4-isopropenyl-1-methylcyclohexene
impurity 3
Reference substance name:
Non identified impurities
Molecular formula:
Not applicable
IUPAC Name:
Non identified impurities
Test material form:
liquid
Details on test material:
Batch No.: 122718
Purity: 97.9%
Name of test material (as cited in study report): TERPINOLENE MONOCONSTITUENT
Physical state: liquid
Storage conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry date: 17 october 2011

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
Source: Janvier, Le Genest-Saint-Isle, France.
Age at study initiation: 8-10 weeks
Weight at study initiation: 18-23 g
Housing: Animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Techniplast 1145T, 435 cm2, 36.9 x 15.6 x 13.2 cm) containing autoclaved sawdust (SICSA, Alfortville, France). In the main test, on Day 6 before the [3H] methyl-thymidine (3H-TdR) injections, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
Diet: SSNIFF R/M-H pelleted diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
Water: Tap water (filtered using a 0.22 µm filter), ad libitum
Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
Temperature: 22 ± 2 °C
Humidity: 30-70 %
Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
The vehicle used for the test item and positive control was a mixture Acetone/Olive Oil (4/1, v/v) (AOO): - acetone: batch No. K41254613, supplied by Merck, - olive oil: batch No. 0001428703, supplied by Sigma
Concentration:
Preliminary test: 10, 25, 50 and 100%
Main test: 2.5, 5, 10, 25 and 50%
No. of animals per dose:
Preliminary screening test: 2 females/dose: right and left ear were treated with different concentrations.
Main test: 4 females/dose
Details on study design:
RANGE FINDING TEST:
Compound solubility: The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at all tested dilutions (10-50%).
Irritation: for 3 consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear). Measurement of the ear thickness (using a micrometer) was performed each day before treatment and 72 hours after the last application. Dryness of the skin and/or eryhema was observed on days 3 and/or 6 in ears of animals 303 and 304 treated at 50% or 100%. In addition, a notable increase in ear thickness was recorded at the concentration of 100%, showing the irritant potential of the test item at this concentration. The highest concentration retained for the main test was therefore 50%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Name of test method: Local Lymph Node Assay
Criteria used to consider a positive response: The results were expressed as disintegration per minute (dpm) per group. Stimulation indices (SI) were calculated according to the following formula: SI = dpm of treated group / dpm of control group. The test item was considered as a skin sensitizer when the SI for a dose group is higher than or equal to 3. Other relevant criteria such as radioactivity levels and ear thickness were also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was administered as a solution in the vehicle and was mixed with the required quantity of vehicle.Dose formulations were prepared by the CIT Pharmacy extemporaneously on the day of each administration. They were stored at between 2 and 8°C and delivered to the study room in brown flasks.
On days 1, 2 and 3, a dose-volume of 25 μL of the appropriate dose formulations was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip. In order to avoid licking, to ensure an optimized application of the test material and to facilitate ear thickness measurement, the animals were placed under light isoflurane anesthesia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.

25 µL of test material at concentrations of 0 (vehicle control), 2.5, 5, 10, 25, and 50 % were applied to the dorsal surface of each ear on Days 1, 2 and 3. On Day 6, 250 µL NaCl 0.9 % containing 20 μCi of 3H-TdR (specific activity of 20 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were killed by a lethal intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph node of each ear was excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical disaggregation in Petri dishes using the plunger of a syringe. LNC were washed with 0.9 % NaCl and precipitated with 5 % (w/v) trichloroacetic acid (TCA) at 4 °C. Pellets were re-suspended in 1 mL TCA and 3 mL of Ultima GoldxR scintillation fluid (Packard) was added in order to measure incorporation of 3H-TdR using β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 8.94). The experiment was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.24
Test group / Remarks:
2.5%
Remarks on result:
other: see table 7.4.1/1
Parameter:
SI
Value:
2.14
Test group / Remarks:
5%
Remarks on result:
other: see table 7.4.1/1
Parameter:
SI
Value:
3.43
Test group / Remarks:
10%
Remarks on result:
other: see table 7.4.1/1
Parameter:
SI
Value:
14.44
Test group / Remarks:
25%
Remarks on result:
other: see table 7.4.1/1
Parameter:
SI
Value:
20.51
Test group / Remarks:
50%
Remarks on result:
other: see table 7.4.1/1
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table 7.4.1/1

Any other information on results incl. tables

Table 7.4.1/1: Results of skin sensitization

Treatment and concentrations

No. of nodes per group

dpm per group

dpm per node

Stimulation index (SI)

Increase in ear thickness (% between Day 1 and Day 6)

Irritation level

EC3 value

Vehicle

8

2706

338.25

-

-1.03

-

-

2.5 %

8

3347

418.38

1.24

-1.00

I

8 %

5 %

8

5781

722.63

2.14

5.15

I

10 %

8

9290

1161.25

3.43

2.04

I

25 %

8

39074

4884.25

14.44

1.01

I

50 %

8

55509

6938.63

20.51

10.00

II

α-hexylcinnamaldehyde

8

24195

3024.38

8.94

-

-

-

                            

dpm = disintegrations per minute

I = non-irritant (increase in ear thickness < 10 %)

II = slightly irritant (increase in ear thickness = 10 to 25 %)

EC3 value = theoretical concentration resulting in a SI value of 3

stimulation index = dpm of treated group / dpm of control group

 

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A dose-related increase in the SI was recorded at concentrations ≥ 2.5%, and the threshold positive value of 3 was exceeded at the concentration of 10%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.
Therefore, terpinolene monoconstituent is classified as sensitising "R43: May cause sensitisation by skin contact" according to Directive 67/548/EEC and skin sensitiser Category 1B according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In a local lymph node assay performed according to OECD guideline No 429 and in compliance with GLP, terpinolene monoconstituent in Acetone/Olive oil (4/1, v/v) was administered to CBA/J 9 weeks old mice.

In a preliminary study, dryness of the skin and/or eryhema was observed on days 3 and/or 6 in ears of 2 animals treated at 50% or 100%. A notable increase in ear thickness was recorded at the concentration of 100%, showing the irritant potential of the test item at this concentration, therefore the highest concentration retained for the main test was 50%.

In a main study, five treated groups of four animals received applications of 25 μL of terpinolene monoconstituent to the external surface of both ears at the concentrations of 2.5, 5, 10, 25, and 50% for 3 consecutive days.

The positive control used was α-hexylcinnamaldehyde which presented a stimulation index of 8.94. Therefore, the positive control gave acceptable positive results and the study can be considered as valid.

No clinical signs and no mortality were observed during the main test. No local reactions and no notable increase in ear thickness were observed at any of the concentrations. Stimulation index for 2.5, 5, 10, 25, and 50 % were 1.24, 2.14, 3.43, 14.44 and 20.51, respectively. The calculated effective concentration inducing a SI of 3 (EC3) was 8%.

A dose-related increase in the SI was recorded at concentrations ≥ 2.5%, and the threshold positive value of 3 was exceeded at the concentration of 10%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

Therefore, terpinolene monoconstituent is classified as sensitising "R43: May cause sensitisation by skin contact" according to Directive 67/548/EEC and skin sensitiser Category 1B according to CLP Regulation (EC) No 1272/2008.