Registration Dossier

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-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of the test item (as cited in the study report): NOPYL ACETATE
IUPAC Name of the test item: (1R,5S)-2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl) ethyl acetate
Synonym: (-) Nopyl Acetate; 6,6-dimethyl -(1R,5S)-bicyclo[3.1.1]hept-2-ene-2-ethanol-2-acetate
Substance type: monoconstituent
Batch No.: 117042
Purity: 99.3%
This composition is within the specifications of the substance identity profile agreed within the SIEF.
Colour: colourless – slightly amber
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 10 April 2013

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: 8-12 weeks
Weight at study initiation: 18.9-23.3 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: 5 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 (non-LLNA)

Inductionopen allclose all
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
not applicable
Challengeopen allclose all
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
not applicable
No. of animals per dose:
not applicable
Details on study design:
not applicable
Challenge controls:
not applicable

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: 5, 10, 25, 50 and 100 %
No. of animals per dose:
Preliminary test: 2 females/dose (right and left ear were treated with different concentrations)
Main test: 4 females/dose and controls
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. No cutaneous reaction and no increase in ear thickness were observed at any concentration up to 100%. The highest concentration retained for the main test was therefore the maximal practicable concentration (100%).


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), 5, 10, 25, 50 and 100 % 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 = 10.08). The experiment was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
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

3942

492.75

-

-1.02

-

-

5 %

8

2883

360.38

0.73

3.09

I

14.11 %

10 %

8

5658

707.25

1.44

3.09

I

25 %

8

28159

3519.88

7.14

3.00

I

50 %

8

14238

1779.75

3.61

-3.06

I

100 %

8

14325

1790.63

3.63

2.06

I

α-hexylcinnamaldehyde 25 %

8

39751

4968.88

10.08

-

-

-

                            

dpm = disintegrations per minute

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

EC3 value = theoretical concentration resulting in a SI value of

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 significant lymphoproliferation (SI > 3) was noted at concentrations ≥ 25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.
Therefore, nopyl acetate is classified as sensitising "R43: may cause sensitisation by skin contact" according to Directive 67/548/EEC and skin sensitiser Category 1B according to Regulation (EC) No 1272/2008 (CLP).
Executive summary:

In a local lymph node assay performed according to OECD Guideline No 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose)

were exposed to 25 µL of nopyl acetate in Acetone/Olive oil (4/1, v/v) at concentrations of 0 (vehicle control), 5, 10, 25, 50 and 100 % (v/v) to the dorsal surface of both ears for three consecutive days. On Day 6, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity, expressed as number of disintegrations per minute (DPM) and stimulation index. The test concentrations for the main study were determined from a preliminary study where no cutaneous reaction and no increase in ear thickness were observed at any concentration up to 100%.

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 tested concentrations. Stimulation Index for 5, 10, 25, 50 and 100 % was 0.73, 1.44, 7.14, 3.61 and 3.63, respectively. The calculated effective concentration inducing a SI of 3 (EC3) was 14.11 %. Positive control (α-hexylcinnamaldehyde) exhibited evidence of sensitisation (SI = 10.08).

A significant lymphoproliferation (SI > 3) was noted at concentrations ≥ 25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

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