2(1H)-Naphthalenone, 3,4-dihydro-5-methoxy-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-AUG-2005 - 17-AUG-2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 April 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(except the pre-test) (d.d. march 2003)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-080-9

EC Name:

-

Cas Number:

32940-15-1

Molecular formula:

C11H12O2

IUPAC Name:

5-methoxy-1,2,3,4-tetrahydronaphthalen-2-one

Test material form:

solid: compact

Details on test material:

- Description: White-yellowish solid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaHsdRcc

Sex:

female

Details on test animals and environmental conditions:

- Source: RCC Ltd, Laboratory Animal Service, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation (main study): 19.1 g - 22.0 g
- Housing: Animals were individually housed in Makrolon type-2 cages
- Diet: Free access to pelleted rodent diet (Pelleted standard Kliba 3433 from Provimi Kliba AG, CH-4303 Kaiseraugst)
- Water: Free access to tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 19 – 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 2.5, 5 and 10% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS
In a pre-test, test substance concentrations of 5, 10, 25 and 50% were tested in two mice. Each concentration was tested on one ear, by a single application.

MAIN STUDY
ANIMAL ASSIGNMENT
Three groups of four animals were treated with one test substance concentration per group. One group of four animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION
- Test substance preparation: The whole sample was melted under nitrogen protection at 87 °C ± 2 °C in a water-bath to get a liquid homogenous sample. After thorough shaking the test item was placed into a volumetric flask on a tared Mettler balance, and vehicle was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained during treatment with the magnetic stirrer.
- Rationale for vehicle: The vehicle was selected based on trial formulations

Performed according to test guidelines:
- Days 1, 2 and 3: Induction (topical treatment of 25 µL/ear)
- Day 6: Injection of 20.4 µCi 3H-methyl thymidine.
Five hours after the injection, all animals were euthanized and the ear lymph nodes were excised and pooled for each experimental group. Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
- Day 7: Radioactivity measurements

Observations:
Mortality/Viability: Once daily from acclimatization start to Day 6
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs and irritation: Once daily on the day of animals delivery and from Days 1 (pre-dose) to Day 6

INTERPRETATION
- Criteria used to consider a positive response: DPM values are presented for each dose group (8 nodes). 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.

Positive control substance(s):

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

Results and discussion

Positive control results:

The reliability check with Alpha-hexylcinnamicaldehyde indicated that the Local Lymph Node Assay as performed at RCC Ltd is an appropriate model for testing for contact hypersensitivity. The SI values calculated for the concentrations 5, 10 and 25% were 2.9, 3.9 and 8.7, respectively. An EC3 value of 5.5% was calculated.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

7.2

Test group / Remarks:

Test substance concentration of 2.5%

Results 2

Key result

Parameter:

SI

Value:

14.9

Test group / Remarks:

Test substance concentration of 5%

Results 3

Key result

Parameter:

SI

Value:

22.1

Test group / Remarks:

Test substance concentration of 10%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The size of the draining lymph nodes of the test substance groups was 2 to 4-fold larger compared to those of the control group.

DETAILS ON STIMULATION INDEX CALCULATION
Mean DPM values for the experimental groups treated with test substance concentrations 2.5, 5 and 10% were 29985, 62299 and 92456 DPM respectively. The mean DPMl value for the vehicle control group was 4182 DPM.

EC3 CALCULATION
An EC3 value could not be determined because this calculation requires a S.I. value of less than 3.

CLINICAL OBSERVATIONS
No mortality occurred.
No clinical signs were observed in any animals of the control group or Group 2 (2.5 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 3 (5 %) and Group 4 (10 %) (for Group 4, a moderate erythema was found on Day 4), persisting for the remainder of the in-life phase of the study (Group 4) or persisting for a total of three days (Group 3). On Day 6, scattered pinhead large lesion was observed at both dosing sites in all mice of Group 4 (10 %).

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Any other information on results incl. tables

Results pre-test

No effects were observed at 10%. One day after the application, a moderate ear erythema was noted at 25 and 50%.

Based on these results, the highest test substance concentration selected for the main study was a 10% concentration.

Applicant's summary and conclusion

Interpretation of results:

other: Category 1 (skin sensitising) based on GHS and CLP criteria

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 (2002) and according to GLP principles, the substance was considered to be a skin sensitiser, as the SI was shown to be >= 3 when tested up to and including 10%.

Executive summary:

An LLNA skin sensitisation study was performed according to OECD 429 (2002) and according to GLP principles with the substance. Reliable positive and negative controls were included. Based on the results of a pre-screen test, three experimental groups of four female mice were treated with test substance concentrations of 2.5, 5 or 10% (w/v) on three consecutive days, by open application on the ears. Four vehicle control animals were similarly treated, but with vehicle alone (acetone/olive oil (4:1 v/v)). No clinical signs were observed in any animals of the control group or Group 2 (2.5 %). On the second application day, a slight ear erythema was observed at both dosing sites in all mice of Group 3 (5 %) and Group 4 (10 %) (for Group 4, a moderate erythema was found on Day 4), persisting for the remainder of the in-life phase of the study (Group 4) or persisting for a total of three days (Group 3). On Day 6, scattered pinhead large lesion was observed at both dosing sites in all mice of Group 4 (10 %). The size of the draining lymph nodes of the test substance groups was 2 to 4-fold larger compared to those of the control group. Mean DPM values for the experimental groups treated with test substance concentrations 2.5, 5 and 10% were 29985, 62299 and 92456 DPM, respectively. The mean DPMl value for the vehicle control group was 4182 DPM. The SI values calculated for the substance concentrations 2.5, 5 and 10% were 7.2, 14.9 and 22.1, respectively. Based on these data, the substance is considered to be a skin sensitiser, as the SI was shown to be >= 3 when tested up to and including 10%. The substance needs to be classified as skin sensitiser (Category 1) according to GHS and CLP. As an EC3 value could not be determined (and the lowest tested substance concentration was 2.5%), the available data are not sufficient for sub-categorisation.