2-benzyl-4-methyl-4a,5,6,7,8,8a-hexahydro-4H-benzo[d][1,3]dioxine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 02 November and 12 December, 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429 without any deviation. The substance is adequately identified, but some data on composition is missing. Therefore validation applies with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2010-12-07 / Signed on 2011-03-07

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-benzyl-4-methyl-octahydro-1,3-benzodioxine

EC Number:

956-606-9

Cas Number:

1373821-23-8

Molecular formula:

C16 H22 O2

IUPAC Name:

2-benzyl-4-methyl-octahydro-1,3-benzodioxine

Test material form:

liquid

Details on test material:

- Physical state: colourless liquid.
- Storage condition of test material: fridge 6°C±3°C, dark

Specific details on test material used for the study:

Date received: 20 October 2011

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 8 weeks
- Weight at study initiation: 18.4 - 21.4 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: food (M20, SDS), ad libitum
- Water: tap water from public distribution system, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 100% in the vehicle Acetone/olive oil
Main test:
Experiment 1: 25%, 50 and 100% in the vehicle Acetone/olive oil (4/1, v/v)
Experiment 2: 5% and 10% in the vehicle Acetone/olive oil (4/1, v/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at 100% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.
- Irritation: No mortality and no signs of systemic toxicity were noted. As no excessive irritation was noted at the concentration of 100%, the concentration of 100% was chosen as the highest concentration for the main study.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of control or test substance was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6. 10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counting using a cell counter (Beckman Coulter Z2). For the run, the lower size selected was 5 μm and the upper size selected was 15 μm (the average size of a lymphocyte is 8 μm).
The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

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

Statistics:

None.

Results and discussion

Positive control results:

Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of α- Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1, v/v) at concentrations of 2.5%, 5% and 10% (v/v). With EC1.4 = 6.75%, α-Hexylcinnamaldehyde is considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.84

Test group / Remarks:

5% (v/v) in Acetone/Olive oil (4:1)

Results 2

Key result

Parameter:

SI

Value:

1.37

Test group / Remarks:

10% (v/v) in Acetone/Olive oil (4:1)

Results 3

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

25% (v/v) in Acetone/Olive oil (4:1)

Results 4

Key result

Parameter:

SI

Value:

2.21

Test group / Remarks:

50% (v/v) in Acetone/Olive oil (4:1)

Results 5

Key result

Parameter:

SI

Value:

2.98

Test group / Remarks:

100%

Results 6

Key result

Parameter:

other: EC1.4

Remarks:

%

Value:

11.36

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

- A stimulation index of more than 1.4 was recorded for three concentrations of the test item (25%, 50% and 100% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 0.84, 1.37, 1.70, 2.21 and 2.98 for the treated group at 5%, 10%, 25%, 50% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 11.36%.

LOCAL IRRITATION
- Slight dryness was noted on day 6 on the treatment site of one animal treated at 50% and of four animals treated at 100%. No significant increase in ear thickness and in ear weight was noted in animals treated at 5%, 10%, 25%, 50% and 100%. Therefore, the test item must be considered non-irritant at the five concentrations.
CLINICAL OBSERVATIONS
- No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

BODY WEIGHTS
- 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.

Any other information on results incl. tables

None.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups of CBA/J (CBA/J@Rj) strain mice (4 females/dose) were treated for three consecutive days (D1, D2, D3) with 50μL (25μL per ear) of the test item at concentrations of 100%, 50% and 25% (first experiment) and 5%, 10% (second experiment) (v/v) in Acetone/Olive oil (4:1). For each experiment, a further group of four animals was treated with Acetone/Olive oil (4:1). At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The test concentrations for the main study were determined from a preliminary study tested at 100 %. Based on the results of preliminary study, the dose levels selected for the main test were 100%, 25% and 50% (v/v) in Acetone/Olive oil (4:1) (first experiment).

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. Slight dryness was noted on day 6 on the treatment site of one animal at 50% and of four animals treated at 100%.

No significant increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item must be considered non-irritant at the three concentrations.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.70, 2.21 and 2.98 for the treated group at 25%, 50% and 100% respectively. The EC1.4 can not be determined.

 

In order to determine the EC1.4, the additional concentrations of 5% and 10% were tested (experiment 2).

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. No significant increase in ear thickness and in ear weight was noted in animals treated at 5% and 10% . Therefore, the test item must be considered non-irritant at the two concentrations.

The Stimulation Index (SI) calculated by pooled approach was respectively 0.84 and 1.37 for the treated group at 5% and 10% respectively. Taking into account the previous results, the EC1.4 determined by linear regression was 11.36%.

 

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B according to the annex I of the Regulation EC No. 1272 /2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin sensitisation endpoint.