6,8-dimethylnon-7-enal

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

30 January to 04 February 2008.

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 details on composition are missing. Therefore validation applies with restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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:

Directive 2004/73/EC

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

11 January 2007.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

7-nonenal, 6,8-dimethyl-, (6S)

Cas Number:

899810-99-2

Molecular formula:

C11 H20 O

IUPAC Name:

7-nonenal, 6,8-dimethyl-, (6S)

Constituent 2

Reference substance name:

7-nonenal, 6,8-dimethyl-,(6R)-

Cas Number:

899810-91-4

Molecular formula:

C11 H20 O

IUPAC Name:

7-nonenal, 6,8-dimethyl-,(6R)-

Test material form:

liquid

Details on test material:

- Physical state: colourless translucent liquid
- Storage condition of test material: Room temperature

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: 18-21 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: foodstuff (M20, SDS), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-24 °C
- Humidity: 33-50 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: from 30 January to 04 February 2008.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Main test: 25%, 50% and 100% in the vehicle Acetone/olive oil (4/1, v/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Undiluted test item was used (100 %)
- Irritation: a preliminary screening test was not performed. Based on the disposal information (results of skin irritation according OECD n°404 & acute toxicity by oral route according OECD n° 423) the dose levels selected for the main test were 25% and 50%(v/v) in Acetone/Olive oil (4:1) and 100%.
Therefore, 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 5%,10% and 25% (v/v). With EC1.4 = 4.86%, 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:

1.4

Test group / Remarks:

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

Results 2

Key result

Parameter:

SI

Value:

1.71

Test group / Remarks:

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

Results 3

Key result

Parameter:

SI

Value:

1.79

Test group / Remarks:

100% (v/v) in Acetone/Olive oil (4:1)

Results 4

Key result

Parameter:

other: EC1.4

Remarks:

%

Value:

25

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

- A stimulation index of more than 1.4 was recorded for two concentration of the test item (50% and 100% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 1.40, 1.71 and 1.79 for the treated group at 25%, 50% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 25%.

LOCAL IRRITATION
- No cutaneous reaction was noted in the treated animals. Only, a slight dryness was registered at D6, in the treated group at 100%.
- A slight increase in ear thickness (+18.9%) and in ear weight (+8.3%) was recorded at the concentration of 100%. Therefore, the test item must be considered “slightly-irritant” at the concentration of 100% and “non-irritant” at the concentrations of 50% and 25%.
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 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 50 μL (25 μL per ear) of the test item at concentrations of 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1). 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 the disposal information (results of skin irritation according OECD n°404 & acute toxicity by oral route according OECD n° 423). Based on the results of preliminary study, the dose levels selected for the main test were 25%, 50% and 100% (v/v) in Acetone/Olive oil (4:1).

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. Only, a slight dryness was registered at D6, in the treated group at 100%. A slight increase in ear thickness (+18.9%) and in ear weight (+8.3%) was recorded at the concentration of 100%. Therefore, the test item must be considered “slightly-irritant” at the concentration of 100% and “non-irritant” at the concentrations of 50% and 25%.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.40, 1.71 and 1.79 for the treated group at 25%, 50% and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 25%.

Under these experimental conditions, the test material is classified as skin sensitiser in category 1B (H317: May cause an allergic skin reaction) 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.