6-(2,4,4-trimethylcyclopentylidene)hexanal

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 15 and 27 May, 2013.

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. The maximum concentration tested was limited to 25% based on analogue data. In this study no sign of irritation were noted in both the preliminary and main tests up to 25%. Therefore, a higher concentration should have been tested. However, this deviation does not affect the outcome - and therefore the validity - of the study as a positive response was already observed at 25%. 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:

2013

Report date:

2013

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:

6 July 2012

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2012-04-23&24 / Signed on 2012-07-18

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(6E)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Cas Number:

1429808-42-3

Molecular formula:

C14 H24 O

IUPAC Name:

(6E)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Constituent 2

Reference substance name:

(6Z)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Cas Number:

1429808-41-2

Molecular formula:

C14 H24 O

IUPAC Name:

(6Z)-6-(2,4,4-trimethylcyclopentylidene)hexanal

Test material form:

liquid

Details on test material:

- Physical state: colourless to light yellow translucent liquid
- Storage condition of test material: fridge 6°C±3°C

Specific details on test material used for the study:

Date received: 22 April 2013

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: 8-9 weeks
- Weight at study initiation: 18.2-22.0 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: food (A04, SAFE), 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 13 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 15 to 27 May, 2013

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

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

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- As information was available regarding irritant potential or systemic toxicity of the analogous substance 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 25% (w/w) 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. Ear thickness was recorded on day 1, day 3 and on day 6. 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. No sign of excessive irritation was noted at the concentration of 25%.
Therefore, the concentration of 25% 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 = 6.25, 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:

1.23

Test group / Remarks:

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

Results 2

Key result

Parameter:

SI

Value:

1.32

Test group / Remarks:

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

Results 3

Key result

Parameter:

SI

Value:

1.94

Test group / Remarks:

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

Results 4

Key result

Parameter:

other: EC1.4

Remarks:

%

Value:

11.94

Test group / Remarks:

Test item

Cellular proliferation data / Observations:

- A stimulation index of more than 1.4 was recorded for one concentration of the test item (25% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 1.23, 1.32 and 1.94 for the treated group at 5%, 10% and 25%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 11.94%.

LOCAL IRRITATION
- No cutaneous reactions were recorded during the main test.
No significant increase in ear thickness and in ear weight was noted in animals treated at 5%, 10% and 25%. Therefore, the test item must be considered as not excessively irritant at the three 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 50 μL (25 μL per ear) of the test item at concentrations of 5%, 10% and 25% (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 a preliminary study tested at 25 %. Based on the results of preliminary study, the dose levels selected for the main test were 5%, 10% and 25% (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. No cutaneous reactions were recorded during the main test. No significant increase in ear thickness and in ear weight was noted in animals treated at 5%, 10% and 25%. Therefore, the test item must be considered as not excessively irritant at the three concentrations. A stimulation index of more than 1.4 was recorded for one concentration of the test item (25% (v/v) in acetone/olive oil). The Stimulation Index (SI) calculated by pooled approach was respectively 1.23, 1.32 and 1.94 for the treated group at 5%, 10% and 25%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 11.94%.

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.