Lorenox™

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-23 February 2009

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:

2009

Report date:

2009

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)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2008-04-14&15 /Signed on 2008-07-16.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1,5-Cyclododecadiene, 9-methoxy-1,6,10-trimethyl-

Cas Number:

900779-74-0

Molecular formula:

C16 H28 O

IUPAC Name:

1,5-Cyclododecadiene, 9-methoxy-1,6,10-trimethyl-

Constituent 2

Reference substance name:

1,5-Cyclododecadiene, 10-methoxy-1,5,9-trimethyl-

Cas Number:

900779-83-1

Molecular formula:

C16 H28 O

IUPAC Name:

1,5-Cyclododecadiene, 10-methoxy-1,5,9-trimethyl-

Constituent 3

Reference substance name:

1,5-Cyclododecadiene, 9-methoxy-2,5,10-trimethyl-

Cas Number:

900779-84-2

Molecular formula:

C16 H28 O

IUPAC Name:

1,5-Cyclododecadiene, 9-methoxy-2,5,10-trimethyl-

Constituent 4

Reference substance name:

1,5-Cyclododecadiene, 9-methoxy-1,5,10-trimethyl-

Cas Number:

900779-85-3

Molecular formula:

C16 H28 O

IUPAC Name:

1,5-Cyclododecadiene, 9-methoxy-1,5,10-trimethyl-

Test material form:

liquid

Details on test material:

- Storage condition of test material: Room temperature

Specific details on test material used for the study:

- Date received: 10 February 2009 (Flask 1); 18 February 2009 (Flask 2)

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).
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 18.4-21.3 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food, ad libitum
- Water: Drinking 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 fifteen changes per hour
- Photoperiod: 12 h dark / 12 h light

- IN-LIFE DATES: 11-23 February 2009

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 100% (undiluted test item)
Main test: 10%, 25% and 50% (v/v) in acetone/olive oil (4:1, v/v)

No. of animals per dose:

Preliminary screening test: One animal/dose
Main test: 4 animals/dose

Details on study design:

PRELIMINARY STUDY
- As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed once daily on Days 1, 2, 3, 4, 5 and 6. 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 on Day 6.
- Irritation: No signs of systemic toxicity were noted. From day 3, dryness was noted on the treated area. Then a scab was recorded on the basis of the ear. Based on this excessive irritation, the dose levels selected for the main test were 10%, 25% and 50% (v/v) in acetone/olive oil (4:1, v/v).

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 = 4.12, 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:

1.38

Test group / Remarks:

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

Results 2

Key result

Parameter:

SI

Value:

2.13

Test group / Remarks:

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

Results 3

Key result

Parameter:

SI

Value:

3.81

Test group / Remarks:

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

Results 4

Key result

Parameter:

other: EC1.4

Remarks:

(%)

Value:

10.4

Test group / Remarks:

test item

Cellular proliferation data / Observations:

ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS
- A stimulation index of more than 1.4 was recorded for two concentrations of the test item (25% and 50% (v/v) in acetone/olive oil).
The Stimulation Index (SI) calculated by pooled approach was respectively 1.38, 2.13 and 3.81 for the treated group at 10%, 25% and 50%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 10.4%.

LOCAL IRRITATION
- Slight dryness was registered on D6 in the treated group at 25%. Slight dryness to dryness was registered on D5 and D6 in the treated group at 50%. An increase in ear thickness (+15.2%) was recorded at 50% and an increase in ear weight (+10.8%, + 12.8%, +56.4%) was recorded at respectively 10%, 25% and 50%. Therefore, the test item must be considered "slightly irritant" at the concentration of 50% and "non-irritant" at the concentrations of 10% 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 item must be classified 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 10%, 25% and 50% (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 100 %. Based on the results of preliminary study, the dose levels selected for the main test were 10%, 25% and 50% (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. Slight dryness was registered on D6 in the treated group at 25%. Slight dryness to dryness was registered on D5 and D6 in the treated group at 50%. An increase in ear thickness (+15.2%) was recorded at 50% and an increase in ear weight (+10.8%, + 12.8%, +56.4%) was recorded at respectively 10%, 25% and 50%. Therefore, the test item must be considered "slightly irritant" at the concentration of 50% and "non-irritant" at the concentrations of 10% and 25%. A stimulation index of more than 1.4 was recorded for two concentrations of the test item (25% and 50% (v/v) in acetone/olive oil). The Stimulation Index (SI) calculated by pooled approach was respectively 1.38, 2.13 and 3.81 for the treated group at 10%, 25% and 50%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 10.4%.

 

Under these experimental conditions, the test item must be classified in category 1B according to the annex I of the Regulation (EC) No. 1272/2008 and to the GHS.

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