Reaction Mass of 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-octamethyldecahydro-1,6-methanonaphthalen-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March to 7 April 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Method of calculation different than proposed in OECD 429.

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:

Different cell count method

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificates - Afssaps - signed the 5 July of 2007

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-octamethyldecahydro-1,6-methanonaphthalen-1-ol

EC Number:

947-570-5

Molecular formula:

Not applicable

IUPAC Name:

Reaction Mass of 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-octamethyldecahydro-1,6-methanonaphthalen-1-ol

Specific details on test material used for the study:

- Name of test material (as cited in study report): Patchouli essential oil
- Physical state: brown liquid
- Analytical purity: not applicable; considered as 100 % for the study
- Origin: Plantae (from Indonesia)
- Binomial family: Labiatae
- Binomial name: Pogostemon cablin

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier, Le genest Saint Isle
- Age at study initiation: 8 weeks
- Weight at study initiation: 22-25 g
- Housing: Animals were housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 41-55 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 100 % w/w in acetone/olive oil 4:1
Main test: 10, 25, 50 % w/w in acetone/olive oil 4:1

No. of animals per dose:

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

Details on study design:

RANGE FINDING TESTS:
- Using available information regarding the 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 test item at concentrations of 100% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).

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 sensitizer if at least one concentration of the test item results in a 1.4 fold or greater increase in 3H Thymidine incorporation compared to control values. Any test item failing to produce a 1.4 fold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3).
Five hours later animals were killed 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 cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainer in 4 mL of PBS 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. The lymphocytescell were counting using a cell counter (Beckman Coulter Z2).
For the run, the lower size selected was 5 µm and the upper size was 15 µm (average 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 relatives to that recorded for the control nodes (Stimulation Index)(Stimulation Index).

The mouse was observed once daily on Days 1, 2, 3, 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- The thickness of each ear was measured using a micrometer, pre-dose on Day 1 and on Day 6

Positive control substance(s):

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

Statistics:

The Stimulation Index (SI) was calculated from the formula SI = cell count of treated group / cell count of control group.

Results and discussion

Positive control results:

A group of 4 mice received the vehicle alone and a further group of four mice received a positive control (alpha-Hexylcinnamaldehyde) diluted at 25% in the vehicle, in the same manner.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.01

Test group / Remarks:

Treated group 10%

Results 2

Parameter:

SI

Value:

1.45

Test group / Remarks:

Test group 25%

Results 3

Parameter:

SI

Value:

2.24

Test group / Remarks:

Test group 50%

Results 4

Parameter:

SI

Value:

2.53

Test group / Remarks:

Test group 100%

Results 5

Parameter:

other: Ec1.4

Value:

23.3

Any other information on results incl. tables

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Bodyweight

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

A moderate (+15.2%) to important (+41.6%) increase in ear thickness was recorded respectively at the concentration of 50% and 100%. An important increase in ear weight (+60.3%) was recorded at the concentration of 100%.

Positive control:

A Stimulation Index (SI) of 2.22 was recorded for the positive control diluted at 25 % in the vehicle.

The SI result was more than 1.4 as expected.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Specific count

Conclusions:

In view of the results, under these experimental conditions, the test item Patchouli EO was not considered to be a sensitiser under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca (CBA/J) strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 10%, 5% or 25% w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone. 

The Stimulation Index (SI) was calculated from the formula SI = cell count of treated group / cell count of control group. 

 

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 2.22, when tested at 25 % v/v. The test system was therefore considered to be more sensitive than planed (2.22 instead of 1.4).

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Under the test conditions, test material should not be classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) .