Reaction mass of [1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene and caryophyllene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 June 2011 - 15 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The LLNA was performed before the REACH regulation came into force requesting in vitro skin sensitisation information first (October, 2016)

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:

2004

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene

EC Number:

207-491-2

EC Name:

[1S-(1α,3aβ,4α,8aβ)]-decahydro-4,8,8-trimethyl-9-methylene-1,4-methanoazulene

Cas Number:

475-20-7

Molecular formula:

C15H24

IUPAC Name:

(1S,3aR,4S,8aS)-4,8,8-trimethyl-9-methylidenedecahydro-1,4-methanoazulene

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J strain mice were supplied by Jackson Laboratories, Bar Harbor, ME 04609. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of six days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail. At the start of the study the animals were in the weight range of 18 to 25 g, and were eight to twelve weeks old.

The animals were group housed per cage. Free access to tap water and food (Harlan Teklad Certified Rodent Chow 2016C) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 24 to 28°C and 16 to 69 %, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

other: 3:1 Diethyl Phthalate:Ethanol (3:1 DEP:EtOH)

Concentration:

Test item concentrations of 2.5%, 5%, 10%, 25% or 50% v/v in vehicle.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Justification for route and dose levels:
The dermal route was selected as this is the route required for this model of hypersensitivity.
In general, the doses have been selected so that the highest concentration maximizes exposure while avoiding systemic toxicity and excessive local irritation. Doses were selected based on known reported uses of the material.
The frequency of dosing is the convention for this type of study.

Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 2.5%, 5%, 10%, 25% or 50% v/v in vehicle. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Approximately 24 ± 2.5 hours separated each application of test substance. A further group of five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, α Hexylcinnamaldehyde, at a concentration of 5%, 15% or 35% v/v in 3:1 Diethyl Phthalate:Ethanol (3:1 DEP:EtOH) was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected i.v. with 250 µL of sterile saline containing 3H methyl thymidine (3HTdR:1mCi/ml, specific activity 5 Ci/mmol, Moravek Biochemicals, Inc.) giving a total of 20 µCi to each mouse.

Observations
Mortality/morbidity: Daily on days 1 to 6.
Clinical Observations: Observations were performed prior to dose administration and following dose administration. Clinical observations were performed once daily on Days 4-6. particular attention was given to the application sites. Any significant alterations to the application sites, and the general appearance of the pinnae, including build up of test article, was recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Dermal irritation: Animals were examined daily for signs of erythema and edema. Irritation was scored and recorded using the Draize scoring system. Scoring was performed prior to dosing on Days 1-3.

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records.

Preparation of Single Cell Suspension: A single cell suspension was prepared from the lymph nodes of each individual mouse (un-pooled). Celle were washed twice with PBS and precipitated with 5% trichloroacetic acid (TCA) overnoght at 2-8°C.

Determination of 3HTdR Incorporation: The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H-thymidine was measured in a β-scintillation counter.

Positive control substance(s):

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

Statistics:

All data was collected manually except for the data generated by the scintillation counter (Beckman LS 6000 SC). SYSTAT version 0.01, developed by SPSS, Inc was used for statistical analysis.

Results and discussion

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, gave a Stimulation Index of greater than 3 when tested at a concentration of 15 and 30 % v/v in 3:1 Diethyl Phthalate:Ethanol (3:1 DEP:EtOH).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

%

Value:

31.4

Results 2

Key result

Parameter:

other: NOAEL

Remarks:

%

Value:

25

Results 3

Key result

Parameter:

SI

Value:

1.5

Remarks on result:

other: 2.5%

Results 4

Key result

Parameter:

SI

Value:

0.9

Remarks on result:

other: 5%

Results 5

Key result

Parameter:

SI

Value:

2

Remarks on result:

other: 10%

Results 6

Key result

Parameter:

SI

Value:

2.1

Remarks on result:

other: 25%

Results 7

Key result

Parameter:

SI

Value:

5.6

Remarks on result:

other: 50%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION
The following SI values were derived at 2.5, 5, 10, 25 and 50%: 1.5, 0.9, 2.0, 2.1 and 5.6, respectively.

EC3 CALCULATION
Based on the results the EC3 is 31.4%.

CLINICAL OBSERVATIONS:
There was no mortality and all animals appeared normal throughout the study.
No erythema or edema was noted in any of the mice in the vehicle group, positive groups or in those treated with test substance concentrations. The ears of the mice treated with 35% HCA appeared wet on Days 2 through 4. There were no other findings.

BODY WEIGHTS
On day 1 there were statistically significant differences in the mean body weights for the groups treated with 5 and 25% Longifolene and 5 and 35% HCA when compared to the vehicle control group (p < 0.01, p < 0.001, p < 0.01, and p < 0.01, respectively). On Day 6 there were statistically significant differences in the mean body weights for the groups treated with 5% Longifolene and 5% HCA when compared to the vehicle control group (p < 0.05 and p < 0.01, respectively). However, when the mean body weight changes were compared, the only statistically significant difference that was observed was an increase for the group treated with 25% Longifoline when compared to the vehicle group (p < 0.001 ). None of these differences were biologically relevant. Therefore, administration of the test article or positive control did not appear to exert any overt toxicity.

Applicant's summary and conclusion

Interpretation of results:

other: Sensitising resulting in Cat 1B

Remarks:

According to Regulation (EC) No 1272/2008 and its updates.

Conclusions:

The SI values calculated for the substance concentrations 2.5, 5, 10, 25 and 50 % were 1.5, 0.9, 2.0, 2.1 and 5.6, respectively. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 31.4% v/v was calculated. A NOEC of 25% is derived. The test isubstance was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the substance has been tested according to OECD TG 429 test guideline and GLP principles. At 2.5, 5, 10, 25 and 50% the substance showed SI values of 1.5, 0.9, 2.0, 2.1 and 5.6, respectively. Reliable negative and positive controls were included. These results show that the test substance could elicit a SI ≥ 3. An EC3 value of 31.4% w/v was calculated. A NOEC of 25% is derived. Based on the results, the substance was considered to be a sensitiser.