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Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The REACH Annex VII information requirements were revised in 2016 to endorse a battery of in vitro assays for skin sensitisation. Conducted prior to the validation and regulatory acceptance of alternative methods, the reliable and GLP compliant in vivo Freund’s Complete Adjuvant Test (FCAT) was considered sufficient to fulfil the information requirement.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The REACH Annex VII information requirements were revised in 2016 to endorse a battery of in vitro assays for skin sensitisation. Conducted prior to the validation and regulatory acceptance of alternative methods, the reliable and GLP compliant in vivo LLNA was considered sufficient for use as a supporting study.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Analytical grade β-Caryophyllene and Caryophyllene oxide (>98%) purchased from Fluka Chemie (Switzerland) were used as reference standards. β-Caryophyllene was purchased from Firmenich (98.6% stated, 95% confirmed by GC MS), for use in the air exposure experiments, in which the test item was exposed to air in an Erlenmeyer flask covered with aluminium foil at standard room temperature. It was stirred for 1 hour, four times per day for 48 weeks. Samples were taken at intermittent time periods to determine the concentrations of components over time.

GC analyses showed that the amount of β-caryophyllene started to decrease immediately upon air exposure. After 5 weeks almost 50% of the original compound was consumed and after 48 weeks only 1% remained. The total concentration of caryophyllene oxide in the different samples of air-exposed β-caryophyllene was found to increase rapidly in the beginning of the oxidation process to reach a concentration of about 40% in the oxidation mixture.

In the photooxidation experiments, β-Caryophyllene (0.77 g, 3.8 mmol, Firmenich), was added to a chloroform solution of tetrabutylammonium salt of Bengal rose (40 ml), and irradiated for 6 hours using a Rayonet reactor with oxygen flow. The solvent was removed under reduced pressure and ether, and the product purified by flash chromatography.
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan (The Netherlands)
- Age at study initiation: 9 weeks old
- Housing: cages with hepa-filtered airflow, under conventional conditions in light-, humidity- and temperature controlled rooms
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Oxidised β-caryophyllene (air exposed for 10 weeks) was tested in concentrations 30%, 10%, and 3% (w/v) (1.36, 0.45, and 0.14 M, calculated from the molecular weight of caryophyllene oxide) and the caryophyllene hydroperoxides (obtained from photooxidation) in concentrations 15%, 5%, and 0.5% (0.63, 0.21, and 0.02 M). The control mice recieved the acteone: olive oil vehicle alone.
No. of animals per dose:
4
Details on study design:
MAIN STUDY
The sensitising potential was investigated according to the local lymph node assay (LLNA) in mice. Nine week old female CBA/Ca mice were administered 25µL of the test item dissolved in acetone:olive oil (4:1) on the dorsum of both ears for three consecutive days. Test solutions were prepared fresh each day. Oxidised β-caryophyllene (air exposed for 10 weeks) was tested in concentrations 30%, 10%, and 3% (w/v) (1.36, 0.45, and 0.14 M, calculated from the molecular weight of caryophyllene oxide) and the caryophyllene hydroperoxides (obtained from photooxidation) in concentrations 15%, 5%, and 0.5% (0.63, 0.21, and 0.02 M). Five days after the initial treatment, all mice were injected intravenously through the tail vein with [methyl-3H]thymidine in 250 µL phosphate-buffered saline (PBS). After 5 h the mice were sacrificed and single-cell suspensions of lymph node cells were prepared. Thymidine incorporation was measured by β-scintillation counting on a Beckman LS 6000TA instrument.

EVALUATION CRITERIA
Results were expressed as mean dpm (disintegrations per minute)/lymph node for each experimental group and as stimulation index (SI), i.e. test group/control group ratio. If the test material at one or more concentrations, caused an SI greater than 3, it was considered to be positive in the LLNA.
Positive control substance(s):
not specified
Statistics:
The EC3 value (the estimated concentration required to induce an SI of 3) was calculated by linear interpolation.
Key result
Parameter:
EC3
Value:
26.2
Test group / Remarks:
Oxidised β-caryophyllene
Parameter:
EC3
Value:
0.6
Test group / Remarks:
Caryophyllene hydroperoxides
Parameter:
SI
Value:
1.4
Test group / Remarks:
Oxidised β-Caryophyllene 3% (air exposed for 10 weeks)
Parameter:
SI
Value:
1.6
Test group / Remarks:
Oxidised β-Caryophyllene 10% (air exposed for 10 weeks)
Parameter:
SI
Value:
3.3
Test group / Remarks:
Oxidised β-Caryophyllene 30% (air exposed for 10 weeks)
Parameter:
SI
Value:
2.7
Test group / Remarks:
Caryophyllene hydroperoxides 0.5%
Parameter:
SI
Value:
12.9
Test group / Remarks:
Caryophyllene hydroperoxides 5%
Parameter:
SI
Value:
23.3
Test group / Remarks:
Caryophyllene hydroperoxides 15%
Cellular proliferation data / Observations:
Oxidised β-Caryophyllene was shown to be weakly positive in the LLNA, and only a 30% concentration produced an SI greater than 3. The EC3 value was calculated to be 26.2% (1.2 M). The caryophyllene hydroperoxides were strongly sensitising, with an EC3 value of 0.6% (0.03 M).

Table 1. LLNA responses for a sample of β-caryophyllene air exposed for 10 weeks and a mixture of caryophyllene hydroperoxides in vehicle (AOO)

 Test material  Concentration  DPM/lymph node  SI
 Oxidised β -caryophyllene  Control  395  -
   3%  554  1.4
   10%  640  1.6
   30%  1310  3.3
 Caryophyllene hydroperoxides  Control  635  -
   0.5%  1730  2.7
   5%  8170  12.9
   15%  14800  23.3
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The EC3 of a sample of β-caryophyllene air exposed for 10 weeks was determined to be 26.2% and the EC3 of a mixture caryphyllene hydroperoxides (produced from the photooxidation with Bengal rose) was determined to be 0.6%.
Caryophyllene has two isolated double bonds that are capable of reacting with dioxygen in triplet state (triplet oxygen) or singlet state (singlet oxygen).
A reaction of caryophyllene with triplet oxygen yielding to autoxidation products requires the presence of light and air and the absence of anti-oxidants. A reaction of caryophyllene with singlet oxygen is a [2+2]-cycloaddition and requires the excitation of triplet oxygen. This excitation can be achieved by UV irradiation of a diluted, cooled solution of caryophyllene in a suitable solvent (e.g. chloroform) containing a sensitiser (e.g. Bengal rose). The solvent must be transparent to the UV radiation and must not quench the excited state of neither the sensitiser nor the singlet oxygen. Water does not have the necessary properties and aqueous preparations of caryophyllene will not produce oxidation products from a reaction with singlet oxygen. For that reason, the EC3 value of 26.2% is considered to be the relevant value for β-caryophyllene.
Executive summary:

The sensitising potential was investigated according to the local lymph node assay (LLNA) in mice. Oxidised β-caryophyllene (air exposed for 10 weeks) was tested in concentrations 30%, 10%, and 3% (w/v) (1.36, 0.45, and 0.14 M, calculated from the molecular weight of caryophyllene oxide) and caryophyllene hydroperoxides (obtained from photooxidation) in concentrations 15%, 5%, and 0.5% (0.63, 0.21, and 0.02 M).

 

Nine week old female CBA/Ca mice were administered 25µL of the test item dissolved in acetone:olive oil (4:1) on the dorsum of both ears for three consecutive days. Five days after the initial treatment, all mice were injected intravenously through the tail vein with [methyl-3H]thymidine in 250 µL phosphate-buffered saline (PBS). After 5 h the mice were sacrificed and single-cell suspensions of lymph node cells were prepared. Thymidine incorporation was measured by β-scintillation counting on a Beckman LS 6000TA instrument. Oxidised β-Caryophyllene was shown to be weakly positive in the LLNA - only a 30% concentration produced an SI greater than 3. The EC3 value was calculated to be 26.2% (1.2 M). The caryophyllene hydroperoxides were strongly sensitising, with an EC3 value of 0.6% (0.03 M). The study demonstrated dose-dependent increases in sensitising potential (stimulation index) with concentration of oxidised β-caryophyllene. 

 

Reported in a peer-reviewed journal, the study was comparable to an OECD Guideline 429 study and considered reliable with restriction (Klimisch 2). The study was considered appropriate for use as a supporting study, evaluating oxidised β-Caryophyllene (i.e. the degradation product).

Data source

Reference
Reference Type:
publication
Title:
The fragrance chemical b-caryophyllene—air oxidation and skin sensitization
Author:
Sköld M, Karlberg AT, Matura M, Börje A
Year:
2006
Bibliographic source:
Food and Chemical Toxicology, 44, 538–545

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
not specified
Type of study:
Freund's complete adjuvant test
Justification for non-LLNA method:
The REACH Annex VII information requirements were revised in 2016 to endorse a battery of in vitro assays for skin sensitisation. Conducted prior to the validation and regulatory acceptance of alternative methods, the reliable and GLP compliant in vivo Freund’s Complete Adjuvant Test (FCAT) was considered sufficient to fulfil the information requirement.

Test material

Constituent 1
Chemical structure
Reference substance name:
Caryophyllene
EC Number:
201-746-1
EC Name:
Caryophyllene
Cas Number:
87-44-5
Molecular formula:
C15H24
IUPAC Name:
(1R,4E,9S)-4,11,11-trimethyl-8-methylidenebicyclo[7.2.0]undec-4-ene
Specific details on test material used for the study:
Analytical grade β-Caryophyllene and Caryophyllene oxide (>98%) purchased from Fluka Chemie (Switzerland) were used in the FCAT test.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Remarks:
ablino
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Sex: Female
- Weight at study initiation: 290 to 380 g
- Housing: Macrolon cages
- Diet: Guinea pig standard diet, ad libitum
- Water: ad libitum

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: FCA/water (1/1) emulsion
Concentration / amount:
Control / 0.1 mL
6.8% (w/w) / 0.1 mL β-caryophyllene
7.3% (w/w) / 0.1 mL caryophyllene oxide
Day(s)/duration:
Day 0
Adequacy of induction:
not specified
Route:
intradermal
Vehicle:
other: FIA/water (1/1) emulsion
Concentration / amount:
Control / 0.1 mL
6.8% (w/w) / 0.1 mL β-caryophyllene
7.3% (w/w) / 0.1 mL caryophyllene oxide
Day(s)/duration:
Days 6 and 10
Adequacy of induction:
not specified
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
Control
1.4, 6.8 and 13.6% β-caryophyllene
0.7, 1.5, 7.3 and 14.7% caryophyllene oxide
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
16 per test item treatment, 15 for the control
Details on study design:
RANGE FINDING TESTS: A pre-test was performed in which five animals received one intradermal injection of FCA, with 0.7, 1.4, 6.8 and 13.6% β-caryophyllene and 0.7, 1.5, 7.3 and 14.7% caryophyllene oxide in non-stabilised petrolatum (w/w). No significant irritation was observed at any test item concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Test groups: 6.8% β-caryophyllene or 7.3% caryophyllene oxide
- Control group: Vehicle control
- Site: Upper back
- Frequency of applications: Days 0, 6 and 10

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21
- Exposure period: 24 hrs
- Test groups: 0.7, 1.4, 6.8 and 13.6% β-caryophyllene or 0.7, 1.5, 7.3 and 14.7% caryophyllene oxide
- Control group: Vehicle (petrolatum)
- Site: Shaved flank
- Evaluation (hr after challenge): 48 and 72 after start of exposure
- Occlusion: Finn chambers

OTHER: The minimum criterion for a positive reaction was a confluent erythema. The results from the FCAT experiment were statistically evaluated using the Fisher exact test. A p-value of <0.05 was considered statistically significant.
Challenge controls:
Vehicle control
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
7.3% test item induction /0.7 % test item challenge
No. with + reactions:
0
Total no. in group:
16
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
7.3 % test item induction / 0.7% test item challenge
No. with + reactions:
0
Total no. in group:
16
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
7.3% test item induction / 1.5% test item challenge
No. with + reactions:
0
Total no. in group:
16
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
7.3% test item induction / 1.5% test item challenge
No. with + reactions:
0
Total no. in group:
16
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
7.3% test item induction / 7.3% test item challenge
No. with + reactions:
3
Total no. in group:
16
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
7.3% test item induction / 7.3% test item challenge
No. with + reactions:
6
Total no. in group:
16
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
7.3% test item induction / 14.7% test item challenge
No. with + reactions:
6
Total no. in group:
16
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
7.3% test item induction / 14.7% test item challenge
No. with + reactions:
8
Total no. in group:
16
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 1. Results from sensitisation experiments on β-caryophyllene and caryophyllene oxide in guinea pigs, using the FCAT method

 Challenge test item concentrations  Number of animals with positive reactions               
   Induction - control (n = 15)   

 Induction - 6.8% β-caryophyllene (n = 16)

 Induction - 7.3% caryophyllene oxide (n = 16)   

 

 48 h

 72 h

 48 h

 72 h

 48 h

 72 h

 Control

 0

 0

 0

 0

 0

 0

 Challenge - 1.4% β-caryophyllene

 0

 0

 0

 0

 0

 0

 Challenge - 6.8% β-caryophyllene

 0

 0

 0

 0

 1

 1

 Challenge - 13.6% β-caryophyllene

 0

 0

 0

 0

 1

 2

 Challenge - 0.7% caryophyllene oxide  0  0  0  0  0  0
 Challenge - 1.5% caryophyllene oxide  0  0  0  0  0  0
 Challenge - 7.3% caryophyllene oxide  0  0  0  0  3  6*
 Challenge - 14.7% caryophyllene oxide  1  1  0  0  6*  8**

* Significantly different from controls, p < 0.05

** Significantly different from controls, p < 0.01

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Animals induced with β-caryophyllene showed no positive reactions following challenge with β-caryophyllene or caryophyllene oxide. However, induction with caryophyllene oxide resulted in 8/16 and 6/16 positive animals at challenge concentrations of 14.7% and 7.3% caryophyllene oxide respectively.
Executive summary:

A modified Freund’s Complete Adjuvant Test (FACT) sensitisation experiment was performed in female Dunkin-Hartley albino guinea pigs. Animals were induced on days 0, 6 and 10 with intradermal injections (0.1 mL) of β-caryophyllene (6.8% w/w, Group A) or caryophyllene oxide (7.3% w/w, Group B), or the vehicle control (FCA/water (1:1) emulsion). Challenge tests were performed on day 21, in which test material was administered to shaved flanks for 24 hours and reactions were assessed at 48 and 72 hours.

 

Challenge with caryophyllene oxide demonstrated dose-dependent sensitisation at dose concentrations of =/>7.3% when intradermally injected with caryophyllene oxide but not with β-caryophyllene.

β-Caryophyllene did not induce positive reactions in exposed animals when intradermally injected with β-caryophyllene but induced positive repsonses when intradrmally injected with caryophyllene oxide.

Therefore, in this test, β-caryophyllene is not considered to be a contact allergen but caryophyllene oxide is.

The study design was similar to the OECD 406 Guideline, used sufficient numbers of animals and was considered to be reliable with restriction (Klimisch 2).

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