Registration Dossier
Registration Dossier
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 201-746-1 | CAS number: 87-44-5
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 06 January 2017 - 03 February 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- Commission Regulation 440/2008, 1st ATP 2009
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- 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
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: Epidermal keratinocytes
- Source strain:
- other: Human
- Details on animal used as source of test system:
- SOURCE ANIMAL
- Source: Human
- Tissue: normal epidermal keratinocytes - Justification for test system used:
- Elicited via a disturbance of the desquamation process and an inflammatory response (i.e. papules, vesicles, bullae and oedema), skin irritation requires penetration of the stratum corneum and elicitation of a biological response. Skin irritation is defined in Section 3.2.1.1 of Annex I to the CLP regulation as “[…] the production of reversible damage of the skin following the application of a test substance for up to 4 hours”
According to Annex VII of the REACH Regulation, if new test data are required these must be derived from in vitro methods only. The EpiDerm™ human skin model (OECD 439) is an accepted in vitro test method to detect skin corrosion/irritation (Category 1 or 2) and/or the absence of effects (not classified under CLP). - Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Reconstructed Human Epidermis
- Source: MatTek Corporation (82105 Bratislava, Slovakia)
- Tissue batch number(s): 23390
- Date of initiation of testing: 06 January 2017
- Pre-warming: Prior to exposure the EpiDerm™ tissues were inspected for quality.
EPIDERM™ QUALITY CRITERIA
- Air bubbles between agarose and insert were not >30% of the total surface
- Liquid on top of the insert was removed with sterile cotton tips
- If moisture was observed on top of the inserts after the pre-incubation or in case of visible defects the respective skin models were discarded.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: DPBS, at least 15 times
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None, protocol dated 29/06/2015
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro, version 4.7.1
- Wavelength: 570 nm
- Filter: 570 nm filter
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Manufacturer MTT assay, 1.581 ± 0.226 OD (acceptable range: 1.0 to 3.0)
- Barrier function: Manufacturer ET-50 assay, 6.79 hrs (acceptable range: 4.77 to 8.72 hrs)
- Contamination: Manufacturer long term antibiotic and antimycotic free culture, sterile (acceptable criteria: no contamination)
NUMBER OF REPLICATE TISSUES: 3
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: The test chemical is considered to be irritant to skin in accordance with UN GHS and EU CLP Category 2 if the tissue viability after exposure and post-treatment incubation is ≤ 50%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 μL (corresponding to 47 μL/cm2)
- Concentration (if solution): Undiluted
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SLS solution in deionised water - Duration of treatment / exposure:
- 60 minutes
- Duration of post-treatment incubation (if applicable):
- Approximately 42 hours
- Number of replicates:
- 3 per test item treatment, positive control and negative control
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- other: relative absorbance
- Run / experiment:
- 1
- Value:
- 99.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- other: relative absorbance
- Run / experiment:
- 2
- Value:
- 103.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- other: relative absorbance
- Run / experiment:
- 3
- Value:
- 114.9
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- other: relative absorbance
- Run / experiment:
- mean
- Value:
- 105.9
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: None reported
- Direct-MTT reduction: Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour.
- Colour interference with MTT: The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water did not led to a change in colour.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Mean OD was 1.490 (criteria: ≥ 0.8 and ≤ 2.8)
- Acceptance criteria met for positive control: Decrease in the relative absorbance as compared to the negative control was 2.2% (criteria: ≤ 20%)
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations between the % variability of the test item, the positive and negative controls in the main test ≤ 9.1% (criteria: ≤ 18%)
- Range of historical values if different from the ones specified in the test guideline: 2.76 to 6.77% range of viabilities for positive control; 1.34 to 2.00 range of absorbance for negative control
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (105.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
- Executive summary:
Skin irritation of the test item was evaluated with the EpiDerm Reconstructed Human Epidermis Model. Cell viability of the multi-layered tissue culture of highly differentiated epidermal keratinocytes topically exposed to the test substance was evaluated using the MTT assay, which measures the conversion of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) into a blue formazan salt.
Undiluted test item was applied to the EpiDerm tissue for 60 minutes, alongside a negative and positive control. The mean relative absorbance value of the test item, corresponding to the cell viability did not decrease (105.9%; threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
The test item passed the MTT- and the Colour Interference pre-tests. Conducted according to OECD Test Guideline 439 and GLP, the study is considered to be reliable without restriction (Klimisch 1).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
