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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:
2017
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

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

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).

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