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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-07-2017 to 21-07-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
LIGHT YELLOW, SOLID MASS
Details on test material:
Name of test material as cited in study report: Cedrol - Cedarwood Texas oil distilled
Mfg Date 01/26/2016
Exp Date 01/25/2019

- Identification: Cedrol, Cedarwood Texas oil distilled
- Appearance: Light yellow, solid mass
- Batch: B-64530
- Purity/Composition: UVCB
- Test item storage: At room temperature
- Stable under storage conditions until: 25 January 2019 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

ODOUR DESCRIPTION: CONFORMS TO STANDARD
Specific details on test material used for the study:
- Identification: Cedrol, Cedarwood Texas oil distilled
- Appearance: Light yellow, solid mass
- Batch: B-64530
- Purity/Composition: UVCB
- Test item storage: At room temperature
- Stable under storage conditions until: 25 January 2019 (expiry date)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item should be equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous sample.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation
Source strain:
other: Keratinocyte strain 00267
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model
- Tissue batch number(s): Lot no.: 26708 Kit L and Kit M
- Production date: 19-07-2017
- Date of initiation of testing: 20-07-2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 min room temperature, 1 hour 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing step: washed with phosphate buffered saline
- Damage: no visual damage

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM)
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The absolute mean OD570 of the two tissues of the negative control should reasonably be within the laboratory historical control data range. The mean relative tissue viability following 1-hour exposure to the positive control should be <15 %.
- Reproducibility: In the range 20 - 100% viability, the Coefficient of Variation (CV) between tissue replicates should be  30%.

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
- 1 experiment with 3 minute application (in duplicate)
- 1 experiment with 1 hour application (in duplicate)

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >= 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
- The test substance is considered to be non-corrosive to skin if
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution):8N
Duration of treatment / exposure:
3 min / 1 hour
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
104
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
116
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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative/positive control:
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit <=2.8) and the laboratory historical control data range.
- Acceptance criteria met for variability between replicate measurements: The mean relative tissue viability following the 1-hour exposure to the positive control was 10%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was <=8.0%, indicating that the test system functioned properly.
- Range of historical values (OD570):

Negative control Positive control Positive control
3-minute treatment 1-hour treatment 3-minute treatment 1-hour treatment 3-minute treatment 1-hour treatment
Range 1.324 – 2.615 1.361 – 2.352 0.0172 – 0.56 0.046 – 0.339 6 – 25 3 – 13
Mean 1.84 1.85 0.19 0.14 11.03 7.45
SD 0.26 0.22 0.09 0.06 4.39 2.51
n 81 83 80 77 38 38

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria
Conclusions:
Under the conditions of the test, the Cedrol, Cedarwood Texas oil distilled was not corrosive. Based on this result, the substance does not need to be classified for skin corrosion in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

Cedrol, Cedarwood Texas oil distilled was evaluated for its ability to induce skin corrosion on a human three dimensional epidermal model according to OECD TG 431.  Cedrol, Cedarwood Texas oil distilled was applied topically for 3 minutes and 1 hour. The test substance was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous liquid sample.  Cedrol, Cedarwood Texas oil distilled (50 µl) was applied directly on top of the skin tissue.  

The positive control had a mean relative tissue viability of 10% after the 1-hour exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥ 0.8 and upper acceptance limit ≤ 2.8) and the laboratory historical control data range.  In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤ 8.0%, indicating that the test system functioned properly.

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Cedrol, Cedarwood Texas oil distilled compared to the negative control tissues was 104% and 116%, respectively.  Because the mean relative tissue viability for Cedrol, Cedarwood Texas oil distilled was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment Cedrol, Cedarwood Texas oil distilled is considered to be not corrosive.

In conclusion, Cedrol, Cedarwood Texas oil distilled is not corrosive in the in vitro skin corrosion test under the experimental conditions described.