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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 Jul 2017 - 21 Jul 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:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
(could crystallize)
Details on test material:
Identification: Cedarwood Oil Virginia
Appearance: Pale yellow to yellow liquid
Batch: 1002960562
Purity/Composition: 100.0% (UVCB)
Test item storage: At room temperature
Stable under storage conditions until: 31 August 2017 (expiry date), extended expery date until: 28 February 2018 (21 Oct 2017)

Purity/composition correction factor: No correction factor required
Chemical name (IUPAC), synonym or trade name: Essential oil of Junipers Virginiana L. (Cupressaceae) obtained from the wood by steam distillation
CAS Number 85085-41-2
Molecular structure: UVCB
Molecular formula: UVCB
Highly reactive to water: Not indicated
Highly reactive to oxygen: Not indicated
Volatile: Not indicated
Solubility in water: Not available
Stability in water: Not available

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1002960562
- Expiration date of the lot/batch: 31 August 2017, extended expery date until: 28 February 2018 (21 Oct 2017)

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none, the liquid test item was applied undiluted (50 µl) directly on top of the tissue.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: MatTek Corporation, Ashland MA, U.S.A.
Source strain:
not specified
Justification for test system used:
Recommended test system in international guidelines (OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature for the 3 hour exposure, 37.0 ± 1.0°C (actual range 36.8 - 37.5°C) for the 1 hour exposure

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: not specified
- Observable damage in the tissue due to washing: none

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:
3 minute exposure: duplicates each measured 3x
1 hour exposure: duplicates each measured 3x

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered corrosive in the in vitro skin corrosion test 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%.
A test item is considered non corrosive in the in vitro skin corrosion test 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
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): Milli-Q undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): control
- Concentration (if solution): 8N KOH
Duration of treatment / exposure:
3 minutes or 1 hour
Duration of post-treatment incubation (if applicable):
After exposure, the DMEM was replaced by 300 µl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2.
Number of replicates:
2 per timepoint

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
94
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:
103
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: no
- Direct-MTT reduction: yes, the non-specific reduction of MTT by the test item was -0.46% and 0.29% of the negative control tissues after 3 minutes and 1 hour respectively.
- Colour interference with MTT: yes

DEMONSTRATION OF TECHNICAL PROFICIENCY: Valid historical control data ranges of the controls have been provided over the period of November 2013 to November 2016.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
Negative control Positive control Positive control
3-minute treatment(OD570) 1-hour treatment(OD570) 3-minute treatment(OD570) 1-hour treatment(OD570) 3-minute treatment(% viability) 1-hour treatment(% viability)
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 corrosive
Remarks:
Based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
Based on the results obtained, it can be concluded that Cedarwood Virginia Oil is not corrosive to skin and does not need to be classified accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin corrosion potential of Cedarwood Virginia Oil was tested according to OECDTG431. A human three dimensional epidermal model (EpiDerm) was exposed topically to 50µL undiluted

Cedarwood Virginia Oil, Milli-Q (negative control), or Potassium hydroxide (positive control) for 3 minutes, or 1 hour. Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 94% and 103%, respectively. Both the negative and the positive control were considered valid. The mean relative tissue viability for Cedarwood Virginia Oil was above 50% after the 3-minute treatment and above 15% after the 1-hour treatment. Based these results, the test 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).