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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:
24-04-2017 to 01-05-2017
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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
pale yellow liquid
Details on test material:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, batch S-71699
- Expiration date of the lot/batch: 14 February 2019
- Purity test date: 14 February 2017

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

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2)
- Tissue batch number(s): 17-EKIN-017
- Production date: 25 April 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (36.3 - 37.4°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: after the exposure period, the tissues were washed with phosphate buffered saline to remove residual test item.
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the
laboratory historical control data range.
- The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

NUMBER OF REPLICATE TISSUES: 3

DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25μL undiluted
NEGATIVE CONTROL
- Amount(s) applied: 25μL undiluted
POSITIVE CONTROL
- Amount(s) applied: 25μL
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main experiment in triplicate
Value:
107
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100%
Positive controls validity:
valid
Remarks:
20%
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 20%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

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: Absorption OD570=0.676-1.336, Mean=1.01, SD=0.016, n=155
Positive control: Absorption OD570=0.036-0.549, Mean=0.16, SD=0.10, n=154
Positive control: Viability %= 2.85-45.43, Mean=15.74, SD=9.22, n=163

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
Based on the results obtained, it can be concluded that Copaiba balsam oil is not considered a skin irritant, and does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin irritation potential of Copaiba balsam oil was tested in accordance to OECD TG 439. Undiluted Copaiba balsam oil was topically applied to EPISKIN-SMTM for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Copaiba balsam oil compared to the negative control tissues was 107%. Since the mean relative tissue viability for Copaiba balsam oil was above 50% after treatment, the testing material it is not considered to be an irritant. Both the positive (20% viability) and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly.

Based on the results obtained, it can be concluded that Copaiba balsam oil is not considered a skin irritant, and does not have to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).