Oils, cinnamon

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-08-2021 to 21-10-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

14 June 2021

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

Essential oil of Cinnamon bark obtained from the bark of Cinnamomum zeylanicum, Lauraceae by distillation

EC Number:

943-043-9

Cas Number:

8015-91-6

IUPAC Name:

Essential oil of Cinnamon bark obtained from the bark of Cinnamomum zeylanicum, Lauraceae by distillation

Test material form:

liquid

Remarks:

Pale yellow to dark yellow or brownish yellow color and powerful diffusive warm-spicy sweat and tenacious odour.

Details on test material:

Identification: Cinnamon Bark Oil (CBO)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: provided by sponsor
- Expiration date of the lot/batch: June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: full tight containers in a cool and dry place protected from heat and light

OTHER SPECIFICS: UVCB

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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 SA, RHE/S/17 (0.50 cm2) (Batch No. 21-RHE-165. Episkin, Lyon, France)
- Tissue batch number: 21-RHE-165
- Production date: 19 October 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: washed with 25x 1 mL of DPBS
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/mL
- Incubation time: 2 hours and 55 minutes between 37.1°C
- Spectrophotometer: BioTek ELx800 absorbance microplate reader (BioTek Gen5 ELISA V1.05.11 software)
- 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.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues: Batch no. 21-RHE-147 and 21-RHE-061. (frozen on 21 September 2021 and 28 April 2021)
- Procedure used to prepare the killed tissues: The tissues were defrozen the day of the treatment and on the same day the insert (filter + epidermis) was gently removed from the agarose. The inserts were placed in a 24 wells culture plate which had been previously filled with 300 µL of growth medium (Episkin SA, batch No. 21 SGM 116) for 2 hours and 33 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA, batch No. 21 SMM 041)
- N. of replicates : 2
- Method of calculation used: True viability % = ((living tissues OD exposed to test item – killed tissues OD exposed to test item (NSMTT)) / living tissues OD exposed to negative control) x 100
DECISION CRITERIA
- The test substance is considered to be non-irritant to skin if the mean percent viability after 42 minutes of exposure and 42 hours of post-treatment incubation >50%.
- The test substance is considered to be irritant / corrosive to skin if the mean percent viability after 42 minutes of exposure and 42 hours of post-treatment incubation ≤50%. (a skin corrosion test is then needed to determine whether the test substance is either irritant or corrosive)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16µL
- Concentration (if solution): 5% SDS. Prepared by weighing 0.5g of SDS (SIGMA - Batch No. STBJ3028) in a 10 mL volumetric flask q.s. 10 mL of distilled water. The preparation was magnetically stirred, just before the treatment to obtain a colourless solution.

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 for Test material, Negative control and Positive control.
2 for NSMTT

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

0.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: yes
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 42 minutes exposure of 1.3%. The OD values of the negative control tissues were 0.969, 0.843 and 0.833. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, for the test item, the negative and positive control 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

Applicant's summary and conclusion

Interpretation of results:

other: irritating/corrosive to skin based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Under the conditions of this test the mean corrected viability of the treated tissues was 0.3%. Based on this result, the substance needs to be classified for skin irritation/corrosion, based on CLP criteria (Annex I of 1272/2008/EC) and a subsequent test was performed (OECD TG 431).

Executive summary:

The skin irritation potential of Cinnamon bark oil was tested in accordance with OECDTG439. Undiluted Cinnamon bark oil was applied to the skin model for 42 minutes. After a 42-hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. Cinnamon bark oil was identified as a direct MTT reducer (2.0% viability of the NSMTT control) but did not cause colour interference. The mean corrected tissue viability obtained after 42 minutes treatment with Cinnamon bark oil compared to the negative control tissues was 0.3%. The positive control had a mean cell viability of 1.3% and the OD values of the negative control tissues were 0.969, 0.843 and 0.833. 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.