Essential oil of Blue tansy obtained from the flowers and leaves of Tanacetum annuum (Asteraceae) by steam distillation

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2019 - July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Essential oil of Tanacetum annuum

IUPAC Name:

Essential oil of Tanacetum annuum

Test material form:

liquid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2, Batch no.: 19-EKIN-019 and 19-EKIN-013), SkinEthic Laboratories, Lyon, France

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): Batch no.: 19-EKIN-019 and 19-EKIN-013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.3 - 37.1°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 (acceptability criteria).

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

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

Main experiment

Run / experiment:

1

Value:

80

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

15

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

Main experiment

Run / experiment:

2

Value:

30

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

15

Results 3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Main experiment

Run / experiment:

3

Value:

109

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

15

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: yes (7.2%)
- Colour interference with MTT: yes (non-specific, 4.4% of negative control tissues)

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 15%. 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 with positive or negative control was < 6%, 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: no - SD to high between test item tissues

Any other information on results incl. tables

Since the individual values of the first experiment were both above and below the criterium of 50% (30, 80 and 109% respectively) the test has to be repeated (planned for July 2019).

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, Blue Tansy Oil is not considered a likely 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). However, the results were spread over two categories (80%, 30% and 109%), which lead to a high standard deviation and therefore this assay will be repeated.

Executive summary:

The skin irritation potential of Blue Tansy Oil was tested in accordance to OECD TG 439. Undiluted test item 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. Both the positive (15% viability) and the negative control were within the historical control data range and therefore considered valid. The standard deviation value of the percentage viability of three tissues treated with positive or negative control was < 6%, indicating that the test system functioned properly. The tissue viability obtained after 15 ± 0.5 minutes treatment with test item compared to the negative control tissues was 30, 80 and 109%.

Since the mean relative tissue viability for Blue Tansy Oil was above 50% after 15 ± 0.5 minutes treatment Blue Tansy Oil could be considered a non-irritant. Therefore, Blue tansy oil 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). Results were however spread over two categories (80%, 30% and 109%), which lead to a high standard deviation and therefore this assay will be repeated.