Artemisia pallens, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April to 29 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.431 and under GLP compliance (the exact composition of the test item cannot be exactly determined because the element is an UVCB substance. This deviation to GLP is under the responsibility of the Sponsor and is considered as without impact on the conclusion of the 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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Dated to 18 June 2019.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Artemisia pallens, ext.

EC Number:

295-155-6

EC Name:

Artemisia pallens, ext.

Cas Number:

91844-86-9

Molecular formula:

not applicable for NCS substances

IUPAC Name:

Essential oil of Artemisia pallens Wall. Ex DC (Asteraceaé) obtained from leaves and stems by steam distillation old name: Essential oil of Artemisia pallens Wall. Ex DC (Compositae) obtained from leaves and stems by steam distillation

Test material form:

liquid

Details on test material:

- Name of test substance: DAVANA ESSENTIAL OIL D0403
- Batch No.: 5030043113
- Storage: room temperature, darkness
- Expiration date of the lot/batch: 22 October 2021

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance: DAVANA ESSENTIAL OIL D0403
- Batch No.: 5030043113
- Expiration date of the lot/batch: 22 October 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage: room temperature, darkness

FORM AS APPLIED IN THE TEST
The test item was used as supplied in the study.

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted epidermis (epiCS, Cell Systems)

Cell type:

other: epiCS, Cell Systems - Batch No.21-13

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems - Batch No.21-13) were received on 28 April 2021. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The four additional killed control tissues (epiCS, Cell Systems – Batch No.21-09, frozen on 29 April 2021) were defrozen the day of the treatment, on 29 April 2021.The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Cell Systems Batch No. 305-AK0844). The culture plates were incubated at 37±2°C, 5% CO2 for 21 hours and 45 minutes before treatment.


EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A purple solution was observed after 1 hour of incubation between 37.1°C and 37.4°C, 5% CO2 in the dark.
> Therefore, the test item was identified as a direct MTT reducer.
That is why four killed control tissue models (two by exposition time) were added to the study, which underwent the entire testing procedure to generate a non-specific MTT reduction control.


SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol (same conditions as in the main test). An orange solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking.
The mean of the corrected OD (blank subtracted) was 0.007 which is lower than 0.08 (value corresponding to approximately twice the OD of the extracting solvent).
> Therefore, the test item will not interfere with the MTT assay and there is no need to add non-specific coloration controls to the study.


TREATMENT
The test item was applied as supplied at the dose of 50 µL to the epidermal surface of the 2 living human skin models for each time during 3 minutes at room temperature and during 1 hour at 37°C +/-1°C, 5% +/-1% CO2. The four killed control tissue models (two by exposition time) were treated in the same manner in order to generate a non-specific MTT reduction.

In the same experimental conditions, a positive control (50µL of 8N KOH - Fisher Scientific, Batch No. A0412420) and a negative control (50µL of distilled water - ADL Prochilab - Batch No. 200908) were carried out.

REMOVAL OF TEST MATERIAL AND CONTROLS
3 minutes and 1 hour after the test item application the human epidermis were washed 20 times with 1 mL of DPBS (DPBS – Dutscher, Batch No. 8061020).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability was quantified by measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal. The skin sample was placed into 300 µL of MTT solution, at the concentration of 1 mg/mL, for 2 hours and 48 minutes at 37°C± 1°C, 5% CO2. The precipitated blue formazan product was then extracted using 2 mL of isopropanol for 2 hours under agitation in the dark, and the concentration of formazan was measured by determining the Optical Density (OD) at 570 nm, just after dilution of the extraction in isopropanol (1:3).
The absorbance was measured in triplicate of MTT extract.
The measured absorbances were proportional to the number of living cells.

The measurement of OD was performed using the Elx800 absorbance microplate reader (controlled and calibrated every year if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = OD test item / OD negative control * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.

As the test follows: item was identified as producing direct MTT reduction, the true tissue viability is then calculated as:
True viability % = [(living tissues OD exposed to test item - killed tissues OD exposed to test item) / living tissues OD exposed to negative control] x 100

PREDICTION MODEL / DECISION CRITERIA:

The OD values obtained for each test sample are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set at 100%. The cut-off values for the prediction of corrosion associated with the epiCS® models are as follows:

Viability measured after exposure time points (t=3 and 60 minutes)

STEP 1 (corrosive or not corrosive)
< 50% after 3 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND < 15% after 60 min exposure ==> Corrosive: Corresponding hazard statement “H314: Causes severe skin burns and eye damage” and signal word “Danger”
≥ 50% after 3 min exposure AND ≥ 15% after 60 min exposure ==> Non-corrosive

STEP 2 (subcategories for items identified as corrosive in step 1)
< 15% after 3 min exposure ==> Optional Sub-category 1A*
≥ 15% after 3 min exposure ==> A combination of optional Sub-categories 1B-and-1C

* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subcategorisation, it was shown that around 33% of the Sub-category 1A results of the epiCS® test methods may actually constitute Sub-category 1B or Sub-category 1C substances/mixtures (i.e. over-classifications).
It must be noted that a limitation of this Test Guideline is that it does not allow discriminating between skin corrosive sub-categories 1B and 1C.

ACCEPTABILITY CRITERIA:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

- Positive Control
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues (8N KOH) was ≤15% relative to the negative control treated tissues for 1 hour exposure time.

- Negative Control
The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues (distilled water) was ≥ 0.8 and ≤ 2.8 for every exposure time.

- Test Item
The assay establishes the acceptance criterion for an acceptable test if in the range 20-100% viability and for ODs ≥ 0.3, the difference of viability between the two tissue replicates was not exceed 30%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

During 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2.

Duration of post-treatment incubation (if applicable):

The skin sample was placed in MTT solution of 1 mg/mL concentration for 3 hours at 37°C± 1°C, 5% CO2.

Number of replicates:

2 living human skin models and 4 additional killed control tissue models (two by exposition time 3 minutes and 1 hour, for the non-specific MTT reduction control)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: Mean corrected percent viability

Run / experiment:

at 3 minutes

Value:

88.46

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

At 1 hour

Value:

79.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean corrected percent viability of the epidermis skins treated with the test item were 88.46% versus 5.43% in the positive control at 3 minutes exposure.
- The mean corrected percent viability of the epidermis skins treated with the test item were 79.50% versus 0.50% in the positive control at 1 hour exposure.

These results are in accordance with the acceptability criteria:

- The mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria was in the range ≥ 0.3 and ≤ 0.9 for the negative control (0.728 for the first experiment and 0.823 for the second experiment).

- The mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, was ≤15% for epiCS model (mean viability 1-hour exposure = 0.5%).

- In the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates was not exceed 30% (2.817% for the first experiment and 1.5% for the second experiment).

Any other information on results incl. tables

Table 7.3.1/1: Individual and average values for the test item, the negative and positive controls

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Negative control	1	0.756 0.728 0.804	0.762	0.728	104.670	100.00	6.605
	2	0.686 0.700 0.698	0.694		95.330
Positive control	3	0.049 0.054 0.058	0.053	0.040	7.280	5.426	2.623
	4	0.025 0.025 0.029	0.026		3.571
Test item	5	0.675 0.689 0.658	0.674	0.660	95.582	90.591	2.817
	6	0.627 0.649 0.659	0.645		88.599
Test item  NSMTT	7	0.016 0.015 0.015	0.015	0.016	2.060	2.129	0.097
	8	0.017 0.017 0.016	0.016		2.198
Test item corrected						88.462

 

Acceptability criteria:

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Viability difference between replicates %
Negative control	9	0.757 0.745 0.772	0.758	0.823	92.2	100.00	11.1
	10	0.906 0.900 0.856	0.887		107.8
Positive control	11	0.004 0.003 0.004	0.003	0.004	0.4	0.5	0.2
	12	0.005 0.006 0.006	0.005		0.6
Test item	13	0.727 0.663 0.620	0.670	0.679	81.5	82.5	1.5
	14	0.730 0.658 0.674	0.687		83.5
Test item  NSMTT	15	0.024 0.027 0.027	0.026	0.025	3.2	3.0	0.3
	16	0.024 0.024 0.023	0.023		2.8
Test item corrected						79.5

Note #: mean of 3 values

OD: optical density

SPL: sample

 

Acceptability criteria:

- Negative control: mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

- Positive control: mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 15% for epiCS model.
- Test item: in the range 20-100% viability, and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Executive summary:

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed.

The test item DAVANA ESSENTIAL OIL D04035 was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (epiCS®, supplied by CellSystems®) for 3 minutes and 1 hour. The application was followed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 2 killed Human skin model surfaces were treated for each time (epiCS®, supplied by CellSystems®) under the same conditions in order to generate non-specific Isopropanol interaction control at the dose of 50 µL.
The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 431 dated 18 June 2019.

 

The quality criteria required for acceptance of results in the test were satisfied.

3 minutes and 1 hour after the test item application, the corrected mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 88.46% and 79.50% versus 5.43% and 0.50% respectively, with the positive control.

 

In accordance with the CLP Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item DAVANA ESSENTIAL OIL D04035 does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.