Artemisia pallens, ext.

Endpoint summary

Description of key information

- Skin irritation: irritating, based on in vitro skin irritation study (OECD 439, GLP, rel.1, K).

- Skin corrosion : not corrosive based on in vitro skin corrosion study (OECD 431, GLP, rel.1, K).

- Serious eye damage/ eye irritant: not irritant based on in vitro eye irritation study (OECD 492, GLP, rel.1, K).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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)

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)

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.

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)

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

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).

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%.

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.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 January 2021 to 11 February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.439 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)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 26 June 2020

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

QA statement signed on 2021-07-06

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.

Test system:

human skin model

Source species:

other: reconstructed epidermises (Episkin SA, RHE/S/17)

Cell type:

other: Episkin SA, RHE/S/17 Batch No. 21-RHE-010

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE

- The 0.50 cm² reconstructed epidermises (Episkin SA, RHE/S/17 Batch No. 21-RHE-010) were received on 09 February 2021. The 2 additional killed Human skin model surfaces (Episkin SA, RHE/S/17 - Batch No. 20-RHE-080 frozen on 21 July 2020) were defrozen the day of the treatment, on 11 February 2021. The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 24 wells culture plate which had been previously filled with 300 μL of growth medium (Episkin SA, batch No. 21 SGM 013) for 2 hours and 20 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. No. 21 SMM 006).

TREATMENT

- The test item was applied as supplied, at the dose of 16 μL to the epidermal surface of 3 living human skin models during 42 minutes at room temperature. To ensure a good contact with the epidermises, during all the treatment period, the test item was covered with a nylon mesh provided by Episkin SA. The test item was identified as a direct MTT reducer. Thereby, the experimentation was repeated on two killed control tissue models which underwent the entire testing procedure to generate a non-specific MTT reduction control.

- In the same experimental conditions, a positive control (16 μL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 μL of distilled water – ADL Prochilab - Batch No. 200908) were carried out. The 5% SDS solution was prepared by weighing 0.500 g of SDS (SIGMA Batch No. STBJ3028) in a 10 mL volumetric flask q.s. 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment to obtain a colourless solution.
To ensure a good contact with the epidermises, during all the treatment period, the control items were covered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS

- 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS (Dutscher - Batch No. 7701020). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues. They were incubated for a 41 hours and 25 minutes post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE

- The cell viability was quantified by the 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 that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.

- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 2 hours and 52 minutes at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).

- The OD of MTT extract was measured in triplicate of MTT extract. The measurement of OD at 570 nm of formazan extracts was performed in triplicate samples using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

VIABILITY CALCULATION:

- The results were expressed as a viability percentage compared with the negative control: viability % = (OD test item / OD negative control) * 100

- As the test item is identified is identified as a direct MTT reducer, true tissue viability is calculated as follows: True viability % = [(living tissues OD exposed to test item - killed tissues OD exposed to test item) / OD of living tissues exposed to 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.

PREDICTION MODEL / DECISION CRITERIA

The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off value of percentage cell viability distinguishing irritant from non-classified test items associated with the SkinEthic RHE model is given below:

- The test item is considered as non-irritant to skin in accordance with UN GHS No Category, if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.

- The test item is identified as requiring classification and labelling according to UN GHS (Category 2), if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non-corrosive”. In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 “Irritant” or in Category 1 “Corrosive”. The corresponding hazard statement is respectively, “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger”.

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 was <40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is lower or equal to 18%.

- Negative Control
The assay establishes the acceptance criterion for an acceptable test if the mean OD for the negative control treated tissues was higher or equal to 0.8 and lower or equal to 3, and the standard deviation value of the percentage viability is lower or equal to 18%.

- Test Item
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is lower or equal to 18%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41 hours and 25 minutes post-incubation period in fresh medium at 37°C, 5% CO2

Number of replicates:

3 living human skin models and controls and 2 killed control tissue models (to generate a non-specific MTT reduction control).

Irritation / corrosion parameter:

other: corrected mean percent viability

Run / experiment:

At 42 minutes exposure

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
As the test item was identified as a direct MTT reducer, the true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the MTT reducer minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same MTT reducer, calculated relative to the negative control run concurrently to the test being corrected (%NSMTT).
> Therefore, the corrected mean percent viability of the treated tissues was 0.6% versus 1.1% in the positive control (5% Sodium Dodecyl Sulfate).

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 439.

ACCEPTANCE OF RESULTS:
These results are in accordance with the acceptability criteria:
- The OD of the negative control was higher or equal to 0.8 and lower or equal to 3, and the standard deviation value of the percentage viability is lower or equal to 18%.
- The relative mean tissue viability for the positive control treated tissues was <40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is lower or equal to 18%.
- The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is lower or equal to 18%.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The corrected mean percent viability of the treated tissues is 0.6%, versus 1.1% in the positive control (5% Sodium Dodecyl Sulfate).
In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

Test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic^TM model) for 42 minutes. The application was followed by a rinse with 25 mL of PBS and a 41 hours and 25 minutes incubation period at 37°C, 5% CO2. In the same experimental conditions, a positive control (16 μL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 μL of distilled water – ADL Prochilab - Batch No. 200908) were carried out. 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 (SkinEthic RHE® model) under the same conditions in order to generate non-specific MTT reduction.
The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 26 June 2020.

 

The quality criteria required for acceptance of results in the test were satisfied.
The corrected mean percent viability of the treated tissues was 0.6% versus 1.1% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 January 2021 to 21 January 2021.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 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).

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted 18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

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.

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0-EA, supplied by MatTek Corporation, batch No. 30692)

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

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

Duration of treatment / exposure:

30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions)

Duration of post- treatment incubation (in vitro):

- Post-exposure immersion period: 12 minutes at room temperature - Post-exposure incubation period: 1 hour and 45 minutes at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

TISSUES CONSTRUCTS
- RhCE tissue construct used, including batch number: 0.60 cm² Reconstructed human Cornea-like Epithelia (EpiOcularTM OCL-212-ver2.0-EA, supplied by MatTek Corporation, batch No. 30692) were received on 19 January 2021. The 2 additional killed Human skin models (EpiOcularTM OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 27009 kit C) had been frozen on 17 October 2017 and defrozen on 27 November 2018 (1st freeze-thaw cycle), frozen again on 28 November 2018 to be defrozen (2nd freeze-thaw cycle) the day of the treatment, on 20 January 2021.

- Pre-incubation of the tissues:
On 19 January 2021, the tissues in their well shipping container were equilibrated to room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium (MatTek Corporation, batch No. 01182ISA) and incubated for 19 hours and 40 minutes at standard culture conditions.

- Indication of controls used for direct MTT-reducers:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL (same conditions as in the main test). A blue and purple solution was observed after 2 hours and 57 minutes of incubation between 35.4°C and 36.8°C, 5% CO2.
> Therefore, the test item was identified as a direct MTT reducer. Two additional killed control tissues were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.

- Indication of controls used for colouring test chemicals:
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). A yellow solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.003 which is less 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.

MAIN TEST
- Pre-treatment:
After the overnight incubation, the tissues were pre-wetted with 50 μL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 5860820). The tissues were incubated at standard culture conditions for 30 minutes.

- Treatment and post-treatment incubation of the tissues:
The test item was applied as supplied, at the dose of 50 μL, to the entire surface of 2 living RhCE tissue replicates during 30 minutes at standard culture conditions. The test item being non-miscible with the DPBS, it was impossible to spread it on all the tissue.
As the test item was identified as a direct MTT reducer. Thereby, the experimentation was repeated on two killed control tissue models which underwent the entire testing procedure to generate a non-specific MTT reduction control.

In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 200824) were carried out. The controls were applied, as supplied, at the dose of 50 μL, to the surface of 2 RhCE tissue replicates during 30 minutes at standard culture conditions.

After the treatment, the test item and control substances were carefully washed from the RhCE tissues by extensive rinsing with Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 5260820). The rinsed tissues were checked for any coloration and noted to be white, comparable coloration to that of the negative control tissues.
This rinsing step was followed by a 12-minute post-exposure immersion period at room temperature in 5 mL of fresh medium to remove any test item absorbed into the tissue.
The RhCE constructs were then incubated for a 1 hour and 45 minutes post-exposure incubation at standard culture conditions in 1 mL of fresh medium at 37°C, 5% CO2.

- MTT viability assay:
Following the exposure to the test item, viability measurements are performed immediately after the post-exposure incubation period of the rinsed tissues in fresh medium. This period allows both for recovery from weak cytotoxic effects and for appearance of clear cytotoxic effects.
The RhCE tissue viability was measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] by the viable cells of the tissue into a blue MTT formazan salt that is quantitatively measured after extraction from tissues.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours and 03 minutes at standard culture conditions.

The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 24 hours and 45 minutes at 7+/-3°C in the dark.
The concentration of formazan was measured by determining the OD at 570 nm, just after dilution of the extractions in isopropanol (1:2).

The OD at 570 nm was measured in triplicate samples of formazan extracts.
The measured OD are proportional to the number of living cells.

The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.

Irritation parameter:

other: % mean corrected percent tissue viability

Run / experiment:

After 30 minutes exposure

Value:

88.61

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
As the test item was identified as a direct MTT reducer, the true tissue viability is calculated as the percent tissue viability obtained with living tissues exposed to the MTT reducer minus the percent non-specific MTT reduction obtained with the killed tissues exposed to the same reducer, calculated relative to the negative control run concurrently to the test being corrected (%NSMTT).
> Therefore, the corrected mean percent viability of the RhCE replicates treated with the test item DAVANA ESSENTIAL OIL D0435 was 88.61% versus 28.80% in the positive control (Methyl acetate).

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 492.

ACCEPTANCE OF RESULTS:
These results are in accordance with the acceptability criteria:
- The OD of the negative control was higher or equal to 0.8 and lower or equal to 2.8,
- The relative mean tissue viability for the positive control treated tissues was < 50% relative to the negative control,

Table 7.3.2/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls after 30 minutes exposure

 

Tissue	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	Viability difference between replicates (%)	Conclusion
Negative Control	1.035 1.094 1.118	1.083	0.979	110.62	100.00	21.25	-
	0.896 0.881 0.846	0.875		89.38
Positive Control	0.323 0.333 0.314	0.324	0.282	33.09	28.80	8.58	UN GHS Category 2 or 1
	0.230 0.243 0.245	0.240		24.51
Test Item	0.828 0.877 0.832	0.846	0.905	86.41	92.44	12.05
	0.973 0.963 0.955	0.964		98.47
Test item NSMTT	0.035 0.035 0.033	0.035	0.038	3.58	3.83	0.51
	0.042 0.039 0.039	0.040		4.09
Test item corrected					88.61		No Category

#: mean of 3 values

OD: optical density

SPL: sample

Interpretation of results:

GHS criteria not met

Conclusions:

The corrected mean percent viability of the RhCE replicates treated with the test item DAVANA ESSENTIAL OIL D0435 was 88.61% versus 28.80% in the positive control (Methyl acetate).
Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item DAVANA ESSENTIAL OIL D04035 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.
No hazard statement and the signal word are required.

Executive summary:

An OECD 492 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular^TM tissue model).

 

Test item was applied as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 30 minutes at 37°C, 5% CO2, (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 1 hours and 46 minutes post-exposure incubation at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 200824) were carried out. The tissue viability was measured by performing an MTT assay. Additionally, 2 killed RhCE (EpiOcularTM tissue model) were treated in the same manner in order to generate non-specific MTT reduction.
The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 492 adopted 18 June 2019.

 

The quality criteria required for acceptance of results in the test were satisfied.
The corrected mean percent tissue viability of the RhCE replicates treated with the test item DAVANA ESSENTIAL OIL D04035 was 88.61% versus 28.80% in the positive control (Methyl acetate).

  

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item DAVANA ESSENTIAL OIL D04035 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.
No hazard statement and the signal word are required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro studies

Skin corrosion (OECD 431, GLP, rel.1, RS, K)

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.

 

Skin irritation (OECD 439, GLP, rel.1, RS, K)

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

Test item was applied as supplied, at the dose of 16 μL, to 3 living Reconstructed Human epidermis (SkinEthic^TM model) for 42 minutes. The application was followed by a rinse with 25 mL of PBS and a 41 hours and 25 minutes incubation period at 37°C, 5% CO2. In the same experimental conditions, a positive control (16 μL of 5% sodium dodecyl sulfate - SDS) and a negative control (16 μL of distilled water – ADL Prochilab - Batch No. 200908) were carried out. 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 (SkinEthic RHE® model) under the same conditions in order to generate non-specific MTT reduction.
The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 26 June 2020.

The quality criteria required for acceptance of results in the test were satisfied.
The corrected mean percent viability of the treated tissues was 0.6% versus 1.1% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

 

Eye irritation (OECD 492, GLP, rel.1, RS, K)

An OECD 492 study was performed to evaluate the eye hazard potential of test item after topical administration on in vitro reconstructed human cornea-like epithelium tissues (EpiOcular^TM tissue model).

Test item was applied as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 30 minutes at 37°C, 5% CO2, (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12 minutes post-exposure immersion period at room temperature and a 1 hours and 46 minutes post-exposure incubation at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate - Sigma-Aldrich, batch No. BCBX8836) and a negative control (distilled water - ADL Prochilab - Batch No. 200824) were carried out. The tissue viability was measured by performing an MTT assay. Additionally, 2 killed RhCE (EpiOcularTM tissue model) were treated in the same manner in order to generate non-specific MTT reduction.
The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 492 adopted 18 June 2019.

The quality criteria required for acceptance of results in the test were satisfied.
The corrected mean percent tissue viability of the RhCE replicates treated with the test item DAVANA ESSENTIAL OIL D04035 was 88.61% versus 28.80% in the positive control (Methyl acetate).

In conclusion, under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item DAVANA ESSENTIAL OIL D04035 does not require classification for eye irritation or serious eye damage. It corresponds to the UN GHS No Category.
No hazard statement and the signal word are required.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self-classification:

Based on an in vitro skin irritation study (OECD 439), the registered substance is classified as skin irritant: Skin Irritant Category 2 (H315: Causes skin irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

Moreover, an assessment of the skin corrosivity has been performed. An in vitro skin corrosion study (OECD 431) has allowed to show that the test item is not considered as a skin corrosive.

Concerning the eye irritation/corrosion, an in vitro eye irritationstudy (OECD 492) has allowed to show that the test item is not considered as irritant to the eyes. No classification is required.