Artemisia pallens, ext.

Eye irritation

Administrative data

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).

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 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)

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.

Test animals / tissue source

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)

Test system

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.

Results and discussion

In vitro

Results

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,

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.