Eucalyptus radiata australiana, ext.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 23 March 2022 to 31 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.492 and under GLP compliance (GLP deviation: the exact composition of the test item which cannot be exactly determined because is an UVCB substance and the stability and homogeneity tests on the test item were not supplied by the Sponsor. Without impact on the conclusion of the study)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

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

1

Reference substance name:

Eucalyptus radiata australiana, ext.

EC Number:

295-995-3

EC Name:

Eucalyptus radiata australiana, ext.

Cas Number:

92201-64-4

Molecular formula:

Not applicable

IUPAC Name:

Essential oil of Eucalyptus radiata (Myrtaceae) obtained from leaves and twigs by steam distillation

Test material form:

liquid

Details on test material:

EC No: 295-995-3
CAS No: 92201-64-4
Color: colorless to pale yellow

Test animals / tissue source

Species:

other: Reconstructed human Cornea-like Epithelium

Details on test animals or tissues and environmental conditions:

Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelium [EpiOcular(TM) OCL-200, OCL-212, supplied by MatTek Corporation, batch No 34959]

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: Undiluted

Duration of treatment / exposure:

30 minutes at standard culture conditions

Duration of post- treatment incubation (in vitro):

Post-exposure immersion period: 12 minutes at room temperature. // Post-exposure incubation period: 2 hours and 05 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
- The 0.60 cm² Reconstructed human Cornea-like Epithelium (EpiOcularTM OCL-200, OCL-212, supplied by MatTek Corporation, batch No. 34959) were received on 29 March 2022.

- Pre-incubation of the tissues:
The same day, 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, ref. OCL-200-ASY, batch No. 032822ISA) and incubated for 20 hours and 45 minutes at standard culture conditions.

- 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 1 mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow solution was observed after 3 hours of incubation between 36.2°C and 36.7°C, 5% CO2 in the dark.
-> Therefore, there is no direct interaction between the test item and MTT and there is no need to add non-specific MTT reduction (NSMTT) controls.

- Coloration potential and spectral analysis of the test item:
In water:The coloration potential of the test item in water was checked by adding 50 μL of the test item to 1 mL of distilled water. A colourless solution was obtained after 1 hour of incubation between 36.2°C and 36.5°C, 5% CO2 in the dark.
In isopropanol:The coloration potential of the test item in isopropanol was checked by adding 50 μL of the test item to 2 mL of isopropanol. A colourless solution was obtained after 2 hours of incubation at room temperature.
> 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 20 μL of Ca2+Mg2+Free-DPBS (Dutscher - Batch No. 7951221). 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.

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. 211021) 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. 7951221). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.
This rinsing step was followed by a 12-minutes 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 2 hours and 05 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 at standard culture conditions.
The precipitated blue formazan product was then extracted from both layers of the tissues by placing each insert in 2 mL of isopropanol for 2 hours and 02 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:

mean percent tissue viability 

Value:

29.1

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 tissue viability of the RhCE replicates treated with the test item was 29.1% versus 40% in the positive control (Methyl acetate).

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none (in water and in isopropanol).

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 > 0.8 and < 2.8. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.4 and < 1.4 for the negative control (mean OD of the negative control = 1.104).
-The relative mean tissue viability for the positive control treated tissues was <50% relative to the negative control treated tissues (mean viability of the positive control = 40%)
-The difference of viability between two tissue replicates is < 20% (difference of viability between two tissue replicates is 8.2% for living tissues exposed to the test item).

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	Well ID	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	SD viability	Viability difference between replicates (%)	Conclusion
Negative Control	SPL 1	1.106 1.122 1.117	1.115	1.104	101.0	100.0	1.4	2.0	No Category
	SPL 2	1.074 1.102 1.101	1.093		99.0
Positive Control	SPL 3	0.449 0.453 0.450	0.451	0.442	40.9	40.0	1.2	1.6	UN GHS Category 2 or 1
	SPL 4	0.436 0.431 0.432	0.433		39.2
Test Item	SPL 7	0.281 0.272 0.275	0.276	0.322	25.0	29.1	5.8	8.2	UN GHS Category 2 or 1
	SPL 8	0.363 0.367 0.370	0.367		33.2

#: mean of 3 values (triplicate of the same extract)

OD: optical density

SPL: sample

Applicant's summary and conclusion

Interpretation of results:

other: to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1

Conclusions:

The mean percent tissue viability of the RhCE replicates treated with the test item EUCALYPTUS RADIATA OIL Batch No. 406007 was 29.1% versus 40% 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 EUCALYPTUS RADIATA OIL Batch No. 406007 has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

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 (EpiOcularTM tissue model).

 

The test item EUCALYPTUS RADIATA OIL Batch No. 406007 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). In the same experimental conditions, a positive control (Methyl acetate) and a negative control (Distilled water) 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 the 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 2 hours and 05 minutes post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. 

 

The mean corrected percent tissue viability of the RhCE replicates treated with the test item EUCALYPTUS RADIATA OIL Batch No. 406007 was 29.1% versus 40% 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 EUCALYPTUS RADIATA OIL Batch No. 406007 has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.