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EC number: 295-995-3 | CAS number: 92201-64-4 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Eucalyptus radiata australiana, Myrtaceae.
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2 022
- 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
- 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
1
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 (EpiOcularTM 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.
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