Eucalyptus radiata australiana, 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:

From 23 March 2022 to 6 May 2022

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 (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 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

Dated to 14 June 2019

Deviations:

no

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

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Reconstructed human epidermises (epiCS, Phenion, Batch No. epiCS 22-15)

Justification for test system used:

Reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstructed epidermises (epiCS, Phenion, Batch No. epiCS 22-15) were received on 05 May 2022. 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 a 6 wells culture plate which had been previously filled with 1 mL of culture medium (Phenion Batch No. 305-AL0854). The culture plates were incubated at 37°C, 5% CO2 during 20 hours and 05 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (epiCS, Henkel-Phenion-Batch No. 305-AL0854).

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 1 hour of incubation between 36.2°C and 36.5°C, 5% CO2 in the dark.
>Therefore, there is no direct interaction between the test item and MTT.

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 300 µL 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 under gentle agitation. No significant coloration appeared.
>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, during 3 minutes at room temperature and during 1 hour at 37±1°C, 5±1% CO2, to the epidermal surface of the 4 living human skin models (two by each exposition time 3 minutes and 1 hour).

In the same experimental conditions, a positive control (50 µL of 8N KOH - Fisher Scientific, Batch No. A0412420) and a negative control (50 µL of distilled water - ADL Prochilab - Batch No. 211021) 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. 7951221).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability was quantified by measurement of the cellular mitochondrial 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 salt in the viable cells.
The skin sample was placed into 300 µL of MTT solution, at the concentration of 1 mg/mL, for 2 hours and 45 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 % = (mean OD test item / mean 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.

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.

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 2 hours and 45 minutes at 37°C± 1°C, 5% CO2.

Number of replicates:

4 living human skin models (two by each exposition time 3 minutes and 1 hour)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

81.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour

Value:

7.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 81.1%, versus 5.5% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 7.7%, versus 1.3% in the positive control.

Any other information on results incl. tables

Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls

 

INDIVIDUAL AND AVERAGE VALUES AFTER 3 MINUTES EXPOSURE

 

	Sample	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	1	0.713 0.709 0.712	0.712	0.716	99.4	100.00	0.8	1.1
	2	0.720 0.712 0.727	0.720		100.6
Positive control	3	0.038 0.037 0.037	0.038	0.040	5.3	5.5	0.3	0.4
	4	0.041 0.040 0.040	0.041		5.7
Test item	7	0.530 0.503 0.514	0.516	0.581	72.1	81.1	12.7	18.0
	8	0.645 0.634 0.656	0.645		90.1

 

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Sample	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	9	0.710 0.690 0.697	0.699	0.670	104.3	100.00	6.1	8.7
	10	0.675 0.644 0.602	0.641		95.7
Positive control	11	0.006 0.005 0.006	0.006	0.009	0.9	1.3	0.5	0.7
	12	0.011 0.011 0.011	0.011		1.6
Test item	13	0.050 0.048 0.048	0.049	0.052	33.0	7.7	0.5	0.7
	14	0.053 0.053 0.054	0.054		43.6

Note #: mean of 3 values, OD: optical density

 

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 with the positive control (8N KOH), 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:

Category 1 (corrosive) based on GHS criteria

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item EUCALYPTUS RADIATA OIL Batch No. 406007 has to be classified at least in Category 1B/1C “Corrosive”.
The corresponding hazard statement is “H314: Causes severe skin burns and eye damage” with the signal word “Danger”.

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 EUCALYPTUS RADIATA OIL Batch No. 406007 was applied as supplied at the dose of 50 μL, during 3 minutes at room temperature and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2, to the epidermal surface of the 4 living human skin models (two by each exposition time 3 minutes and 1 hour). In the same experimental conditions, a positive control (50 µL of 8N KOH) and a negative control (50 µL of distilled water) were carried out. The application was followed by a rinse with 20 mL of PBS. 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.

 

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

3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item was 81.1 % and 7.7%, versus 5.5% and 1.3%, respectively, with the positive control item (potassium hydroxide 8N).

 

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that the test item EUCALYPTUS RADIATA OIL Batch No. 406007 has to be classified at least in Category 1B/1C “Corrosive”. The corresponding hazard statement is “H314: Causes severe skin burns and eye damage” with the signal word “Danger”.