[No public or meaningful name is available]

Endpoint summary

Description of key information

- Skin irritation/corrosion : Skin irritant cat.2, based on the rules of the CLP Regulation for classification of mixtures but not corrosive based on in vitro skin corrosion study (OECD 431, GLP, rel.1, K).

- Serious eye damage/ eye irritant: Eye irritant cat.2, based on in vitro eye corrosion study (OECD 437, GLP, rel.1, K).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation / corrosion, other

Remarks:

Classification based on calculation rules for mixtures of the CLP Regulation

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline required

Principles of method if other than guideline:

Classification based on calculation rules for mixtures of the CLP Regulation

Irritation parameter:

other: classification

Remarks on result:

other: skin irritant category 2

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The registered substance has more than 1% of its constituents classified as Skin corrosive Sub-Category 1A, 1B, 1C or Category 1 but less than 5% and should be classified as a Skin irritant Category 2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Executive summary:

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant was based on existing data on constituents (additivity principles): the registered substance has more than 1% of its constituents classified as Skin corrosive Sub-Category 1A, 1B, 1C or Category 1 but less than 5% and should be classified as a Skin irritant Category 2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

 

Constituent	CAS	Classification	Source
(R)-3,7-dimethyl-1,6-octadien-3-ol (l-linalool)	126-91-0	H314	ECHA self-classification: https://echa.europa.eu/fr/registration-dossier/-/registered-dossier/27524/2/1

Source: ECHA disseminated dossiers or self classification

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 03 February 2022 to 23 February 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

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
- Source and lot/batch No. of test material: 0000765018
- Date received : 25 January 2022
- Expiration date of the lot/batch: 15 December 2022
- Manufacture date : 15 December 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 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, Phenion)

Cell type:

other: epiCS, Cell Systems

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Phenion - Batch No.epiCS 22-05) were received on 22 February 2022. 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. The culture plates were incubated at 37°C, 5% CO2 for 20 hours and 48 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 yellow liquid was observed after 1 hour of incubation between 36.5°C and 37.9°C, 5% CO2 in the dark.
> Therefore, there is no direct interaction between the test item and MTT.

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 liquid was obtained after 1 hour of incubation between 36.5°C and 36.9°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 liquid 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 to the epidermal surface of the 2 living human skin models during 3 minutes at room temperature and during 1 hour at 37±1°C, 5±1% CO2.
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. 201117) 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. 3730921).

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 45 minutes at 37±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±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±1°C, 5% CO2.

Number of replicates:

2 living human skin models for each time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 3 minutes

Value:

83.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

at 60 minutes

Value:

46.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 83.9%, versus 10.9% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 46.3 %, versus 0.7% in the positive control.

ACCEPTANCE CRITERIA
- Negative control: 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 should be in the range ≥ 0.3 and ≤ 0.9 for the negative control. As the mean OD of the negative control is 0.673 for 3-minute of exposure and 0.667 for 1-hour of exposure, therefore, the criteria is met.
- Positive control: the 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%. As the mean viability is 0.7%, therefore, the criteria is met.
- 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%. As the maximum difference of viability between the two tissue replicates is 14.6%, therefore, the criteria is met.

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

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	1	0.748 0.723 0.694	0.722	0.673	107.3	100.00	10.3	14.6
	2	0.630 0.609 0.632	0.624		92.7
Positive control	3	0.082 0.085 0.084	0.084	0.074	12.5	10.9	2.2	3.1
	4	0.061 0.063 0.065	0.063		9.4
Test item	5	0.555 0.540 0.538	0.544	0.565	80.8	83.8	4.3	6.1
	6	0.577 0.579 0.600	0.585		86.9

 

 

 

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	SD viability	Viability difference between replicates %
Negative control	9	0.706 0.690 0.693	0.696	0.667	104.3	100.00	6.1	8.7
	10	0.638 0.619 0.658	0.638		95.7
Positive control	11	0.006 0.006 0.007	0.006	0.005	0.9	0.7	0.2	0.3
	12	0.004 0.004 0.003	0.004		0.6
Test item	13	0.315 0.324 0.317	0.319	0.309	47.8	46.3	2.1	3.0
	14	0.299 0.299 0.299	0.299		44.8

Note #: mean of 3 values OD: optical density

SPL: sample

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 aim of the study was to evaluate the possible corrosive effects of the test item LITSEA CUBEBA TERPENES L62840 after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by Phenion).

The test item LITSEA CUBEBA TERPENES L62840 was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (epiCS®, supplied by Phenion) for 3 minutes at room temperature and for 1 hour at 37°C ± 1°C, 5% ± 1% CO2. 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 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.

 

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 corrected percent viability of the epidermis skins treated with the test item were 83.9% and 46.3%, versus 10.9% and 0.7%, 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 LITSEA CUBEBA TERPENES L62840 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.

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:

20 to 26 April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 437

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 26 June 2020

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle obtained from the slaughterhouse (Vitelco's Hertogenbosch, The Netherlands)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: not specified
- Indication of any antibiotics used: not specified
- Selection and preparation of corneas: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
Then, the isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32±1°C. The corneas were incubated for the minimum of 1 hour at 32±1°C.
- Quality check of the isolated corneas: After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Vehicle:

unchanged (no vehicle)

Remarks:

The test material was tested neat.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL onto the epithelium of the cornea.
- Concentration (if solution): The test material was tested neat.
VEHICLE
- Amount(s) applied (volume or weight with unit): not applicable

Duration of treatment / exposure:

10±1 minutes at 32±1°C

Duration of post- treatment incubation (in vitro):

120±10 minutes at 32±1°C

Number of animals or in vitro replicates:

Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

Details on study design:

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control (Ethanol) or test material was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test material over the entire cornea. Corneas were incubated in a horizontal position for 10±1 minutes at 32±1°C.

RINSING OF THE CORNEAS
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test material on the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120±10 minutes at 32±1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity= [(I0/I)-0.9894]/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each cornea treated with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90±5 minutes at 32±1°C.
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

SCORING SYSTEM AND INTERPRETATION OF RESULTS
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro irritancy score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value)

Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
- If the test item induces an ≤ 3: UN GHS No Category
- If the test item induces an > 3; ≤ 55: No prediction can be made
- If the test item induces an > 55: UN GHS Category 1

ACCEPTANCE CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Run / experiment:

Positive control

Value:

43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Run / experiment:

Negative control

Value:

2.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Remarks:

Mean

Run / experiment:

Test material

Value:

7.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

IN VITRO IRRITANCY SCORE:
- The test material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.3 after 10 minutes of treatment. Individual IVIS values of test item-treated corneas were: 10, 5.5 and 6.5.
On the basis of these concordant results obtained on the three corneas (IVIS > 3 ≤ 55), no additional experiment was performed and no prediction on the classification can be made for eye irritation or serious eye damage.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The individual opacity scores for the negative controls ranged from 1.3 to 3.3. The individual permability scores (uncorrected) for the negative controls ranged from -0.003 to -0.001.The corneas treated with the negative control material were clear after the 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range (opacity: -2.5 to 3.70; permeability: -0.020 to 0.065) indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: The individual positive control in vitro irritancy scores ranged from 34 to 56. The corneas treated with the positive control material were turbid after the 10 minutes of treatment. The mean in vitro irritancy score of the positive control (Ethanol) was 43 and within two standard deviations of the current historical positive control mean (28-86).

It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
No pH effects of the negative and positive control were observed on the rinsing medium.

Table 7.3.2/1: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean IVIS^1, 2
Negative control	2.6	0.000	2.6
Positive control (Ethanol)	22	1.387	43
Test material	4.2	0.208	7.3

¹Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and test material.

²In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:

other: No prediction can be made

Conclusions:

Under the experimental conditions of this study, since Litsea Cubeba oil, Terpenes fraction L62840 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP. The objective of this study was to evaluate the eye hazard potential of Litsea Cubeba oil, Terpenes fraction L62840 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage potential of the test material was tested through topical application for 10 minutes on isolated bovine corneas obtained from freshly slaughtered calves. The test material was applied as supplied (750 µL) directly on top of the corneas. Chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 120±10 minutes at 32±1°C. Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

 

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 43 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.3 after 10 minutes of treatment. Individual IVIS values of test item-treated corneas were: 10, 5.5 and 6.5.

 

In conclusion, under the experimental conditions of this study, since Litsea Cubeba oil, Terpenes fraction L62840 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion

Calculation rules

The NCS is composed of several identified constituents and in that, it can be considered as a mixture according to the definition of the CLP Regulation.

The decision logic for classification of mixtures from the ECHA Guidance on the Application of the CLP Criteria (2017) was used to determine the skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant was based on existing data on constituents (additivity principles): the registered substance has more than 1% of its constituents classified as Skin corrosive Sub-Category 1A, 1B, 1C or Category 1 but less than 5% and should be classified as a Skin irritant Category 2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

Constituent	CAS	Classification	Source
(R)-3,7-dimethyl-1,6-octadien-3-ol (l-linalool)	126-91-0	H314	ECHA self-classification: https://echa.europa.eu/fr/registration-dossier/-/registered-dossier/27524/2/1

Source: ECHA disseminated dossiers or self classification

 

In vitro study

Skin corrosion (OECD 431, GLP, rel.1, S)

An in vitro skin corrosion test according to the OECD Guideline OECD 431 and in compliance with GLP was performed. The aim of the study was to evaluate the possible corrosive effects of the test item LITSEA CUBEBA TERPENES L62840 after topical administration on in vitro human reconstituted epidermis (epiCS®, supplied by Phenion).

The test item LITSEA CUBEBA TERPENES L62840 was applied as supplied, at the dose of 50 μL to 2 living Human skin model surfaces for each time (epiCS®, supplied by Phenion) for 3 minutes at room temperature and for 1 hour at 37°C ± 1°C, 5% ± 1% CO2. 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 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.

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 corrected percent viability of the epidermis skins treated with the test item were 83.9% and 46.3%, versus 10.9% and 0.7%, 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 LITSEA CUBEBA TERPENES L62840 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.

 

Eye irritation/corrosion

In vitro study

Eye corrosion (OECD 437, GLP, rel.1, K)

An ex vivo eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP. The objective of this study was to evaluate the eye hazard potential of Litsea Cubeba oil, Terpenes fraction L62840 as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage potential of the test material was tested through topical application for 10 minutes on isolated bovine corneas obtained from freshly slaughtered calves. The test material was applied as supplied (750 µL) directly on top of the corneas. Chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 120±10 minutes at 32±1°C. Three corneas for each treated series (test item formulation, positive control and vehicle control) were used.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 43 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

The test material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 7.3 after 10 minutes of treatment. Individual IVIS values of test item-treated corneas were: 10, 5.5 and 6.5.

In conclusion, under the experimental conditions of this study, since Litsea Cubeba oil, Terpenes fraction L62840 induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

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 the available information and typical composition provided by the Lead Registrant, the registered substance is classified as skin and eye irritant: Skin Irritant Category 2 (H315: Causes skin irritation) and Eye irritant category 2 (H319: Causes serious eye irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP). To reach this conclusion on the classification of the substance, an assessment of the eye and skin corrosivity has been performed: the in vitro skin corrosion study (OECD 431) enable to conclude that the test item does not have to be classified in Category 1 “Corrosive” and the in vitro eye corrosion study (OECD 437) did not allow to discriminate between the corrosion potential of the substance and the non-classification ("no prediction can be made").

 

These studies are considered sufficient with the following arguments:

• Skin irritation/corrosion:
  1/ Calculation rules have allowed to classify the substance as Skin irritant.
  2/ no constituents classified as "skin corrosive" are present above the threshold for classification as H314 (≥ 5%).
  3/ the skin corrosion study enable to conclude that the test item does not have to be classified in Category 1 “Corrosive”.


• Eye irritation/corrosion:
  1/ the eye corrosion study confirmed the absence of corrosive or severe irritating effects and did not indicate that the test item was corrosive to the eyes, from a sample where the H318 constituents (3.77 % of l-linalool) were above the threshold for classification as H318 (≥ 3%).
  2/ the skin corrosion study did not indicate that the test item was corrosive to the skin, therefore, the substance is not expected to be corrosive to the eyes.

According to these arguments, it can therefore be concluded that the test item is not considered as an eye and skin corrosive but the registered substance is classified as Skin Irritant Category 2 (H315: Causes skin irritation) and Eye irritant category 2 (H319: Causes serious eye irritation) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).