Lavender, Lavandula hybrida, ext.

Endpoint summary

Description of key information

- Skin irritation/corrosion : irritating, 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, S).

 

- Serious eye damage/ eye irritant: irritating, based on the rules of the CLP Regulation for classification of mixtures.

A supporting in vitro eye corrosion study (OECD 438, GLP, rel.1, S) did not confirm the corrosivity of the test item.

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 10% of its constituents classified as Skin irritant Category 2 and should be classified as a skin irritant 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 (2015) 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 10% of its constituents classified as Skin irritant Category 2 and should be classified as a skin irritant without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Constituent	CAS	Classification	Source
Terpinol-4	562-74-3	H315	ECHA C&L inventory–self classification
Linalool	78-70-6	H315	ECHA C&L inventory - self classification
Linalyl acetate	115-95-7	H315	ECHA C&L inventory - self classification

Source: ECHA disseminated dossiers or self classification

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 March 2020 to 02 April 2020

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 due to the exact composition of the test item which cannot be exactly determined because is an UVCB substance. Without impact on the conclusion of the study)

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)

Remarks:

14 October 2019

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 100481020
- Date received : 14 March 2019
- Expiration date of the lot/batch: 15 November 2019
- Manufacture date : 17 August 2017
- Purity test date: 26 February 2019

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, Cell Systems)

Cell type:

other: epiCS, Cell Systems

Vehicle:

unchanged (no vehicle)

Details on test system:

HUMAN SKIN MODEL
The 0.6 cm² reconstituted epidermis (epiCS, Cell Systems - Batch No.100-AJ0612-1) were received on 01 April 2020. Two additional living human skin model surfaces were added. The insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The insert was placed in a 6 wells culture plate which had been previously filled with 1 mL of culture medium. The culture dishes were incubated at 37±2°C, 5% CO2 during 18 hours and 15 minutes before treatment. Just before the treatment, the culture medium was replaced by a new culture medium (Cell Systems Batch No. 305-AJ0650).

EVALUATION OF DIRECT INTERACTION WITH MTT
The direct interaction of MTT with the test item was checked by adding 50 mg and 50.2 mg of the test item previously applied on a nylon mesh to 1mL of the solution of MTT at 1 mg/mL (same conditions as in the main test). A yellow solution with the test item at the bottom of the well was observed after 1 hour of incubation between 37.1°C and 37.5°C, 5% CO2.
Therefore, there is no direct interaction between the test item and MTT.

SPECTRAL ANALYSIS OF THE TEST ITEM IN ISOPROPANOL
The spectral properties at 570nm of the test item in ispopropanol were checked by adding 50.2 mg of test item previously applied on a nylon mesh to 2mL of isopropanol (same conditions as in the main test). A yellow solution with test item at the bottom of the well was obtained after 2 hours of incubation at ambient temperature with gentle shaking.
The mean of the corrected OD (blank subtracted) was 0.081 which is higher than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore, the test item was identified as causing colour interference with the viability assay and two viable control tissues were added to the study in order to generate non-specific Isopropanol interaction control.

TREATMENT
The test item was applied on a nylon mesh at the dose of 40.88 mg and 37.52 mg during 1 hour and at the dose of 39.60 mg and 40.19 mg during 3 minutes in order to cover the entire surface of the epidermis. The test item was subsequently applied to the entire surface of 2 living human skin models previsouly moistened with 15 µL of distilled water, during 3 min at RT and during 1 hour at 37°C ± 1°C, 5% ± 1% CO2. The 2 additional living human skin model surfaces were treated in the same manner in order to generate non-specific Isopropanol interaction control at the dose of 41.17 mg and 41.52 mg (1h) and 40.26 mg and 40.21 mg (3min).

In the same experimental conditions, a positive control (50µL of 8N KOH - Fisher Scientific, Batch No. H3410) and a negative control (50µL of distilled water - ADL Prochilab - Batch No. 180907) 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. 3241019). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues.

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 in MTT solution of 300µL concentration for 3 hours at 37°C± 1°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 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): approximately 40 mg
- 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 3 hours at 37°C± 1°C, 5% CO2.

Number of replicates:

2 living human skin models and 2 additional living human skin models

Irritation / corrosion parameter:

% tissue viability

Value:

83.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 3 minutes

Irritation / corrosion parameter:

% tissue viability

Value:

24.04

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: at 60 minutes

Other effects / acceptance of results:

MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues, after 3 minutes exposure, was 83.89%, versus 43.83% in the positive control.
- The mean percent viability of the treated tissues, after 1 hour exposure, was 24.04 %, versus 0.52% in the positive control.

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 %	Standard deviation (SD)
Negative control	1	0.529 0.592 0.622	0.581	0.556	104.59	100.00	9.2
	2	0.526 0.517 0.547	0.530		95.41
Positive control	3	0.274 0.262 0.274	0.270	0.244	48.60	43.83	9.5
	4	0.180 0.215 0.257	0.217		39.06
Test item	5	0.524 0.490 0.501	0.505	0.468	90.91	84.25	13.3
	6	0.427 0.442 0.423	0.431		77.59

Test item  living control	7	0.003 0.002 0.002	0.002	0.002	0.36	0.36	0.0
	8	0.002 0.002 0.002	0.002		0.36

Test item corrected
	Mean viability (%) = 83.89

INDIVIDUAL AND AVERAGE VALUES AFTER 1 HOUR EXPOSURE

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	9	0.560 0.583 0.622	0.588	0.572	102.80	100.00	5.6
	10	0.549 0.551 0.569	0.556		97.20
Positive control	11	0.003 0.003 0.003	0.003	0.003	0.52	0.52	0.0
	12	0.004 0.003 0.003	0.003		0.52
Test item	13	0.121 0.122 0.119	0.121	0.139	21.15	24.30	6.3
	14	0.152 0.156 0.162	0.157		27.45

Test item  living control	15	0.003 0.002 0.002	0.002	0.002	0.35	0.26	0.2
	16	0.002 0.001 0.001	0.001		0.17

Test item corrected
	Mean viability (%) =24.04

Note #: mean of 3 values OD: optical density

OD: Optical density

Acceptability criteria:

- The mean OD of negative control tissues for the treatment of 3 minutes and for the treatment of 1 hour were respectively 0.556 and 0.572 instead of ≥ 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.

- The mean viability of positive control tissues for the treatment of 1 hour was 0.52 %.It should be < 20% for epiCS model.

 

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 A “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.

Test item was applied as supplied at the average dose of 40 mg to 2 living Human skin model surfaces (epiCS^®, CellSystems^®) during 3 minutes and 1 hour. As the test item was identified as potentially causing a colour interference with the viability assay, two additional viable control tissues were added to the study. 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.

 

3 minutes and 1 hour after the test item application, the corrected mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 83.89% and 24.04% versus 43.83% and 0.52% respectively, with the positive control.

 

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 A “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

Reference 1

Endpoint:

eye irritation, 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: Eye damage category 2

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The registered substance has more than 10% of its constituents classified as Eye irritant Category 2 and should be classified as a skin irritant 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 (2015) was used to determine the serious eye damage/ eye irritant hazard of the registered substance. The decision of classification as irritant to the eyes was based on existing data on constituents (additivity principles):the registered substance has more than 10% of its constituents classified as Eye irritant Category 2 and should be classified as a skin irritant without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Constituent	CAS	Classification	Source
Terpinol-4	562-74-3	H319	ECHA C&L inventory–self classification
Linalool	78-70-6	H319	ECHA C&L inventory - self classification
Linalyl acetate	115-95-7	H319	ECHA C&L inventory - self classification

Source: ECHA disseminated dossiersor self classification

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No.438 and under GLP compliance (GLP deviation due to the exact composition of the test item which cannot be exactly determined because is an UVCB substance. Without impact on the conclusion of the study)

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Dated to 14 october 2019

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES

- Source: The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.

- Characteristics of donor animals (e.g. age, sex, weight): The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).

- Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 24 February 2020 at 8:10 am.

- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Phycher on 24 February 2020 at 9:45 am.

- Indication of any existing defects or lesions in ocular tissue samples: None

- Indication of any antibiotics used: None

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): 30 mg

- Concentration (if solution): Test item was used as supplied

Duration of treatment / exposure:

Test item was applied for 10 seconds to the cornea

Number of animals or in vitro replicates:

1eye for negative control and 3 eyes for positive control and test item

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES

- The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

- The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus were at a controlled temperature between 31.9 °C.

- After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

- Once all eyes had been examined and approved, the eyes were incubated between 45 and 56 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

APPLICATION DOSE AND EXPOSURE TIME

- Immediately following the zero reference measurements, the eye (in its holder) was removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied on three gauzes to the cornea of 3 enucleated chicken eyes, for 10 seconds, such that the entire surface of the cornea was evenly covered with the test item.

REMOVAL OF TEST SUBSTANCE

- After exposure, test item was rinsed twice from the eye with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position.

OBSERVATION PERIOD

- Treated corneas were evaluated before the pre-treatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:

- All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.

- The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after exposure to the test item) were determined at each of the above time points.

SCORING SYSTEM:

- Mean corneal swelling (%): Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope.

Corneal swelling (%) = ((corneal thickness at time t - corneal thickness at time = 0) / (corneal thickness at time = 0)) x 100

The mean percentage of corneal swelling for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for the test item.

- Mean maximum opacity score: Corneal opacity was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all tested eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.

0: No opacity

0.5: Very faint opacity

1: Scattered or diffuse areas; details of the iris clearly visible

2: Easily discernible translucent area; details of the iris are slightly obscured

3: Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible

4: Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment: The mean fluorescein retention value for all tested eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.

0: No fluorescein retention

0.5: Very minor single cell staining

1: Single cell staining scattered throughout the treated area of the cornea

2: Focal or confluent dense single cell staining

3: Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA:

- Results from corneal opacity, swelling, and fluorescein retention were evaluated separately to generate an ICE class for each endpoint. The ICE classes for each endpoint were then combined to generate an Irritancy Classification for the test item.

- Once each endpoint was evaluated, ICE classes were assigned based on a predetermined range. Interpretation of corneal thickness, opacity, and fluorescein retention using four ICE classes was done according to the table 7.3.2/1, 7.3.2/2, 7.3.2/3.

Irritation parameter:

cornea opacity score

Run / experiment:

maximal mean score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean score

Value:

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:

OCULAR REACTIONS:

- maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;

- mean score of fluorescein retention: 2.0, corresponding to ICE class III;

- maximal mean corneal swelling: 3%, corresponding to ICE class I.

The combination of the three endpoints for test item was 2 x I, 1 x III.

ACCEPTANCE OF RESULTS:

- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "No prediction can be made", as defined by the OECD guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.
Based on the results from the calculations rules, the test item can be classified as skin irritant (Category 2).

Executive summary:

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item was applied on three gauzes in order to cover the entire surface of the cornea. The gauzes were subsequently applied to 3 enucleated eyes at the dose of 30 mg, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;

- mean score of fluorescein retention: 2.0, corresponding to ICE class III;

- maximal mean corneal swelling: 3%, corresponding to ICE class I.

The combination of the three endpoints for test item was 2 x I, 1 x III.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "No prediction can be made", as defined by the OECD guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 (2015) was used to determine the Eye and skin irritation/corrosion hazard of the registered substance. The decision of classification as skin irritant and eye irritant was based on existing data on constituents (additivity principles):the registered substance has more than 10% of its constituents classified as Skin irritant Category 2 and classified as Eye irritant Category 2 and should be classified as a skin irritant and eye irritant without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008

Skin irritation

Constituent	CAS	Classification	Source
Terpinol-4	562-74-3	H315	ECHA C&L inventory–self classification
Linalool	78-70-6	H315	ECHA C&L inventory - self classification
Linalyl acetate	115-95-7	H315	ECHA C&L inventory - self classification

Eye irritation

Constituent	CAS	Classification	Source
Terpinol-4	562-74-3	H319	ECHA C&L inventory–self classification
Linalool	78-70-6	H319	ECHA C&L inventory - self classification
Linalyl acetate	115-95-7	H319	ECHA C&L inventory - self classification

Source: ECHA disseminated dossiersor self classification

In vitro studies

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.

Test item was applied as supplied at the average dose of 40 mg to 2 living Human skin model surfaces (epiCS^®, CellSystems^®) during 3 minutes and 1 hour.As the test item was identified as potentially causing a colour interference with the viability assay, two additional viable control tissues were added to the study.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.

3 minutes and 1 hour after the test item application, the corrected mean percent viability of the epidermis skins treated with the test item and treated with the positive control item (potassium hydroxide 8N) were respectively 83.89% and 24.04% versus 43.83% and 0.52% respectively, with the positive control.

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 inCategory A “Corrosive”.The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Eye corrosion (OECD 438, GLP, rel.1, S)

An ex vivo eye irritation study was performed according to the OECD Guideline 438 and in compliance with GLP to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item was applied on three gauzes in order to cover the entire surface of the cornea. The gauzes were subsequently applied to 3 enucleated eyes at the dose of 30 mg, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.0, corresponding to ICE class I;

- mean score of fluorescein retention: 2.0, corresponding to ICE class III;

- maximal mean corneal swelling: 3%, corresponding to ICE class I.

The combination of the three endpoints for test item was 2 x I, 1 x III.

The combination of the three endpoints for the positive control, 5% Benzalkonium chloride, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected. The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category "No prediction can be made", as defined by the OECD guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

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 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).

Moreover, an assessment of the skin and eye corrosivity has been performed. About the skin corrosion, an in vitro study (OECD 431) has allowed to show that the test item is not considered as a skin corrosive.

Concerning the eye corrosion, the in vitro study (OECD 438) did not allow to discriminate between the corrosion potential of the substance and the non-classification ("no prediction can be made"). However, the following arguments can be taken into consideration:

- no constituants were classified as "corrosive"

- Calculation rules have allowed to classify the substance as Eye irritant

- the substance is not a skin corrosive

In addition to these arguments, the eye corrosion study did not confirm that the test item was a corrosive substance.

According to the weight of evidence, it can therefore be concluded that the test item is not considered as an eye corrosive.