Essential oil of Citrus sinensis (Rutaceae) peel, terpene-free

Endpoint summary

Description of key information

Skin irritation/corrosion (WoE)

- Skin irritation study (OECD 439, WoE, Rel.1)

The mean corrected percent viability of the treated tissues was 0.0%, versus 2.2% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008, the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

- Calculation rules (WoE, Rel.1)

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 cat.2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

Eye irritation/corrosion (WoE)

- Eye irritation (OECD 492, WoE, Rel.1)

The mean corrected percent tissue viability of the RhCE replicates treated with the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 was 54.17% versus 35.98% 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

- Eye corrosion (OECD 438, WoE, Rel.1)

The combination of the three endpoints for test item was 2 x II. 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 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. 

- Calculation rules (WoE, Rel.1)

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 Eye irritant cat.2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 16 July 2020 to 23 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 18 June 2019

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstructed epidermises

Cell type:

non-transformed keratinocytes

Cell source:

other: foreskin

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE

- 0.50 cm² reconstructed epidermis were received, and on 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 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 05 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).

TREATMENT

- The test item was applied as supplied, at the approximate dose of 16 mg, on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.

- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.

REMOVAL OF TEST MATERIAL AND CONTROLS

- 42 minutes after the test item application, the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be slightly brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 41 hours post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE

- The cell viability was quantified by the 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 that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.

- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).

- The OD of MTT extract was measured in triplicate. 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.

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 were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:

- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.

- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger".

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41 hours post-incubation period at 37°C, 5% CO2

Number of replicates:

3 living human skin models

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Value:

0

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 viability of the treated tissues is 0.0%, versus 2.2% in the positive control (5% Sodium Dodecyl Sulfate).

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: Yes, two additional killed control tissues were used to generate non-specific MTT reduction
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the negative control OD of the 3 replicates is >= 0.8 and <= 3.0 for every exposure time. The OD was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range >= 0.4 and <= 1.5 for the negative control.
- Acceptance criteria met for standard deviation: yes, the standard deviation should be <= 18%.

Interpretation of results:

other: to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”

Conclusions:

The mean corrected percent viability of the treated tissues is 0.0%, versus 2.2% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Executive summary:

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

Test item was applied as supplied, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE^®model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours post-incubation period at 37°C, 5% CO₂. 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.Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) under the same conditions in order to generate non-specific MTT reduction.

 

The mean corrected percent viability of the treated tissues was 0.0%, versus 2.2% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008, the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

Reference 2

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:

weight of evidence

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

Executive summary:

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

 

Constituent	CAS	Classification according to the Regulation (EC) No. 1272/2008 (CLP)	Source
Linalool	78-70-6	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification
Geranial	141-27-5	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification
Alpha-terpineol	98-55-5	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification
(Z)-3,7-dimethylocta-2,6-denial	106-26-3	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification

 

Source: ECHA disseminated dossiers or self classification

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 438 without any deviation.

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

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed 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 22 June 2020 at 8:08 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 22 June 2020 at 09:27 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 µL of the pure test substance.
- 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:

1, 3 and 3 eyes for negative & positive control and test item, respectively.

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 of 32.0 °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 65 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 µL of the test item was applied for 10 seconds to the cornea 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 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:
- The Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a % and was calculated from corneal thickness measurements according to the following formula:
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

- Morphological effects include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the the interpretation of the investigator.

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:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

mean score

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

maximal mean score

Value:

8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OCULAR REACTIONS:

- maximal mean score of corneal opacity: 1.0, corresponding to ICE class II;

- mean score of fluorescein retention: 1.2, corresponding to ICE class II;

- maximal mean corneal swelling: 8%, corresponding to ICE class II.

The combination of the three endpoints for test item was 3 x II.

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. It is also within the results obtained in the historical negative control data.

- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, sodium hydroxide was 3 x IV. Therefore, the positive control is classified as "Corrosive/Severe Irritant", as expected. It is also similar to the results obtained in the historical control data.

Interpretation of results:

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

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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 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.

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.

Test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 was applied as supplied , at the dose of 30 µL, to 3 enucleated chicken eyes 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: 1.0, corresponding to ICE class II;

- mean score of fluorescein retention: 1.2, corresponding to ICE class II;

- maximal mean corneal swelling: 8%, corresponding to ICE class II.

The combination of the three endpoints for test item was 2 x II.

The combination of the three endpoints for the positive control, sodium hydroxide, 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 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. 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 27 July 2020 to 30 July 2020.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation.

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)

Species:

other: Reconstructed human Cornea-like Epithelia

Details on test animals or tissues and environmental conditions:

Description of the cell system used: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation]

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 37°C, 5% CO2, 95% humidity (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 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
- RhCE tissue construct used, including batch number: 0.60 cm² Reconstructed human Cornea-like Epithelia [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 30640] were received on 15 January 2020. The 2 additional killed Human skin models [EpiOcular(TM) OCL-212-ver2.0, supplied by MatTek Corporation, batch No. 30614] had been frozen on 02 July 2019 and defrozen on 13 November 2019 (1st freeze-thaw cycle), refrozen on 14 November 2019 to be defrozen the day of the treatment, on 16 January 2020 (2nd freeze-thaw cycle).

- 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 culture medium and incubated during 20 hours and 40 minutes, at standard culture conditions.

- Indication of controls used for direct MTT-reducers:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of solution of MTT at 1 mg/mL. A purple suspension was observed after 3 hours of incubation between 36.6°C and 37.5°C, 5% CO2.
> Therefore, the test item was identified as a direct MTT reducer. Two additional killed control tissues were added to the study which underwent the entire testing procedure to generate a non-specific colour control (NSC control).

- Indication of controls used for colouring test chemicals:
The spectral properties at 570 nm of test item in isopropanol were checked by adding 50 µL of the test item to 2 mL of isopropanol. A yellow solution was obtained after 2 hours and 1 minute of incubation at ambient temperature with gentle shaking. The mean of the corrected OD was 0.071 which is lower than 0.08.
> 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. 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 and 2 killed RhCE (EpiOcular(TM) tissue model) (NSC control) 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. 190521) 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. 2190919). The rinsed tissues were checked for any coloration and noted to be yellowish.
This rinsing step was followed by a 12-minute 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 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 2 hours and 50 minutes at standard culture conditions.

The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 19 hours and 03 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.

Irritation parameter:

other: % mean corrected percent tissue viability

Value:

54.17

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 54.17% versus 35.98% in the positive control (Methyl acetate).

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: Yes, two additional killed control tissues were used to generate non-specific MTT reduction
- Colour interference with MTT: none.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Proficiency chemicals were tested according to the OECD TG 492.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for mean tissue viability: yes, tissues treated with the positive control substance showed a mean tissue viability < 50% (value of 35.98%).
- Acceptance criteria met for negative control: yes, the negative control OD of the 2 replicates is > 0.8 and < 2.5 for every exposure time. As the extract was diluted at 50% just before the OD measurement, the acceptability criteria should be in the range > 0.8 and <2.5 for the negative control.
- Acceptance criteria met for variability between replicate measurements: yes, less than 20% between two tissue replicates (value of 4.44%).

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	OD	Mean OD/ disc (#)	Mean OD / Product	Viability (%)	Mean viability (%)	Viability difference between replicates (%)	Conclusion
Negative Control	0.952 0.979 1.002	0.977	0.924	105.74	100.00	11.47	-
	0.829 0.878 0.906	0.871		94.26
Positive Control	0.316 0.333 0.331	0.326	0.333	35.28	35.98	1.41	UN GHS Category 2 or 1
	0.323 0.345 0.351	0.339		36.69
Test Item	0.508 0.540 0.554	0.534	0.555	57.79	60.01	4.44
	0.535 0.587 0.604	0.575		62.23
Test item NSMTT	0.063 0.059 0.056	0.059	0.054	6.39	5.84	1.08
	0.050 0.048 0.049	0.049		5.30
Test item corrected					54.17		UN GHS Category 2 or 1

#: mean of 3 values

OD: optical density

SPL: sample

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 corrected percent tissue viability of the RhCE replicates treated with the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 was 54.17% versus 35.98% 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 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 (EpiOcular^TM tissue model).

 

Test item was applied as supplied, at the dose of 50 µL, to 2 living DPBS pre-treated RhCE (EpiOcular^TM tissue model) during 30 minutes at 37°C, 5% CO₂, 95% humidity (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 post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 killed RhCE (EpiOcular (TM) tissue model) were treated in the same manner in order to generate non-specific MTT reduction.

 

The mean corrected percent tissue viability of the RhCE replicates treated with the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 was 54.17% versus 35.98% 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

Reference 3

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:

weight of evidence

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

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 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 Eye irritant cat.2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

Constituants	Cas Number	Classification (CLP 1272/2008)	Source
Linalool	78-70-6	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
Decanal	112-31-2	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
Geranial	141-27-5	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
Alpha-terpineol	98-55-5	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
(Z)-3,7-dimethylocta-2,6-denial	106-26-3	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification

Source: ECHA disseminated dossiers or self classification

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 (2017) was used to determine the skin irritation/corrosion and the serious eye damage/ eye irritant hazards of the registered substance. The decision of classification as skin 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 as Eye irritant Category 2. Therefore, the test item should be classified as a skin irritant cat.2 and an Eye irritant cat.2 without further testing according to the rules for classification of mixtures of Regulation (EC) No 1272/2008.

 

- Skin irritation/corrosion

Constituent	CAS	Classification according to the Regulation (EC) No. 1272/2008 (CLP)	Source
Linalool	78-70-6	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification
Geranial	141-27-5	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification
Alpha-terpineol	98-55-5	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification
(Z)-3,7-dimethylocta-2,6-denial	106-26-3	Skin Irrit. 2 - H315	ECHA C&L inventory–self classification

Source: ECHA disseminated dossiers or self classification

 

- Eye irritation/corrosion

Constituants	Cas Number	Classification (CLP 1272/2008)	Source
Linalool	78-70-6	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
Decanal	112-31-2	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
Geranial	141-27-5	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
Alpha-terpineol	98-55-5	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification
(Z)-3,7-dimethylocta-2,6-denial	106-26-3	Eye Irrit. 2 - H319	ECHA C&L inventory - self classification

Source: ECHA disseminated dossiers or self classification

 

IN VITRO STUDIES

Skin irritation (OECD 439, GLP, rel.1, WoE)

An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.

Test item was applied as supplied, at the dose of 16 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE®model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 41 hours post-incubation period at 37°C, 5% CO2. 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.Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) under the same conditions in order to generate non-specific MTT reduction.

The mean corrected percent viability of the treated tissues was 0.1%, versus 2.2% in the positive control (5% Sodium Dodecyl Sulfate). In accordance with the Regulation EC No. 1272/2008, the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 has to be classified in Category 2 “Irritating to skin” or in Category 1 “Corrosive”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” or “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are required.

 

Eye irritation (OECD 492, GLP, rel.1, WoE)

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).

Test item 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, 95% humidity (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 post-exposure incubation at standard culture conditions. The tissue viability was measured by performing an MTT assay. Additionally, 2 killed RhCE (EpiOcular (TM) tissue model) were treated in the same manner in order to generate non-specific MTT reduction.

The mean corrected percent tissue viability of the RhCE replicates treated with the test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 was 54.17% versus 35.98% 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 has to be identified as potentially requiring classification and labeling according to UN GHS Category 2 or Category 1.

 

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

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.

Test item ORANGE ESS TYPE BRESIL DETERP 100% O15231 was applied as supplied , at the dose of 30 µL, to 3 enucleated chicken eyes 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: 1.0, corresponding to ICE class II;

- mean score of fluorescein retention: 1.2, corresponding to ICE class II;

- maximal mean corneal swelling: 8%, corresponding to ICE class II.

The combination of the three endpoints for test item was 2 x II.

The combination of the three endpoints for the positive control, sodium hydroxide, 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 ORANGE ESS TYPE BRESIL DETERP 100% O15231 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 substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

 

Self classification:

Skin and eye irritation/corrosion

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).

Moreover, in vitro studies were available to support the previous classification.

About the eye irritation/corrosion, an in vitro study (OECD 492) did not allow to discriminate between the UN GHS Category 2 ("irritant") or Category 1 ("corrosive") and 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 cat.2.

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.

 

About the skin corrosion, no study on corrosivity has been performed but the following arguments can be taken into consideration:

- no constituants were classified as "corrosive".

- the substance is not considered as an eye corrosive.

- Calculation rules have allowed to classify the substance as skin irritant cat.2.

- An in vitro study (OECD 439) has allowed to show that the test item is a skin irritant.

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

 

Respiratory irritation:

No data was available regarding respiratory irritation.