Reaction mass of 1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptane and cineole

Endpoint summary

Description of key information

According to the results of a study conducted according to OECD Guideline 439 and in accordance with GLP, the substance has to be classified as skin irritant.

According to the results of two studies conducted according to OECD Guidelines 438 and 492 and in accordance with GLP, the substance has to be classified as eye irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August - 15 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 439 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

dated 23 July 2009

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Test system:

human skin model

Source species:

human

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 (Episkin SA, RHE/S/17 Batch No. 16-RHE-098) were received on 13 September 2016.
- 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, batch No. 16 MPE 094) during 2 hours and 5 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, batch No. 16 MA 051).

TREATMENT
- The test item was applied, as supplied, at the dose of 16 µL, to the epidermal surface of 3 living skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS – PAN BIOTECH GmbH - Batch No. 4710416) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) in a 10 mL volumetric flask (QS 10 mL of distilled water). Then, the preparation was magnetically stirred, just before the treatment.

REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the human epidermis were washed with 25 x 1 mL of DPBS (PAN BIOTECH GmbH, Batch No. 4710416). The rinsed tissues were checked for any coloration and noted to be whitish, comparable coloration to that of the negative control tissues. They were incubated for a 41-hour and 41-minute post-treatment incubation-period in fresh medium at 37°C, 5% CO2. Then, the epidermis 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.
- 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 measured OD were 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.

VIABILITY CALCULATION:
- 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. The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100

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%. 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”.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

Duration of treatment / exposure:

42 minutes at room temperature

Duration of post-treatment incubation (if applicable):

41-hour and 41-minute post-treatment incubation period in fresh medium at 37°C, 5% CO2

Number of replicates:

3 replicates of living human skin models

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

main test (duration of exposure: 42 minutes)

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
No blue or purple coloration was noted after 3 hours of incubation between 37.4°C and 37.8°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colorless solution was obtained in water after 3 hours of incubation between 37.4°C and 37.8°C, 5% CO2. A colorless solution was obtained in isopropanol after 2 hours of incubation at room temperature. No significant coloration appeared. Therefore the test item was considered to be not interfering with the MTT assay.


MTT VIABILITY ASSAY RESULTS
The mean percent viability of the treated tissues was 1.4%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate).

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

 

	Skin	OD	Mean OD / disc (#)	Mean OD / product	Viability %	Mean viability %	Standard deviation (SD)
Negative control	1	0.829	0.885	1.053	84.0	100.0	14.2
		0.912
		0.916
	2	1.059	1.102		104.7
		1.127
		1.121
	3	1.146	1.172		111.3
		1.165
		1.205
Positive control	1	0.012	0.012	0.015	1.1	1.4	0.2
		0.012
		0.013
	2	0.015	0.015		1.4
		0.016
		0.016
	3	0.017	0.017		1.6
		0.017
		0.017
Test item	1	0.014	0.014	0.014	1.3	1.4	0.1
		0.015
		0.015
	2	0.014	0.015		1.4
		0.015
		0.016
	3	0.014	0.014		1.3
		0.014
		0.015

#: mean of 3 values (triplicate of the same extract)

OD: optical density; measured after a 1:2 dilution of the formazan extracts in isopropanol.

 

Acceptability criteria: SD ≤ 18%

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

In accordance with Regulation EC No. 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. It is also classified in Category 2 according to UN GHS regulation.
Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or in Category 2 “Irritant”.

Executive summary:

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

The test substance was applied as supplied, at the dose of 16 µL, to 3 living Reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 41-minute 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.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean corrected percent viability of the treated tissues was 1.4%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

Therefore, in accordance with Regulation EC No. 1272/2008, the test substance has to be classified in Category 2 “Irritating to skin”. The hazard statement “H315: Causes skin irritation” with the signal word “Warning” are required. It is also classified in Category 2 according to UN GHS regulation.

Considering the results obtained, an in vitro test of skin corrosivity has to be conducted to know if the test item has to be classified in Category 1 “Corrosive” or in Category 2 “Irritant”.

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:

key study

Study period:

19 May - 02 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study performed according to OECD Guideline 492 without any deviation

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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 28 July 2015

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Species:

human

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, batch No. 23712] were received on 31 May 2016.

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): 50 µL
- Concentration (if solution): 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: 1 hour and 50 minutes at standard culture conditions

Number of animals or in vitro replicates:

Test item, negative and positive controls were applied on duplicate tissues.

Details on study design:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
The direct interaction of MTT with the test item was checked by adding 50 µL of the test item to 1 mL of MTT solution at 1 mg/mL.
- Test for the detection of the colouring potential of the test item:
The colouration potential of the test item in water or isopropanol was checked by adding 50 µL of the test item to 1 mL of distilled water or 2 mL of isopropanol, respectively.


MAIN TEST
- Pre-incubation of the tissues:
On the day of receipt, the 12 tissues in their 24-well shipping container were equilibrated at room temperature for 15 minutes. Then the inserts (filter + epithelium) were gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. They were placed in 6 wells culture plate which had been previously filled with 1 mL of 37°C pre-warmed assay medium and incubated during 21 hours and 05 minutes at standard culture conditions.
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 during 30 minutes at standard culture conditions. In the same experimental conditions, a positive control (Methyl acetate), and a negative control (distilled water) were used. The controls were applied, as supplied, at the dose of 50 µL, to the surface of 2 viable 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. The rinsed tissues were checked for any colouration and noted for comparable colour with the negative control. 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 1-hour and 50-minute 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 by Optical Density (OD) after extraction from tissues. The measured OD was proportional to the number of living cells.
The RhCE constructs were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at standard culture conditions. The precipitated blue formazan product was then extracted from the tissues by placing each insert in 2 mL of isopropanol during 17 hours and 05 minutes at 6±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 measurement of formazan extracts at 570 nm was measured in triplicate samples 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 viability of the tissues

Run / experiment:

Main test

Value:

39.06

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution with colourless deposit of test item in the bottom of the well was observed after 3 hours of incubation between 36.5°C and 38.2°C, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Test for the detection of the colouring potential of the test item:
A colourless solution with colourless deposit of test item in the bottom of the well was obtained in water after 1 hour of incubation between 36.5°C and 38.2°C, 5% CO2. A colourless solution with colourless deposit of test item in the bottom of the well was obtained in isopropanol after 3 hours of incubation at room temperature. No significant colouration appeared, therefore the test item was considered to be not interfering with the MTT assay.

MAIN TEST
- MTT assay results: The mean percent tissue viability of the RhCE replicates treated with the test substance was 39.06%, versus 34.89% in the positive control (Methyl acetate). Refer Tables 7.3.2/1 for more details.

Table 7.3.2/1: Main test - Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

	Tissue	OD	Mean OD/disc (#)	Mean OD/product	Viability %	Mean viability %	Difference of viability %
Negative control	1	1.261	1.214	1.223	99.30	100.00	1.39
		1.180
		1.203
	2	1.274	1.231		100.70
		1.215
		1.205
Positive control	1	0.471	0.462	0.427	37.79	34.89	5.81
		0.461
		0.455
	2	0.393	0.391		31.98
		0.389
		0.392
Test item	1	0.503	0.500	0.478	40.90	39.06	3.68
		0.492
		0.506
	2	0.458	0.455		37.22
		0.461
		0.447

#: mean of 3 values (triplicate of the same extract)

OD: optical density

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In accordance with Regulation EC No. 1272/2008, the test substance was identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1. The results of an eye corrosivity study are necessary to conclude on the classification of the test substance.

Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ 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-minute post-exposure immersion period at room temperature and a 1-hour and 50-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test substance was 39.06%, versus 34.89% in the positive control (Methyl acetate).

Therefore, in accordance with Regulation EC No. 1272/2008, the test substance was identified as potentially requiring classification and labelling according to UN GHS Category 2 or Category 1. The results of an eye corrosivity study are necessary to conclude on the classification of the test substance.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

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 06 February 2017 at 8:05 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 06 February 2017 at 9:30 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
- 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 between 32.3 °C and 32.7 °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 64 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.

NUMBER OF REPLICATES
- 1, 3 and 3 eyes for negative & positive control and test item, respectively.

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: 5% Benzalkonium chloride

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, as supplied, to the cornea 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 from the eye with 20 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:

Mean of three replicates /10 seconds exposure

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no prediction can be made

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean of three replicates /10 seconds exposure

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Irritation parameter:

percent corneal swelling

Run / experiment:

Mean of three replicates /10 seconds exposure

Value:

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:

OCULAR REACTIONS:
- maximal mean score of corneal opacity: 0.5, 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 1 x III, 2 x I.

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.

Table 7.3.2/5: Individual and average values for evaluation of corneal lesions after treatment

 

Endpoint measured	Eye No.	Time (minutes)
		0	30	75	120	180	240
Corneal opacity	4	0	0	0	0	0	0
	5	0	0	0	0.5	1	1
	6	0	0	0	0.5	0.5	0.5
Mean		0.0	0.0	0.0	0.3	0.5	0.5
ICE class		I
Fluorescein retention	4	0.5	1	-	-	-	-
	5	0.5	3	-	-	-	-
	6	0.5	2	-	-	-	-
Mean		0.5	2.0		-	-	-
ICE class		III
Corneal thickness	4	0.54	0.54	0.55	0.55	0.55	0.55
	5	0.58	0.58	0.60	0.60	0.61	0.61
	6	0.58	0.58	0.60	0.60	0.60	0.60
Corneal swelling (%)	4	-	0	2	2	2	2
	5	-	0	3	3	5	5
	6	-	0	3	3	3	3
Mean		-	0	3	3	3	3
ICE class		I
Combination of the three endpoints	1 x III, 2 x I
Classification	No prediction can be made

Note: From 30 minutes, significant background noise not preventing the fluorescein quotation.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In accordance with Regulation (EC) No. 1272/2008, the obtained results lead to category “no prediction can be made”, as defined by OECD Guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or is not classified for eye irritation/serious eye damage (No category) in the Isolated Chicken Eye test.

Executive summary:

An ex vivo eye irritation study was performed according to 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, 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 are listed below.

- maximal mean score of corneal opacity: 0.5 , 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 the test item was 1 x III, 2x I.

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 category “no prediction can be made”, as defined by OECD guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or is not classified for eye irritation/serious eye damage (No category) in the Isolated Chicken Eye test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

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

The test substance was found not to have direct MTT reducing properties or colouring potential. The mean corrected percent viability of the treated tissues was 1.4%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validate the experiment.

According to the results of this study, the registered substance should be classified in category 2 for skin irritation (H315) according to Regulation (EC) No. 1272/2008 (CLP) and GHS.

Eye irritation:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.The mean percent tissue viability of the RhCE replicates treated with the test substance was 39.06%, versus 34.89% in the positive control (Methyl acetate).

An ex vivo eye irritation study was performed according to 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 ocular reactions observed in eyes treated with the test item are listed below.

- maximal mean score of corneal opacity: 0.5 , 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 the test item was1 x III, 2x I.

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.

Therefore, the test item is not predicted as causing serious eye damage (Category 1) or is not classified for eye irritation/serious eye damage (No category) in the Isolated Chicken Eye test.

According to the results of those studies, the registered substance should be classified in category 2 for eye irritation (H319) according to Regulation EC No. 1272/2008 (CLP) and GHS.

Justification for classification or non-classification

Based on the results of an OECD 439 study and according to Regulation (EC) No. 1272/2008 and GHS, the registered substance is classified in category 2 for skin irritation (H315).

Based on the results of an OECD 438 and an 492 studies and according to Regulation (EC) No. 1272/2008 and GHS, the registered substance is classified in category 2 for eye irritation (H319).