Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 20 February 2018 Experimental completion date: 02 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD guideline and performed under GLP
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% suspension (w/v) in saline using sonication for 10 minutes.

VEHICLE (Saline)
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 0.9% NaCl in deionised water


POSITIVE CONTROL (Benzalkonium chloride)
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 10% (w/v) in 0.9% (w/v) NaCl (saline) using sonication for 10 minutes.
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 for test itema and each control
Details on study design:
Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The corneae were directly used in the BCOP test on the same day.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.
At the end of the incubation period, the basal opacity was determined (t0).
The basal opacity of all corneae was recorded. Each cornea with a value of the basal opacity > 7 was discarded. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

Exposure of the Corneae to the Test Groups
The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
Afterwards, the test item or the control items, respectively, were each rinsed off from the according application sides with saline, and fresh incubation medium was added into the anterior compartment and opacity was measured (t240).
In the second step of the assay, permeability of the cornea was determined.

Opacity Measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
After exposure of the corneae to the different test groups, and after rinsing the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
2.74
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
This in vitro study was performed to assess the corneal damage potential of beta-carotene by means of the BCOP assay using fresh bovine corneae.
After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test item beta-carotene, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t240).
After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
The experiment has to be repeated twice since the values of the controls did not fulfil the acceptance criteria.
For the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 1.03).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =108.55) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item beta-carotene did not cause a relevant increase of the corneal opacity. The calculated mean IVIS was 2.74 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorized.

Results after 240 Minutes Treatment Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Proposedin vitroIrritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

1

0.33

0.045

0.046

1.68

1.03

No Category

0

0.044

0.66

0

0.050

0.75

Positive Control

95.67*

0.627*

105.07

108.55

Category 1

103.67*

0.735*

114.69

98.67*

0.483*

105.91

beta-carotene

2.67*

0.030*

3.11

2.74

No Category

1.67*

0.035*

2.19

2.67*

0.017*

2.92

*corrected values

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, beta-carotene is not classified for eye irritation (GHS).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of beta-carotene by means of the BCOP assay using fresh bovine corneae. Testing was performed under GLP and in compliance with the test guidelines OECD 437 and EC B.47.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspensionin saline (0.9% (w/v) NaCl in deionised water) of the test item beta-carotene as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae andopacity was measured again (t240).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed.

The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (CLP/EPA/GHS (Cat 1)).

 

Relative to the negative control, the test item beta-carotene did not cause a relevant increase of the corneal opacity. The calculated mean in vitro irritancy score was 2.74. According to OECD 437 the test item is not classified for eye irritation (GHS).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 14 August 2017 Experimental completion date: 01 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
human
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Test System and Supporting Information
Lot No.: 27012 + 27025

Cell Culture
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELL, 10 mm Ø).
EpiOcular™ tissues were received at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL fresh Assay Medium at 37 °C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (16 -24 hours).
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 mg

POSITIVE CONTROL (Methyl acetate)
- Amount(s) applied (volume or weight with unit): 50 µL

NEGATIVE CONTROL (Deionised water)
- Amount(s) applied (volume or weight with unit): 50 µL
Duration of treatment / exposure:
6 hours
Duration of post- treatment incubation (in vitro):
25 minutes post-soak intended to remove any test item absorbed into the tissue.
18 hours post-treatment incubation
Number of animals or in vitro replicates:
conducted on duplicate tissues
Details on study design:
Experimental Performance
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.
After the 30 minute Ca2+Mg2+free-DPBS pre-treatment, the test and control item were tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 °C, 5 ± 0.5% CO2, 95% RH) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca2+Mg2+-free DPBS (brought to room temperature).
Three clean beakers containing a minimum of 100 mL each of Ca2+Mg2+-free DPBS were used per test item. The test item utilized a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts are mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The culture was then be rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).
Since it was not possible to remove the visible test item completely, this was noted in the study file. No further rinsing was done.
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 minutes immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. The plates were sealed with parafilm (between the plate cover and upper edge of the wells) or a standard plate sealer, and were immediately extracted (shaken for 2 to 3 hours at room temperature). The tissues were not pierced. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Colorant Controls for Assessment of Coloured or Staining Test Items
Since the test item showed to have or to develop relevant colour, which could interact with the MTT measurement, an additional test had to be performed to determine the amount of colour bound to and extracted from the tissues. For this purpose the coloured test item was applied to two additional tissues (= colorant controls (CC)), and were treated in the same way previously described. In contrast to the normal viability test, no MTT assay was performed. The bound colour was extracted and the absorbance of the isopropanol extracts was measured identically as in the MTT assay for coloured test items (as described for the MTT assay with 2 mL extraction solution in 6-well plates without piercing the tissue, and starting with a 180 min incubation in medium instead of MTT solution addition). The amount of extracted colour was subtracted from the results of the viability assay.
Since residues of the test item were on the tissues even after isopropanol extraction, this was documented in the remarks.
Irritation parameter:
other: cell viability (%)
Run / experiment:
1
Value:
57.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: borderline positive
Irritation parameter:
other: cell viability (%)
Run / experiment:
2
Value:
17.9
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Results first experiment
The optical pre-experiment (colour interference pre-experiment) to investigate the test item’s colour change potential in water or isopropanol led to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue/purple colour. Therefore, an additional test with freeze-killed tissues was not necessary.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 57.9% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.494 and 1.569).
• The mean relative viability of the positive control is below 50% of the negative control viability (28.6%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% and 14.3%) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the additional viable tissues (without MTT addition) which were calculated as percent values related to the viability of the relating negative control.

Results second experiment
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 17.9% (threshold for irritancy: ≤ 60%), consequently the test item was irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.833 and 1.965).
• The mean relative viability of the positive control is below 50% of the negative control viability (38.6%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 0.0% and 6.0%) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the additional viable tissues (without MTT addition) which were calculated as percent values related to the viability of the relating negative control.

Results first experiment

Results after the treatment for 6 hours withbeta-caroteneand the controls

Treatment Group

Tissue No.

OD 570 nm
Well 1

OD 570 nm
Well 2

Mean OD of 2 Wells

Mean OD

of 2 Wells blank

corrected

Mean

OD

of Treatment Group

blank corrected

Rel. Viability [%] Tissue
1, 2 *

Absolute Value of the Difference of Rel. Viability 
Tissue 1,2
[%]

Mean Rel. Viability

[%]**

Blank

 

0.035

0.034

0.035

 

 

 

 

 

Negative Control

1

1.543

1.494

1.518

1.484

1.507

98.5

3.0

100.0

2

1.569

1.560

1.564

1.530

101.5

Positive Control

1

0.493

0.479

0.486

0.451

0.431

30.0

2.7

28.6

2

0.446

0.446

0.446

0.411

27.3

Test Item

1

1.024

1.019

1.022

0.987

0.880

65.5

14.3

57.9***

2

0.817

0.797

0.807

0.772

51.3

Blank

 

0.035

0.034

0.035

 

Negative Control
Viable Tissues

1

0.042

0.042

0.042

0.007

0.006

0.5

0.1

0.4

2

0.040

0.041

0.041

0.006

0.4

Test Item Viable Tissues

1

0.042

0.044

0.043

0.008

0.008

0.5

0.0

0.5

2

0.042

0.043

0.042

0.008

0.5

* Relative viability [rounded values]: (100 x (absorbance test item / positive control / negative control)) / (mean absorbance negative control)

 

** Mean relative viability [rounded values]: (100 x (mean absorbance test item / positive control / negative control)) / (mean absorbance negative control)

 

*** corrected value True viability [%]: mean absorbance test item– mean absorbance additional viable tissue test item treated– (mean absorbance test item treated killed control – mean absorbance negative control of freeze killed tissues)

Results second experiment

Results after the treatment for 6 hours withbeta-caroteneand the controls

Treatment Group

Tissue No.

OD 570 nm
Well 1

OD 570 nm
Well 2

Mean OD of 2 Wells

Mean OD

of 2 Wells blank

corrected

Mean

OD

of Treatment Group

blank corrected

Rel. Viability [%] Tissue
1, 2 *

Absolute Value of the Difference of Rel. Viability 
Tissue 1,2
[%]

Mean Rel. Viability

[%]**

Blank

 

0.037

0.036

0.036

 

 

 

 

 

Negative Control

1

1.895

1.833

1.864

1.828

1.859

98.3

3.3

100.0

2

1.965

1.887

1.926

1.890

101.7

Positive Control

1

0.715

0.683

0.699

0.662

0.718

35.6

6.0

38.6

2

0.828

0.794

0.811

0.774

41.7

Test Item

1

0.383

0.369

0.376

0.340

0.349

18.3

0.9

17.9***

2

0.396

0.391

0.393

0.357

19.2

Blank

 

0.037

0.036

0.036

 

Negative Control
Viable Tissues

1

0.042

0.041

0.041

0.005

0.005

0.3

0.0

0.2

2

0.041

0.040

0.040

0.004

0.2

Test Item Viable Tissues

1

0.056

0.049

0.052

0.016

0.015

0.9

0.1

0.8

* Relative viability [rounded values]: (100 x (absorbancetest item / positive control / negative control)) / (mean absorbancenegative control)

 

** Mean relative viability [rounded values]: (100 x (mean absorbancetest item / positive control / negative control)) / (mean absorbancenegative control)

 

*** corrected value True viability [%]: mean absorbancetest item– mean absorbanceadditional viable tissue test item treated– (mean absorbancetest item treated killed control– mean absorbancenegative control of freeze killed tissues)

Interpretation of results:
other: eye irritating potential
Conclusions:
In conclusion, it can be stated that in this study and under the experimental conditions reported, beta-carotene possesses an eye irritating potential.
Executive summary:

This in vitro study was performed to assess the eye irritation potential of beta-carotene by means of the Human Cornea Model Test.

The test item did not prove to be an MTT reducer in the MTT pre-test. Its intrinsic colour was not intensive but it proved to dye water and isopropanol in the colour interference pre-test. Therefore, additional tests with viable tissues (without MTT addition) had to be performed. The viability values resulted in this additional test were used to correct the values gained in the normal tests.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with thenegative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Since the result of the first experiment was borderline positive, the test was repeated. The second result was clearly below the threshold and confirms the first result.

Irritating effects were observed following incubation with beta-carotene. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (to 57.9% in the first experiment, to 17.9% in the second experiment).

In conclusion, it can be stated that in this study and under the experimental conditions reported, beta-carotene possesses an eye irritating potential.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

The eye irritancy potential of the substance was assessed according to the Bovine corneal opacity and permeability test method (OECD 437) and the reconstructed human cornea-like epithelium test method (OECD 492).

In the OECD 437 study, a substance is considered not to have eye irritancy potential if an in vitro irritancy score (IVIS) of ≤ 3 is reported. Values that fall in the range >3; ≤ 55 can not be used to make an accurate prediction of eye irritancy potential, and values > 55 would lead to classification as Category 1 eye damage. In this study an IVIS of 2.74 was reported, and the substance was not classified for eye irritation under the conditions of this test. Two previous BCOP tests which were invalidated due to the negative and then positive controls being out of range reported IVIS of 1.86 and 4.66 respectively. If valid, these results would have given classifications of non-irritating and unable to make a prediction.

In the OECD 492 study the threshold for a negative result is 60 % tissue viability. Therefore, a tissue viability of > 60 % after treatment with the test substance would lead to no classification for eye irritation. In the first run of this test a value of 57.9% was a reported, which was considered to be a borderline result. The second run gave a clearly positive result of 17.9 % and it was concluded that the substance possesses eye irritation potential.

The difference in results between the two OECD studies could be due to the difference in incubation time (4 hours in OECD 437 and 6 hours in OECD 492), but also due to the test item being difficult to completely remove from the cornea tissue in the OECD 492 study. As stated in the study report, there was still residues of the test item on the tissues even after isopropanol extraction. Therefore, the tissues in the OECD 492 study may have been exposed to the test item for more than the stated 6 hours due to the difficulty in completely removing the test item after the exposure duration.

Considering that in the BCOP study a negative result was reported in the valid study and in the EpiOcular study the first test gave a borderline positive result, and the positive result in the second test may have been due to the difficulty in removing the test item from the cornea, a precautionary classification of Eye Irritation Category 2 was concluded.