Registration Dossier

Administrative data

Description of key information

2 studies performed for skin irritation/corrosion:

Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate: In Vitro Skin Corrosivity Test in the EPISKINTM(SM) Model

Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate: In Vitro Skin Irritation Test in the EPISKINTM(SM) Model

1 study performed for eye irritation:

Sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate: In Vitro Eye Irritation Test in Isolated Chicken Eyes

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human SkinModel Test”,
Version / remarks:
Official Journal of the European Union No. L142 (31 May 2008)
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult humanderived epidermal keratinocytes
Justification for test system used:
The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Details on test system:
Kit Contents
Units: EPISKINTM(SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM(SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 18 MAIN3 004; Exp. Date: 07 February 2018)
A flask of sterile “Assay Medium”
(Batch No.: 18 ESSC 004; Exp. Date: 07 February 2018)

Number of Replicate Wells
In this assay, two replicates for test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD
evaluation.

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM(SM) kits were kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test item was applied as supplied, no formulation was required (although it was grounded to fine powder).

Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere.

Application (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact
with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of powdered test item was applied evenly to the epidermal surface of each of two test item treated skin units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the
epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin
units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
Note: The negative and positive controls were also part of a concurrent study (Citoxlab study code: 17/361-039B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.
The plates with the treated epidermis units were incubated for 4 hours at room temperature (23.1-25.2°C) covered with the plate lids.
Duration of treatment / exposure:
1 day
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
optical density (OD) measured at 570 nm
Value:
104.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the two negative control tissues was in the recommended range (0.928).

The two positive control treated tissues showed -0.2% viability* demonstrating the proper performance of the assay.

*Note: The positive control material completely destroyed the skin cells. Thus the minor negative viability values were considered acceptable, and the result were considered to meet the validity criterion.

The difference of viability between the two test item-treated tissue samples in the MTT assay was 1.0%.

The difference of viability between the two negative control tissue samples in the MTT assay was 5.1 %.

The mean OD value of the blank samples (acidified isopropanol) was 0.046. All these parameters were within acceptable limits and therefore the study was considered to be valid.

INTERPRETATION OF TEST RESULTS

The prediction model below corresponds to the methods agreed by EU regulatory agencies in line with OECD No. 431 (OECD, 2016).

The cut-off value of 35% and classification method was validated in an international validation study of this kit (Fentem, 1998).

For 2 disks:

If both disks have mean viability of ≥35% = Non Corrosive

If both disks have mean viability of <35% = Corrosive (at the corresponding incubationperiod)

For more than 2 disks:

If the mean value is ≥35% and the variability is less than 50% = Non Corrosive

If the mean value is <35% and the variability is less than 50% = Corrosive

Otherwise:

If the classification is not made with these criteria, retest with 2 more disks. Take the mean of the 4 disks to classify as above or below 35%. Outlier values may be excluded where there are scientific reasons, such as where application or rinsing is difficult and that the Study Director considers that a result is not representative.

VIABILITY RESULTS

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2 (see attached). The mean OD value for the test item treated skin samples showed a 104.6% relative viability compared to the negative control.

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H- 1-benzopyran-7-yl)oxy]acetate, the mean cell viability was 104.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN™(SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-corrosive to the skin.
Executive summary:

An in vitro skin corrosivity test of sodium [(6-hydroxy-4-methyl-2-oxo-2H-1 -benzopyran-7-yl)oxy]acetate test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5 - Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with powdered sodium [(6-hydroxy- 4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control).

Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7 - yl)oxy]acetate, the mean cell viability was 104.6% compared to the negative control.

This is above the threshold of 35%, therefore the test item was considered as being non corrosive.

The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-corrosive to the skin.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: from adult donors
Justification for test system used:
The EPISKINTM (SM) model has been validated for irritation testing in an international validation study [10] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this
study.
Details on test system:
Kit Contents
Units: EPISKINTM (SM) plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EPISKINTM (SM) biopsy punch for easy sampling of epidermis
Medium: A flask of sterile “Maintenance Medium”
(Batch No.: 18 MAIN3 010; Exp. Date: 07 March 2018)
A flask of sterile “Assay Medium”
(Batch No.: 18 ESSC 008; Exp. Date: 07 March 2018)

Number of Replicate Wells
In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Kit Reception
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- orange colour = good
- yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40°C (the colour change is irreversible, independent of the length of the period above 40°C):
- white colour = good
- grey or black colour = not acceptable
The kits were found to be in good order at reception.

Storage
The EPISKINTM (SM) kit was kept in their packaging at 37°C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2-8°C until the initiation of the test.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The test item was applied as supplied, no formulation was required (although it was grounded to fine powder).


Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis
into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere.

Application and rinsing (Day 0)
Test Item
First an appropriate amount (10 μL) distilled water was applied to the epidermalsurface in order to improve further contact between test item and epidermis and then
20 mg of the powdered test item was applied evenly to the epidermal surface. The test item was spread gently on the skin surface with a pipette tip without damaging the epidermis units. The amount was sufficient to cover the epidermal surface.

Negative and positive controls
50 μL of negative control (PBS) or positive control (5% (w/v) SDS solution) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with
the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Note: The negative and positive controls were also part of a concurrent studies (Citoxlab study codes: 17/360-043B, 18/009-043B and 18/011-051B) performed in the same experimental period using the same batch of chemicals and same batch of skin units.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (23.9-25.5°C).
After the 15 minutes incubation time, the EPISKINTM (SM) units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal
surface with a pipette (without touching the epidermis).
Duration of treatment / exposure:
2 days
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Optical density measured at 570 nm
Value:
97.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

VALIDITY OF THE TEST

After receipt, the two indicators of the delivered kits were checked. Based on the observed colours, the epidermis units were in proper conditions.

The mean OD value of the three negative control tissues was in the recommended range (0.810). Standard deviation of the viability results for negative control samples was 3.5%.

The positive control treated tissues showed 4.5% viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.0%.

The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 3.3%.

The mean OD value of the blank samples (acidified isopropanol) was 0.047.

All these parameters met the acceptability criteria, therefore the study was considered to be valid.

INTERPRETATION OF TEST RESULTS

The irritation potential of test items can be classified according to the United Nations Globally Harmonized System of Classification and Labelling of Chemicals, and a similar system is used in CLP. In the present study, the irritancy potential of test items is predicted by the mean tissue viability of tissues exposed to the test item. The test item considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control. The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability after 15 minutes of exposure and 42 hours post incubation is more than (˃) to 50% of the negative control.

VIABILITY RESULTS

The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2 (see attached). The mean OD values for the test item treated skin samples showed 97.2% relative viability compared to the negative control.

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7- yl)oxy]acetate, the mean cell viability was 97.2% compared to the negative control.
This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKINTM (SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category (since the test item is known not to be corrosive).
Executive summary:

An in vitro skin irritation test of sodium [(6-hydroxy-4-methyl-2-oxo-2H-1 -benzopyran-7-yl)oxy]acetate test item was performed in a reconstructed human epidermis model. EPISKINTM (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5 - Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

The irritation potential of the test item was evaluated according to the OECD No. 439 guideline.

Disks of EPISKINTM (SM) (three units) were treated with the powdered test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2, in a >95% humidified atmosphere. The viability of each disk was assessed by incubating the

tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere, protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test item. For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes of exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7 - yl)oxy]acetate, the mean cell viability was 97.2% compared to the negative control.

This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid. In conclusion, in this in vitro EPISKINTM (SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-irritant to skin, UN

GHS Classification: No Category (since the test item is known not to be corrosive).

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

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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)
GLP compliance:
yes
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
In each experiment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
Duration of treatment / exposure:
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Duration of post- treatment incubation (in vitro):
The all cornea surfaces were cleared at 30 minutes after the post-treatment rinse.
Details on study design:
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye.
The cornea thickness of the eyes should not change by more than 5% within the -45min and the zero time. No changes in thickness (0.0%) were observed in the eyes in each experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

Treatment
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea. In each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the test item and another three with powdered Imidazole in each experiment.

Test item removal
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible.Additional gentle rinsing with at least 20 mL saline was performed at each time point when the test item or positive control material remaining on the cornea was observed.
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 75 min
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 240 min
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
I
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 75 min
Run / experiment:
II
Value:
0.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
percent corneal swelling
Remarks:
Mean maximum corneal swelling at up to 240 min
Run / experiment:
II
Value:
1.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
II
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Run / experiment:
II
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate showed no corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results.
Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (09 October 2017).

In each experiment after the zero reference measurements, the eye was held in horizontal position and powdered 30 mg test item was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in each experiment. Thus, the study was considered to be valid.

Experiment I: No corneal swelling was observed during the four-hour observation period on test item treated eyes. No cornea opacity change and no fluorescein retention change was observed on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The all cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The all cornea surfaces were cleared at 30 minutes after the post-treatment rinse.

Based on these in vitro eye irritation assays in isolated chicken eyes with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, the test item was non-irritant, UN GHS Classification: No Category.

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

Additional information

Justification for classification or non-classification

Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H- 1-benzopyran-7-yl)oxy]acetate, the mean cell viability was 104.6% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™(SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-corrosive to the skin.

Following exposure with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7 - yl)oxy]acetate, the mean cell viability was 97.2% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKINTM (SM) model test with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate (Batch number: 44034), the results indicate that the test item is non-irritant to skin, UN GHS Classification: No Category (since the test item is known not to be corrosive).

Based on these in vitro eye irritation assays in isolated chicken eyes with sodium [(6-hydroxy-4-methyl-2-oxo-2H-1-benzopyran-7-yl)oxy]acetate, the test item was non-irritant, UN GHS Classification: No Category.