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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The test was performed on 17 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
The positive control is 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline since the laboratory historical control data is established with this chemical.
Qualifier:
according to guideline
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) No 1152/2010: B. 47. Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants (Official Journal of the European Union, L 324, 9.12.2010).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (Reach).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 13 until 16 April 2015, Date of signature: 14 September 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)
EC Number:
233-357-8
EC Name:
Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)
Cas Number:
10127-27-2
Molecular formula:
C16H11CrN5O8S.Na
IUPAC Name:
sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

Test animals / tissue source

Species:
other: Freshly isolated bovine cornea (at least 9 month old donor cattle)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was tested as a 20% suspension (w/v) in saline.
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
90 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
Details on study design:
The anterior compartment received the test item suspension, the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae via open chamber method, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath.
The incubation time lasted 240 minutes.
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Since the test item proved to be difficult to remove by the rinsing method, the front cover of the holder was opened and the cornea was carefully washed using a gentle stream of incubation medium and eye wipers. The corneae still remained stained orange to red and the test item remained on the surface. The corneae were given a final rinse with cMEM without phenol red. Fresh cMEM was added into the anterior compartment and opacity was measured (t240).

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae and displays a numerical opacity value. 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 both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 5 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).


DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation

The following formula is used to determine the IVIS of the negative control:

IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.

Depending on the IVIS score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):

≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1


Criteria for Determination of a Valid Test

The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

A single testing run composed of at least three corneae should be sufficient for a test chemical when the resulting classification is unequivocal. In cases of borderline results in the first testing run, a second testing run will be considered, as well as a third one in case of discordant mean IVIS results between the first two testing runs. A result in the first testing run is considered borderline if the predictions from the 3 corneae are non-concordant, such that:
• 2 of the 3 corneae give discordant predictions from the mean of all corneae, or,
• 1 of the 3 corneae gives a discordant prediction from the mean of all 3 corneae, and the discordant result is >10 IVIS units from the cut-off threshold of 55.
• If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean IVIS value), then a final decision can be taken without further testing. If the repeat testing run results in a non-concordant prediction from the initial testing run (based upon the mean IVIS value), then a third and final testing run should be conducted to resolve equivocal predictions, and to classify the test chemical. It may be permissible to waive further testing for classification and labelling in the event any testing run results in a UN GHS Category 1 prediction.


Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
main experiment
Value:
58.55
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
Category 1
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: fulfilled, no category (IVIS 1.25)
- Acceptance criteria met for positive control: filfilled, category 1 (IVIS 104.82)

Any other information on results incl. tables

Results after 240 Minutes Treatment Time

Test Group

Opacity #

value

Permeability ##

 

IVIS

Mean IVIS

Proposed in vitro Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

0

 

0.062

 

0.93

 

 

Negative Control

0

0.33

0.068

0.061

1.02

1.25

No Category

Negative Control

1

 

0.054

 

1.81

 

 

Positive Control

95.67*

 

0.583*

 

104.41

 

 

Positive Control

88.67*

 

0.474*

 

95.77

104.82

Category 1

Positive Control

104.67*

 

0.642*

 

114.29

 

 

Aluminium Orange G

46.67*

 

1.100*

 

63.16

 

 

Aluminium Orange G

37.67*

 

0.976*

 

52.30

58.55

Category 1

Aluminium Orange G

44.67*

 

1.035*

 

60.19

 

 

# Opacity value = Difference (t240 - t0) of Opacity

## Permeability at 490 nm (OD490)

*corrected values

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, the test item is serious eye damaging (EU CLP/UN GHS Category 1).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of the test item 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 (0.9% (w/v) NaCl in deionised water) of the test item 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 was 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 (EU CLP/UN GHS Category 1).

 

Relative to the negative control, the test item caused an increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 58.55.

According to OECD 437 the test item is classified as serious eye damaging (EU CLP/UN GHS Category 1).

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