Registration Dossier

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 26 February 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
other: OECD Guideline for Testing of Chemicals 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 (July, 2013).
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 25 April 2012, 23 ,25,Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

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: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany

Test system

Vehicle:
physiological saline
Amount / concentration applied:
The test item was tested as a 20% suspension (w/v) in saline. The test item was crushed in mortar with a pistil to improve its consistency. The test item could not be suspended or solved homogeneously, therefore, each 0.75 mL of the prepared stock was distributed to each cornea.
Duration of treatment / exposure:
240 minutes
Details on study design:
Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the stest item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.


SCORING SYSTEM:
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 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 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
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 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.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
the test item did not cause an increase of the corneal opacity or permeability.The calculated mean IVIS was 1.15 (Decission Criteria ≤ 3: no category, > 3; ≤ 55: no prediction can be made, > 55: Category 1).
Value:
1.15
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
Relative to the negative control, the test item 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol did not cause an increase of the corneal opacity or permeability
Other effects:
The calculated mean IVIS was 1.15 (Decission Criteria ≤ 3: no category, > 3; ≤ 55: no prediction can be made, > 55: Category 1). Based on these findings and according to OECD 437 the test item does not require classification for eye irritation or serious eye damage (UN GHS No Category).

Any other information on results incl. tables

Results after 240 Minutes Incubation Time


Test Group

Opacity value = Difference (t240-t0) of Opacity

Permeability at 490 nm (OD490)

IVIS

Mean IVIS

Classification
UN GHS

 

 

Mean

 

Mean

 

 

 

Negative Control

0

0

0.080

0.079

1.20

1.19

No Category

0

0.075

1.13

0

0.082

1.23

Positive Control

140.00*

0.016*

140.24

128.59

Category 1

130.00*

0.038*

130.57

115.00*

-0.003*

114.96

1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol

3.00*

-0.019*

2.72

1.15

No Category

1.00*

-0.002*

0.97

0.00*

-0.016*

-0.24

*corrected values

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol does not require classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

This in vitro study was performed to assess the corneal damage potential of1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphtholby means of the BCOP assay using fresh bovine corneae.

After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v)suspensionin saline of the test item 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphthol, the positive, and the negative controls were applied to corneae and incubated for 240 minutes at 32± 1 °C. 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 (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS =1.19).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS =128.59) corresponding to a classification asserious eye damaging(CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item 1-[(4-Methoxy-2-nitrophenyl)azo]-2-naphtholdid not cause an increaseof the corneal opacityor permeability.The calculated mean IVIS was1.15(Decission Criteria ≤ 3: no category, > 3; ≤ 55: no prediction can be made, > 55: Category 1). Based on these findings and according to OECD 437 the test item does not require classification for eye irritation or serious eye damage (UN GHS No Category).