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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
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)
Deviations:
no
Principles of method if other than guideline:
Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 25 April 2012, 23 ,25, Date of inspection: 13-16 July 2015, Date of Signature: 14 September 2015

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(methylamino)anthraquinone
EC Number:
201-417-2
EC Name:
1-(methylamino)anthraquinone
Cas Number:
82-38-2
Molecular formula:
C15H11NO2
IUPAC Name:
1-(methylamino)anthraquinone
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
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.

Amount(s) applied (volume or weight with unit): 0.75 mL
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
3 corneae per group (test item, negative control, positive control)
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.

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 (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 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:
1
Value:
ca. 0.09
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.09 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is not categorized.

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

Proposed

in vitro

Irritancy Score

 

 

Mean

 

Mean

 

 

 

Negative Control

-1

-0.33

0.071

0.71

0.06

0.73

Not categorized

0

0.078

1.17

0

0.064

0.96

Positive Control

106.33*

0.156*

108.67

119.23

Category 1

102.33*

0.165*

104.81

142.33*

0.125*

144.21

Test item

0.33*

0.004*

0.039

0.09

Not categorized

0.33*

0.010*

0.48

-0.67*

0.003*

-0.62

*corrected values

Applicant's summary and conclusion

Interpretation of results:
other: Not categorized
Conclusions:
According to the current study and under the experimental conditions reported, the test item is not categorized (CLP/EPA/GHS (Cat 1) (GHS)).
Executive summary:

This in vitro study was performed to assess the corneal 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) suspensionin saline of the test item Solvaperm-Rot PFS, 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 thecorneae could be observed (mean IVIS = 0.73).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity anddistinctive permeability of the corneae (mean IVIS = 119.23) corresponding to a classification asserious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item did not cause an increase of the corneal opacity or permeability. The calculated mean IVIS was 0.09 (threshold for serious eye damage: IVIS ≥ 55). According to OECD 437 the test item is not categorized.

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