Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 17, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Required by REACH

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
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)
Version / remarks:
October 9, 2017
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:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
9.12.2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.
Version / remarks:
April 1997
Deviations:
no
Qualifier:
according to
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).
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity: 99.7%
Physical description: white powder
Storage conditions: room temperature
Expiration date: March 20, 2020

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Collection of Bovine Eyes
Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Buffered Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during 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.
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 medium was changed before basal opacity measurement (t0).
At the end of the incubation period, the basal opacity was determined (t0).
Only corneae with a value of the basal opacity < 7 were used. Sets of three corneae were used for treatment with the test item and for the negative and positive controls, respectively.

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
20%
Duration of treatment / exposure:
240 minutes
Duration of post- treatment incubation (in vitro):
Following to the opacity readings, the permeability endpoint 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).
Number of animals or in vitro replicates:
3
Details on study design:
This in vitro study was performed to assess the corneal irritation and damage potential of DOPO-HQ 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 DOPO-HQ 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 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.
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 DOPO-HQ did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.37. According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (EU CLP/UN GHS No Category).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
>= 2.02
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
>= 1.08
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Negative control
Value:
>= 1.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
>= 93.35
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
>= 95.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Positive control
Value:
>= 95.76
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
DOPO-HQ
Value:
>= 1.77
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
DOPO-HQ
Value:
>= 0.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
DOPO-HQ
Value:
>= 1.68
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Negative control
Value:
>= 1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Negative control
Value:
>= 0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control
Value:
>= 84.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control
Value:
>= 86.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control
Value:
>= 85.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
DOPO-HQ
Value:
>= 1.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
DOPO-HQ
Value:
>= 0.67
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, according to the current study and under the experimental conditions reported, DOPO-HQ is not categorized (EU CLP/UN GHS No Category).
Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of DOPO-HQ 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 DOPO-HQ 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 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.

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 DOPO-HQ did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.37. According to OECD 437 (see table in chapter 3.8.3) the test item is not categorized (EU CLP/UN GHS No Category).