Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-14 to 2018-09-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
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:
09 October 2017
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany
- Number of animals: not specified
- Characteristics of donor animals: 6-12 months old
- Storage, temperature and transport conditions of ocular tissue: To minimise deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing antibiotics.
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used.
The quality of each cornea was also evaluated at later steps in the assay. Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): 750 µLof a 20% suspension in 0.9% sodium chloride solution (w/v)

VEHICLE
- Concentration (if solution): 750 µL 0.9% sodium chloride solution
- Lot/batch no. (if required): 180618002; B. Braun Melsungen AG, 34212 Melsungen, Germany
Duration of treatment / exposure:
4h
Duration of post- treatment incubation (in vitro):
90 ± 5 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAE
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse, containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL. Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation. Only corneas from eyes free of defects were used. The quality of each cornea was also evaluated at later steps in the assay. Corneae that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: 3 corneae incubated with 0.9% sodium chloride solution
POSITIVE CONTROL USED: 3 corneae incubated with 20% imidazole in 0.9% sodium chloride solution
APPLICATION DOSE AND EXPOSURE TIME: each 750 µL were applied for each dosing for 240 min
TREATMENT METHOD: open chamber
POST-INCUBATION PERIOD: yes, for 90 min
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
After correcting the opacity and mean permeability (OD 490 ) values for background opacity and the negative control permeability OD 490 values, the mean opacity, and permeability OD 490 values for each treatment group were combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 x mean permeability OD 490 value)
DECISION CRITERIA: As recommended by the guideline.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneae (test group)
Value:
0.472
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneae (negative control)
Value:
0.93
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3 corneae (positive control)
Value:
95.182
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, opacity and permeability are below the upper limits for background  opacity  and  permeability  values  for  bovine  corneae  treated  with  the respective negative or solvent/vehicle control.
- Acceptance criteria met for positive control: yes, the positive control gives an IVIS that falls within two standard deviations of the current historical mean.

Any other information on results incl. tables

Table 1: Opacity values

 

Cornea No.

Opacity [Opacity Units]

Corrected Opacity

 

Mean of group

Standard deviation

0.9% NaCl

1

1.315

0.585

0.585

1.163

2

1.196

3

-0.757

20% TA-3

4

0.638

0.053

0.717

0.580

5

1.713

1.128

6

1.554

0.969

20% Imidazol

7

66.972

66.387

57.702

9.732

8

60.120

59.535

9

47.769

47.184

Table 2: Permeability OD Values (490 nm)

 

Corne a no.

Permeability

[OD]  

Mean of

Tripli- cates

Corrected Permeability [OD]

 

Per Cornea

Per Group

 

 

 

 

 

Mean

SD

Mean

SD

0.9% NaCl

1

0.039

0.039

-

 

 

0.039

 

 

0.001

0.023

0.014

0.038

-

0.039

-

2

0.017

0.016

-

0.016

0.001

0.016

-

0.016

-

3

0.013

0.014

-

0.014

0.001

0.013

-

0.015

-

20% TA-3

4

0.004

0.004

-0.019

-0.019

0.001

-0.016

0.002

0.003

-0.020

0.004

-0.019

5

0.008

0.008

-0.015

-0.015

0.001

0.008

-0.015

0.007

-0.016

6

0.008

0.008

-0.015

-0.015

0.001

0.008

-0.015

0.009

-0.014

20% Imidazol

7

2.544

2.557

2.521

2.534

0.057

2.499

0.384

2.508

2.485

2.619

2.596

8

2.946

2.887

2.923

2.864

0.055

2.838

2.815

2.877

2.854

9

2.112

2.121

2.089

2.098

0.013

2.115

2.092

2.136

2.113

Table 3: IVIS score

 

Cornea No.

Opacity

Permeability

 

IVIS

Per Cornea

Per Group

Mean

SD

0.9% NaCl 

1

1.315

0.039

1.900

0.930

1.300

2

1.196

0.016

1.436

3

-0.757

0.014

-0.547

20% TA-3

4

0.053

-0.019

-0.232

0.472

0.615

5

1.128

-0.015

0.903

6

0.969

-0.015

0.744

20% Imidazol

7

66.387

2.534

104.397

95.182

14.345

8

59.535

2.864

102.495

9

47.184

2.098

78.654

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the present test conditions, the test item tested in the in vitro BCOP test method, had an IVIS value of 0.472, which is below the cut-off value of 3. Therefore, the test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not a severe irritant or corrosive to the eye.