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Toxicological information

Eye irritation

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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:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
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:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Physical state/ Appearance:liquid/yellowish, clear
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 0016044582
- Content: 91.4 /100 g (100 g/100 g minus water content)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: No analysis of test substance preparation was performed because the test substance was applied minimally moistened with deionized water or PBS.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
pH value: Ca. 6 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
Physical state / color: Liquid, high viscous to solid, waxy / colorless to light yellowish to white
Handling of the test substance: The solid test substance was grinded with pestle and mortar before application.
Storage conditions: Room temperature

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Schlachthof (slaughterhouse) Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Characteristics of donor animals: age minimum 12 months, maximum 60 months
- indication of any existing defects or lesions in ocular tissue samples: none

Test system

Vehicle:
water
Remarks:
deionized
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750µL
- Concentration: 20% (w/v)
Duration of treatment / exposure:
4 hours
Duration of post- treatment incubation (in vitro):
The negative control and the positive control were removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION, PREPARATION AND QUALITY CHECK OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32°C for at least 1 hour.

TREATMENT METHOD: [closed chamber / open chamber]
The test-substance preparation (non-surfactant) was applied directly to the epithelial surface of the cornea by using a pipette (open chamber method).

POST-INCUBATION PERIOD: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was spectrophotometrically measured. Three aliquots per cornea were transferred to a 96 well microtiter plate (Sunrise Absorbance Reader) and the optical density (OD490) was determined. An aliquot was diluted 1:5 in Eagle’s MEM (without phenol red) and analogously measured (positive control and test substance).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria were modified and amended with a borderline evaluation based on additional internal validation data: The borderline evaluation was statistically determined by using historic BASF data and hence considers the variance of the test method.

IVIS Prediction
< 1.5 No classification for eye irritation
1.5 – 4.5 Borderline
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required
45 - 65 Borderline
> 65 Ocular corrosive or severe irritant

The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437. There is no relevance for the outcome of this study since the IVIS is greater than 65.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: mean opacity value
Run / experiment:
Mean value from three replicates.
Value:
0
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: mean permeability value
Value:
0.001
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
other: In Vitro Irritancy Score
Value:
0
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Opacity score of the test substance, the NC and the PC

 

Test

substanceidentification

 

Cornea-No.

 

Initialopacity

 

Finalopacity

 

OpacityChange

Corrected

Opacity Change*

 

 

Mean

 

 

SD

 

7

6.3

3.7

-2.5

0.0

 

 

16/0277-1

8

5.2

3.9

-1.4

0.0

0.0

0.0

 

9

5.4

5.4

0.0

0.0

 

 

 

1

4.5

18.6

14.2

NA

 

 

NC

2

4.4

13.3

9.0

NA

16.2

8.4

 

3

3.1

28.5

25.4

NA

 

 

 

4

3.9

105.7

101.7

85.6

 

 

PC

5

3.9

97.1

93.2

77.0

83.8

6.0

 

6

6.8

111.7

104.8

88.7

 

 

 

  Permeability score of the test substance, the NC and the PC

 

Test

substance identification

 

Cornea- No.

 

Mean OD490

 

Dilution Factor

Mean

Corrected OD490*

 

 

Mean

 

 

SD

 

7

0.001

1

0.000

 

 

16/0277-1

8

0.002

1

0.001

0.001

0.001

 

9

0.004

1

0.002

 

 

 

1

0.001

1

NA

 

 

NC

2

0.002

1

NA

0.002

0.001

 

3

0.003

1

NA

 

 

 

4

0.457

5

2.283

 

 

PC

5

0.386

5

1.926

1.787

0.578

 

6

0.231

5

1.153

 

 

 

* Negative values are set to zero for further calculation

In Vitro Irritancy score (IVIS) of the test substance, the NC and the PC

Test substance

identification

Cornea No

Opacity per Cornea

Permeability per cornea

             IVIS

per cornea     per group 

              mean        SD

16/0277-1

7

0.0

0.000

0.0

 

 

8

0.0

0.001

0.0

0.0

0.0

9

0.0

0.002

0.0

 

 

NC

1

14.2

0.001

14.2

 

 

2

9.0

0.002

9.0

16.2

8.4

3

25.4

0.003

25.5

 

 

PC

 

4

85.6

2.283

119.8

 

 

5

77.0

1.926

105.9

110.6

8.0

6

88.7

1.153

106.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results observed for the BCOP test, the test substance does not cause irreversible eye effects