Registration Dossier

Diss Factsheets

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
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. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Peptones, casein
EC Number:
293-428-4
EC Name:
Peptones, casein
Cas Number:
91079-40-2
Molecular formula:
Not applicable
IUPAC Name:
Peptones, casein
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
other: Bos primigenius Taurus (fresh bovine corneas)
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2.4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hank's balanced salt solution (supplemented with 0.01 % streptomycin and 0.01 % penicillin). Then, the corneas were dissected and incubated in medium at 32 ± 1°C in an incubation chamber for 1 hour.

Test system

Vehicle:
other: 0.9 % sodium chloride solution
Controls:
yes
Amount / concentration applied:
750 µl of the test item solution (concentration of 10 % in a 0.9 % sodium chloride solution) were used.
Duration of treatment / exposure:
Exposition time on the corneas was 10 minutes at 32°C.
Observation period (in vivo):
2 hours post-incubation after the removal of the test item.
Number of animals or in vitro replicates:
no data
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): thorough rinsing with cMEM (complete Minimum Essential Medium) with phenol red and final rinsing with cMEM without phenol red
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- Calculation of Opacity Value: Opacity was calculated from the measured absorption at 570 nm following:
0 = 1/10^(-A) with 0 = Opacity / A= Absorption at 570 nm
The difference between opacity before and after exposition is used for further evaluation.
- Correction of Measured Absorption at 490 nm: As cuvettes with a pathlength of 0.2 cm are used in the measurement of the Fluorescein­Na solution in the spectral photometer, the pathlength must be corrected to 1 cm. Coefficient: 1/0.2 = 5: all absorptions were multiplied with this coefficient.
- Calculation of IVIS (In Vitro Irritancy Score): The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD490 value)
The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference - mean opacity difference of the negative controls) + [15 x (corrected OD490 - mean corrected OD490 of the negative controls)]
Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

TOOL USED TO ASSESS SCORE: fluorescein-Na solution

Results and discussion

In vitro

Results
Irritation parameter:
cornea opacity score
Value:
ca. 1.64
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Opacity and permeability values:

The absorption (570 nm) and opacity values which were measured before and after exposition are given in the following table:

Table 1-a. Absorption and Opacity Values Negative Control

Parameter

Negative Control

Absorption before exposition

0.1561

0.1562

0.1497

Absorption after exposition

0.2539

0.2604

0.2306

Opacity before exposition

1.4325

1.4328

1.4116

Opacity after exposition

1.7943

1.8214

1.7006

Opacity Difference

0.3618

0.3885

0.2890

Mean opacity difference of the negative control is 0.3465

Table 1-b. Absorption and Opacity Values Test Item and Negative Control

Parameter

Test Item peptone casein

Positive Control

Absorption before exposition

 

0.1235

 

0.1466

 

0.2209

 

0.1564

 

0.1368

 

0.1905

Absorption after exposition

 

0.1634

 

0.1943

 

0.2778

 

1.9956

 

1.9713

 

1.7888

Opacity before exposition

 

1.3289

 

1.4015

 

1.6630

 

1.4335

 

1.3703

 

1.5506

Opacity

after exposition

 

1.4568

 

1.5642

 

1.8958

 

98.9920

 

93.6052

 

61.4894

Opacity Difference

 

0.1279

 

0.1627

 

0.2328

 

97.5585

 

92.2350

 

59.9388

For the permeability measurement, three replicates for each treatment group were measured. The optical density values at 490 nm are given in the following table:

Table 1-c. Optical density at 490 nm

Repl.

Negative Control

Test Item peptone casein

Positive Control

Measured

-0.0051

-0.0096

-0.0095

-0.0135

-0.0132

-0.0147

0.7970

0.6769

0.7647

Corrected

-0.0255

-0.0480

-0.0475

-0.0675

-0.0660

-0.0735

3.9850

3.3845

3.8235

Mean

-0.0403

--

IVIS was then calculated using the values in tables 1-a, 1-b and 1-c and the following equation: IVIS = (opacity difference - mean opacity difference of the negative controls) + [15 x (corrected OD490 - mean corrected OD490 of the negative controls)].

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Table 2: IVIS

 

Test Group

 

IVIS

 

Mean IVIS

Relative Standard

Deviation IVIS

 

Negative Control 0.9% NaCI

-0.021

 

-0.259

 

-81.5%

-0.331

-0.423

 

Test Item

-0.627

 

-0.603

 

-4.9%

-0.569

-0.612

 

Positive Control 10% NaOH

157.591

 

139.467

 

14.5%

143.260

117.549

According to OECD Guideline no.437(2009), a substance that induces an IVIS >= 55.1 is defined as a corrosive or severe irritant. Substances with IVIS < 55.1 may be considered as non corrosive resp. not severely irritant.

ln the negative control, no signs of eye irritation were observed. The positive control showed very severe eye irritation. The test item peptone casein showed no severe eye irritation (values lay below negative control).

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A mean IVIS of - 0.603 was calculated for peptone casein. According to ICCVAM classification and to OECD Guideline no. 437 (2009), the substance is not eye irritant.

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