Peptones, casein

Eye irritation

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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.