Peptones, beef

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 February 2013 to 28 February 2013

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, beef

EC Number:

293-426-3

EC Name:

Peptones, beef

Cas Number:

91079-38-8

Molecular formula:

The substance is an UVCB. The molecular formula as well as the molecular weight are not defined.

IUPAC Name:

Peptones, beef

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:

in vitro irritation score

Run / experiment:

mean

Value:

0.048

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Relative Standard Deviation IVIS: 294.7%

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	0.1356	0.1498	0.1373
Absorption after exposition	0.1787	0.1936	0.1994
Opacity before exposition	1.3665	1.4119	1.3718
Opacity after exposition	1.5090	1.5617	1.5827
Opacity Difference	0.1426	0.1498	0.2109

Mean opacity difference of the negative control is 0.1678.

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

Parameter	Test Item peptone beef			Positive Control
Absorption before exposition	0.1315	0.1334	0.1378	0.1533	0.1296	0.1529
Absorption after exposition	0.2074	0.1443	0.1841	2.0205	1.8584	2.1306
Opacity before exposition	1.3536	1.3596	1.3734	1.4233	1.3477	1.4220
Opacity after exposition	1.6121	1.3941	1.5279	104.8335	72.1772	135.0828
Opacity Difference	0.2585	0.0346	0.1545	103.4102	70.8295	133.6608

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 beef			Positive Control
Measured	0.0037	0.0034	0.0014	0.0027	0.0032	0.0053	0.7925	0.4768	0.6390
Corrected	0.0185	0.0170	0.0070	0.0135	0.0160	0.0265	3.9625	2.3840	3.1950
Mean	0.0142			--

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	0.420	0.380	14.8%
	0.405
	0.316
Test Item	0.080	0.048	294.7%
	-0.106
	0.171
Positive Control 10% NaOH	162.467	149.960	26.0%
	106.209
	181.205

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 0.048 was calculated for peptone beef. According to ICCVAM classification and to OECD Guideline no. 437 (2009), the substance is not eye irritant.

Executive summary:

A mean IVIS of 0.048 was calculated for peptone beef. According to ICCVAM classification and to OECD Guideline no. 437 (2009), the substance is not eye irritant.