Soybean meal, protein extn. residue

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2018

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 guideline

Test guideline 1

Qualifier:

according to guideline

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:

2017

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)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Soybean meal, protein extn. residue

EC Number:

293-693-6

EC Name:

Soybean meal, protein extn. residue

Cas Number:

91081-84-4

Molecular formula:

UVCB substance

IUPAC Name:

Not determined

Test material form:

solid: particulate/powder

Details on test material:

- Physical state/Appearance: Beige-yellow/tan powder
- Storage Conditions: Room temperature in the dark

Specific details on test material used for the study:

- The test material was prepared as a 20 % w/v solution in sodium chloride 0.9 % w/v.
- The test material was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals: adult cattle (typically 12 to 60 months old)
- The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Eyes were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

other: sodium chloride 0.9 % w/v

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 20 % w/v

VEHICLE
- Amount(s) applied: 0.75 mL
- Concentration: 0.9 % w/v
- Lot No.: 3012487

Duration of treatment / exposure:

240 minutes

Duration of post- treatment incubation (in vitro):

90 minutes with sodium fluorescein

Number of animals or in vitro replicates:

Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control item.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
- The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
- The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 °C for 75 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
- The medium from both chambers of each holder was replaced with fresh complete EMEM.
- A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES
- Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test material and three corneas to the positive control material.

NEGATIVE CONTROL USED: Sodium chloride 0.9 % w/v

POSITIVE CONTROL USED: Imidazole, used as a 20 % w/v solution in sodium chloride 0.9 % w/v.

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL for 240 minutes

TREATMENT METHOD:
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test material preparation or control materials were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the material over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 °C for 240 minutes.

REMOVAL OF TEST SUBSTANCE
- At the end of the exposure period the test material and control materials were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A post-treatment opacity reading was taken after removal of the test material and each cornea was visually observed. The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader. A 1:5 dilution was performed on two of the positive controls. The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
- Others: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin. No histopathology was required for this study.

SCORING SYSTEM:
- In Vitro Irritancy Score (IVIS):
The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test material induced a response through only one of the two endpoints.
- The condition of the cornea was visually assessed post treatment.

DECISION CRITERIA:
The test material was classified according to the following prediction model:
IVIS ≤ 3: No Category
IVIS >3; ≤ 55: No prediction can be made
IVIS > 55: Category 1

ACCEPTABILITY OF THE TEST
- The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2017 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 71.2 to 132.9.
- The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2017 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.3 and for permeability ≤0.044.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL EPITHELIUM CONDITION
- The corneas treated with the test material were clear post treatment. The corneas treated with the negative control material were clear post treatment. The corneas treated with the positive control material were cloudy post treatment.

ACCEPTABILITY OF THE TEST
- The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Any other information on results incl. tables

Table 1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		IVIS
		Pre-treatment	Post-treatment	Post-treatment – Pre-treatment	Corrected Value		Corrected Value
Negative Control	1	6	8	2		0.015
	2	4	6	2		0.000
	3	3	4	1		0.000
				1.7*		0.005♦		1.7
Positive Control	4	7	83	76	74.3	1.540	1.535
	5	4	76	72	70.3	1.805	1.800
	6	4	66	62	60.3	1.201	1.196
					68.3•		1.510•	91.0
Test Material	7	4	6	2	0.3	0.040	0.035
	9	5	6	1	0.0	0.007	0.002
	10	4	6	2	0.3	0.003	0.000
					0.2•		0.012•	0.4

OD = Optical density

* = Mean of the post-treatment − pre-treatment values

♦ = Mean permeability

• = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU criteria

Conclusions:

Under the conditions of this study, the test material induced an in vitro irritancy score of 0.4 and therefore does not require classification as an eye irritant.

Executive summary:

The eye irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 437 and EU Method B.47, under GLP conditions.

The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test material is assessed by quantitative measurements of changes in corneal opacity and permeability.

The test material was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. Negative and positive control materials were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The positive control In Vitro Irritancy Score was within the range of 71.2 to 132.9. The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤2.3 and permeability ≤0.044. The negative control acceptance criteria were therefore satisfied.

Under the conditions of this study, the test material induced an in vitro irritancy score of 0.4 and therefore does not require classification as an eye irritant.