Disodium L-tyrosinate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 January 2017 - 17 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

2013

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:

2010

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Disodium L-3-(4-hydroxyphenyl)alaninate hydrate

Molecular formula:

C9H9NNa2O3 • xH2O

IUPAC Name:

Disodium L-3-(4-hydroxyphenyl)alaninate hydrate

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals: 18 - 24 months
- Storage, temperature and transport conditions of ocular tissue: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. Transport medium: HBSS (Hanks' Balanced Salt Solution)
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20%

VEHICLE CONTROL
- Amount applied: 750 µL
- Concentration: 0.9%

POSITIVE CONTROL
- Amount applied: 750 µL
- Concentration: 20%

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

90 min (determination of permeablity)

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1 °C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES
3

NEGATIVE/SOLVENT CONTROL USED
0.9% sodium chloride solution

POSITIVE CONTROL USED
20% (w/v) Imidazole in 0.9 % sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME
750 µL, 20%, 4 h

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD:
No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The corneal surface was washed three times with wash medium. Incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement.

- POST-EXPOSURE INCUBATION:
90 min (determination of permeablity)

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany).
- Corneal permeability: microtiter plate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany) (at OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
Opacity:
The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values.
From the individual corrected opacity values, a mean corrected opacity value was calculated for each group
Permeability:
For each cornea either treated with the positive control or the test item, an individual corrected OD490 value was calculated by subtracting the average negative control permeability value from each individual permeability reading. From the individual corrected permeability values, a mean corrected permeability value was calculated for each group.
In Vitro Irritancy Score (IVIS) Calculation:
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)

The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)

The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values

DECISION CRITERIA:he following IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were applied. IVIS score below or equal to 3: UN GHS No Category
IVIS score above 3 and less or equal to 55: No prediction can be made
IVIS score above 55: UN GHS Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1, Tissue 1

Value:

62.34

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

1, Tissue 2

Value:

84.605

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

1, Tissue 3

Value:

87.325

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

1, Mean

Value:

78.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Study Acceptance Criteria:

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.4 - 3.4). After treatment with the positive control (20% Imidazole) the calculated IVIS was 102.3 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 79.9 - 134.6). Therefore, the study fulfilled the acceptance criteria.

The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Results:

	Opacity	Permeability	IVIS
			Per Cornea	Per Group (Mean)	SD
Negative Control	0.7	0.001	0.715	1.1	0.4
	1.3	0.000	1.300
	1.4	0.001	1.415
Positive Control	68.5	1.487	90.805	102.3	10.5
	72.7	2.142	104.830
	80.6	2.044	111.260
Test Substance	24.0	2.556	62.340	78.1	13.7
	20.6	4.267	84.605
	19.0	4.555	87.325

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In an ex vivo Bovine Corneal Opacity and Permeability Assay (BCOP) according to OECD Guideline 437, the test item showed serious eye damaging properties.

Executive summary:

In an ex vivo Bovine Corneal Opacity and Permeability Assay (BCOP) according to OECD Guideline 437, the eye damaging potential ofthe test item was examined. To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test substance as a 20% (w/v) solution in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the dissolved test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the corneas were rinsed and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control the calculated IVIS was 1.1 (study acceptance criteria range: -1.4 - 3.4). Treatment with the positive control revealed an IVIS of 102.3 (study acceptance criteria range: 79.9 - 134.6). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test substance was 78.1 and, thus, > 55, i.e. according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1).