[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11th November 2016 to 11th November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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)

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:

D-fructose and D-glucose and reaction products of D-fructose and D-glucose and hexamethylenediamine

IUPAC Name:

D-fructose and D-glucose and reaction products of D-fructose and D-glucose and hexamethylenediamine

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS /TISSUE
- Source: Local abattoir
- Age at slaughter: 12-60 months
- Acclimation period : After slaughter eyes were stored in HBSS supplemented with antibiotics, refrigerated and used within 24 hours

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- 0.75 mL of test material
- Concentration: Neat

Duration of treatment / exposure:

10 minutes (at 32 ±1 °C)

Duration of post- treatment incubation (in vitro):

120 minutes (at 32 ±1 °C)

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS

Bovine eyes were macroscopically examined and only corneas free of damage were used. Corneas from each eye were removed leaving 2-3 mm rim of sclera for handling. The cornea were then isolated and immersed in HBSS before mounting on BCOP holders.

QUALITY CHECK OF THE ISOLATED CORNEAS

Pre-treatment opacity readings were performed on a calibrated opacitometer. Only corneas with opacity values close to the median value of all corneas were used for the test.

NUMBER OF REPLICATES

3 replicates

NEGATIVE CONTROL USED

Sodium chloride (0.9 %m/v) was used as a negative control (3 replicates)

POSITIVE CONTROL USED

Ethanol (>99.8 %) was used as a positive control (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME

0.75 mL of test item was applied for 10 minutes

TREATMENT METHOD: [closed chamber / open chamber]

Closed Chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After exposure the test item was removed from the chamber. The corneas and chamber were washed 3 times with complete EMEM containing p henol red, followed by a further wash with EMEM without phenol red.
- POST-EXPOSURE INCUBATION:
120 minutes at 32 ±1 °C before taking a final opacity reading.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea was calculated by subtracting the initial opacity from the final reading. These values were corrected by subtracting the average change in th e opacity observed for the negative control corneas. The mean opacity value for each treatment gro up was the calculated by averaging the corrected opacity value for each cornea for that treatment group. All opacity measurements were made using a calibrated opacitometer.

- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of microplate reader OD4902 . 1 mL of sodium fluorescien solution (4 mg/mL) was added to the previously emptied anterior chamber and left to incubate for 90 minutes at 32 ±1 °C. After incubation the posterior chamber was decanted and the container retained for OD measurement.
- Others (e.g, pertinent visual observations, histopathology): Corneas retained for histopathology but no observations noted. Corneas visually observed post treatment and post incubation. The negative control and the test item corneas were clear for all replicates at all observations and the positive control was cloudy for all replicates at all observations points.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

IVSI = mean opacity + (15 x mean permeability OD492 value)
DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
IVIS is ≤3 the substance is not requiring classification for eye irritancy
IVIS is ≥3 ; ≤55 no prediction of eye irritancy can be made
IVIS >55 Cat 1. UN GHS or EU CLP causes serious eye damage

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

ca. 1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤2.9 and permeability ≤0.103. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 29.6 to 52.0. The positive control acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No category, not requiring classification

Executive summary:

The BCOP method was used to assess the potential for the substance pre-reacted hexamethylenediamine and glucose/fructose in a high solids binder to induce eye irritancy. The guideline method was followed and the in a fully GLP-compliant study.

Pre-reacted hexamethylenediamine and glucose/fructose in a high solids binder produced an IVIS result of 1.2 during the study and as such did not meet the requirements for classification as an eye irritant according to Regulation (EC) N. 1272/2008.