Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-12-2017 to 06-02-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
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
Qualifier:
equivalent or similar 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

Test material

Constituent 1
Reference substance name:
Reaction products of 2-hydroxyethyl methacrylate and diphosphorous pentoxide and water
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)
IUPAC Name:
Reaction products of 2-hydroxyethyl methacrylate and diphosphorous pentoxide and water
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) container protected from light
- Other: Yellow liquid

Test animals / tissue source

Species:
other: bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Recognised supplier)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): Young cattle.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Not reported.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: None.
- Housing: The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (recognised supplier) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 32 ± 1

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 microlitres
- Concentration (if solution): undiluted

Duration of treatment / exposure:
10 ±1 minutes at 32 ± 1ºC.
Duration of post- treatment incubation (in vitro):
After treatment the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed.
Number of animals or in vitro replicates:
Three (3) per test item, or negative or positive controls, respectively.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. Following mounting: the anterior and posterior chambers of each BCOP holder were filled with complete Earle’s Minimum Essential Medium (cMEM) and the holders were incubated at 32 ± 1 ºC for a minimum of 1 hour. After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were examined for defects macroscopically. Only corneas with opacity ≥ 7.0 are discarded, in accordance with the guideline.

NUMBER OF REPLICATES: 3 (Triplicate)

NEGATIVE CONTROL USED: physiological saline

SOLVENT CONTROL USED (if applicable): Not applicable.

POSITIVE CONTROL USED: Ethanol ; > 99.9% purity

APPLICATION DOSE AND EXPOSURE TIME: 0.75 mL and 10 minutes

TREATMENT METHOD: Closed chamber

POST-INCUBATION PERIOD: Yes. Following exposure the holders were incubated, for 120 ± 10 minutes at 32 ± 1°C. A post-treatment opacity reading was taken and each cornea was visually observed.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the
anterior chamber and the cornea was rinsed with fresh MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
- POST-EXPOSURE INCUBATION: Following the final opacity measurement the posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 4 mg Na-fluorescein/ml cMEM solution Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Measured through light transmission through the cornea quantitatively using an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): Any other pertinent visual observations would be recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:
In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value). A test item that induces an In Vitro Irritancy Score ≥ 55.1 is defined as an ocular corrosive or severe irritant. A test item with an ≤ IVIS 3.0 is predicted to be not irritating to the eye (UN GHS and/or CLP Regulation (EC) 1272/2008 as amended).

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean (n=3)
Value:
150
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Yes. The corneas treated with the test item were turbid post treatment. A pH effect was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were turbid post treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The test laboratory has validated the specified assay.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes. The test was conducted twice (details of first test not reported). The first test was rejected due to an inappropriate positive control response not in accordance with the acceptability criteria. The second test is reported.
- Range of historical values if different from the ones specified in the test guideline:
1. Ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the current historical control data (HCD) mean. ACTUAL: PC IVIS range of 43 to 56. Mean = 51.0.
2. Physiological saline solution was used for negative control purposes. 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 for bovine corneas treated with the respective negative control of the current historical control data (HCD). When testing liquids the negative control limit for opacity should be ≤ 3.0 and for permeability -0.034 to 0.100. ACTUAL: NC IVIS = 1.2, opacity ≤ 2.1 and permeability -0.018.

Any other information on results incl. tables

Table 1.0 - Summary of opacity, permeability and in vitro scores

Treatment

Mean

Opacity #1

Mean

Permeability #1

Mean In vitro Irritation Score #1, #2

Negative control

1.4

-0.018

1.2

Positive control

(Ethanol)

28

1.536

51

Test item

130

1.361

150

#1: Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

#2: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this in vitro study, the test item is considered to be severely damaging/irritating to the eye. The test item caused a mean IVIS score 150 after 10 minute exposure and would be classified under Regulation (EC) 1272/2008: eye damage/irritation - category 1.
Executive summary:

The study was performed to OECD TG 437 and EU Method B.47 to assess the irritancy potential of the test item to the eye following exposure to bovine corneas in accordance with GLP. A total of 3 corneas per treatment group were used. A volume of 750 microlitres of the test item was placed the cornea. The negative control group received physiological saline and the positive control group received neat ethanol. For each group the corneas were incubated for 10 ± 1 minutes at 32 ± 1°C. After the incubation the solutions were removed and the corneas were washed with MEM with phenol red (Earle’s Minimum Essential Medium) and thereafter with cMEM. Possible pH effects of the test item the corneas were recorded. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 51.0 and was within two deviations of the historical positive control data mean and within the historical positive control range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The corneas treated with the test item showed opacity values ranging from 108 to 142 and permeability values ranging from 0.935 to 1.177 and in vitro irritancy scores ranged from 137 to 157. The test item induced severe ocular damage/irritation through both endpoints, resulting in a mean in vitro irritancy score of 150 after 10 minutes of treatment. Based on these results the test item is considered to be severely damaging/irritating to the eye in the Bovine Corneal Opacity and Permeability test.