Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
-Batch: PO 4924335980 - ML 2005975931
- Analytical purity: 100% UVCB
- Physical state: Solid, violet
- Homogeneity: homogeneous by visual inspection
- Storage conditions: Room temperature
- The stability of the test substance under storage conditions is guaranteed until Apr 2019.
-pH of the substance: Ca. 5 (undiluted test substance, moistened with de-ionized water)

Test animals / tissue source

Species:
other: in vitro test on isolated bovine cornea
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
The test system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

Test system

Vehicle:
water
Controls:
other: not applicable
Amount / concentration applied:
TEST MATERIAL

Form of application: 750 µL of the 20% (w/v) test-substance preparation
Duration of treatment / exposure:
4 hours
Observation period (in vivo):
not applicable (in vitro test)
Number of animals or in vitro replicates:
not applicable (in vitro test); each treatment group consisted of 3 corneas
Details on study design:
TEST SYSTEM:
- Isolated bovine cornea: Target system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

OBJECTIVE:
- Corneal opacity was measured quantitatively as the amount of light transmission through the cornea.
- Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea.
- Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas.

EXPERIMENTAL PROCEDURE:
- Preparation of the bovine corneas and measurement of initial corneal opacity:
• The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle's MEM (containing phenol red) and once with Eagle's MEM (without phenol red) . Both chambers were then refilled with fresh Eagle's MEM (without phenol red).
• After the equilibration period the medium in both chambers was replaced with fresh medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units were discarded.

- Application of the test substance and washing
• Before application the medium in the anterior chamber was removed using a syringe.
750 μL of the 10% (w/v) test-substance preparation (surfactant) was applied into the anterior chamber.

Control tissues:
• Negative control, NC: 750 μL of de-ionized water
• Positive control, PC: 750 μL of 100% ethanol I 100% dimethylformamide

• After the incubation period, NC and PC were removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
The epithelium of the test substance treated corneas was rinsed with the open chamber method.

- Measurement of final corneal opacity
• Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

- Determination of permeability
• For determination of permeability the medium in the anterior chamber was replaced by 1 ml of sodium fluorescein solution (4 mg/mL for liquid test substances) and incubated for 90 ± 5 min at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS).

- Calculation of the corneal opacity value:
First, the opacity was calculated using the opacitometer specific algorithm:
• opacity value = a * Io/I + b
a and b: device specific;
Io: illuminance (lux) through the empty corneal holder with windows and liquid
I: illuminance (lux) through the holder with the cornea

Then the opacity change per cornea was calculated by subtracting the initial from the final
• opacity (opacity change per cornea = final opacity - initial opacity).

Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control
• corrected opacity change = opacity change - mean opacity change of NC).

Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group
• mean opacity value = mean of all corrected opacity changes per group).

- Calculation of permeability value:
First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea.
• OD490 value = OD490 - mean blank OD490

If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value:
• OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490)

Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control.
• corrected OD490 value = OD490 value - mean OD490 value of NC

Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.
• mean OD490 value = mean of all corrected OD490 values per group

- Calculation of the In Vitro Irritancy Score (IVIS)
The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined:
• IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change
• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

ACCEPTANCE CRITERIA
- In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
• A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
• The negative control responses should result in opacity and permeability values that are less than the established upper limits.
• Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS
- Rules for assessment:
IVIS > 55: risk of serious damage to the eyes
IVIS ≤ 55: no risk of serious damage to the eyes

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
2.2
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
It is referred to table 1.

Any other information on results incl. tables

Table 1: results

  Mean Opacity Value Mean Permeability Value Mean  In VitroIrritancy
Score
test substance 2.2 0.001 2,2
water 6.3 0.005 6.4
20% imidazole in water 76.1 3.061 122

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met