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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-21 October 2015
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 01, 2014 / signed on October 07, 2015)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Pale yellow, slightly viscous liquid
- Storage condition of test material: Room temperature in the dark in a 50-mL amber glass jar with a screw cap

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: supplied by Joseph Morris Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (last excised at 12:30 hours, 20 October 2015).
- Number of animals: not reported
- Characteristics of donor animals (e.g. age, sex, weight): cattle aged under 30 months.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle.
- Time interval prior to initiating testing: The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 14:36 hours, 20 October 2015).
- indication of any existing defects or lesions in ocular tissue samples: none; Instructions were given to avoid damaging the corneas during excision.
- Indication of any antibiotics used: HBSS containing 1% (v/v) penicillin/streptomycin solution

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 μL of test item was applied on each cornea.
- Concentration (if solution): The test item was tested undiluted.
- The pH of the test substance was approximately 7.0.
Duration of treatment / exposure:
10 minutes (± 30 seconds) at 32 ± 1 °C in horizontal position.
Duration of post- treatment incubation (in vitro):
2 hours ±10 minutes at 32 ± 1 °C in a vertical position.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with HBSS plus 1% (v/v) penicillin/streptomycin, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. The holders were then plugged and incubated, in an upright position, overnight at room temperature (approximately 20 °C). After overnight incubation at room temperature (approximately 20 °C) the HBSS plus 1% (v/v) penicillin/streptomycin was removed from both chambers. Both chambers were filled with cMEM (posterior chamber first), taking care to exclude air bubbles and plugged. For equilibration, the corneas in the holder were incubated in the upright position for 60 minutes ± 5 minutes at 32 ± 1 °C in a water-bath. At the end of the 60 minutes incubation period, the medium was removed and refilled with fresh cMEM and then, the basal opacity was determined.

QUALITY CHECK OF THE ISOLATED CORNEAS : Throughout the assay the corneas were examined for opaque spots or other irregularities.

NUMBER OF REPLICATES : 3 corneas/group

NEGATIVE CONTROL USED : 0.9% sodium chloride solution

POSITIVE CONTROL USED : Ethanol

APPLICATION DOSE AND EXPOSURE TIME :
The anterior compartment received the test item or negative or positive control at a volume of 750 μL on the surface of the corneas. The corneas were incubated in a horizontal position for 10 minutes (± 30 seconds) at 32 ± 1 °C in the water-bath.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times or until the wash medium (EMEM with phenol red) was clear and there was no discolouration. The test substance required four washes.
- POST-EXPOSURE INCUBATION: yes, after the test item was rinsed off from the application site by washing, the corneas were incubated at 32 ± 1 °C for 2 hours ± 10 minutes in an upright position.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer measured the light transmission through the centre of each mounted cornea, displaying a numerical opacity value (arbitrary unit). The opacity of each cornea was measured by reading each holder in the right-hand chamber of the calibrated opacitometer. Corneas with an opacity value greater than 7 units were discarded. The mean basal opacity value of the corneas was then calculated and three corneas with opacity values close to the mean value were chosen for use as negative control corneas.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490). Following the final opacity measurement, the incubation medium was removed from the anterior compartment of the holder and replaced by 1 mL of sodium fluorescein solution (4 mg/mL). Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred to a 1 cm path length cuvette and the optical density at 490 nm (OD490) was determined with a spectrophotometer.
- Others (e.g, pertinent visual observations, histopathology): Throughout the assay the corneas were examined for opaque spots or other irregularities. Moreover an estimate of the pH of the test substance as 10% v/v solution in 0.9% sodium chloride solution, was determined using pH test sticks and recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean/3 corneas incubated 2 hours
Value:
39.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
39.4 ± 8.2
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Following treatment with test substance, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas, treated with the negative control, 0.9% sodium chloride solution, were clear. It was noted during the post opacity measurement that cornea number 1, treated with the negative control, 0.9% sodium chloride solution, had an opaque line in the centre of the cornea. As cornea no. 1 was visibly damaged the opacity and permeability results were not included in the calculation of the mean In Vitro Irritancy Score for test substance and the positive control ethanol.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:
The positive control should elicit an In Vitro Irritancy Score that falls within two standard deviations of the historical mean, 2 x SD = 25.5 – 50.3, for the laboratory.
The negative control mean opacity change value should be ≤3.0 and the permeability mean value ≤0.1 (historical period August 2010 to August 2015).

Any other information on results incl. tables

Table 7.3.2/1: Results of the BCOP assay

 

Sample

Opacity ± SD

Permeability ± SD

In vitro irritancy Score ± SD

In vitro

classification

Test substance

18.667 ± 4.619

1.382 ± 0.349

39.4 ± 8.2

No prediction can be

made

Ethanol

16.667 ± 1.528

0.713 ± 0.240

27.4 ± 2.7

No prediction can be

made

0.9% Sodium

chloride solution

2.000 ± 0.000

0.015 ± 0.001

Not applicable

Not applicable

 

 

ASSAY VALIDITY

The positive control, ethanol, elicited an In Vitro Irritancy Score of 27.4. This value was within the historical range (mean ± 2 x SD = 25.5 – 50.3) for the assays performed to date.

The negative control, 0.9% sodium chloride solution, opacity mean change value was 2.0 which was below the maximum acceptance value of 3.0. The permeability mean of the negative control was 0.015, which was below the maximum acceptance value of 0.1.

Applicant's summary and conclusion

Interpretation of results:
other: No prediction can be made
Conclusions:
With an IVIS of 39.4, no prediction can be made as the result is outside the decision criteria (i.e.≤3 or >55).
Executive summary:

An in vitro eye irritation study was performed according to the OECD Guideline 437 and in compliance with GLP to assess the corneal damage potential of test substance by means of the BCOP assay using fresh bovine cornea.  750 µL of test item was applied to cornea for 10 minutes followed by an incubation period of 2 hours at 32 ± 1 °C and corneal opacity was measured. Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score.   Following treatment with test substance, the corneas were noted as clear. The corneas treated with the positive control, ethanol, were opaque and the corneas, treated with the negative control, 0.9% sodium chloride solution, were clear. It was noted during the post opacity measurement that cornea number 1, treated with the negative control, 0.9% sodium chloride solution, had an opaque line in the centre of the cornea. As cornea no. 1 was visibly damaged the opacity and permeability results were not included in the calculation of the mean In Vitro Irritancy Score for test substance and the positive control ethanol. The negative and positive controls met the acceptance criteria for this assay.   The calculated mean in vitro irritancy score for test substance and positive control were 39.4 ± 8.2 and 27.4 ± 2.7, respectively.  

With an IVIS of 39.4, no prediction can be made as the result is outside the decision criteria (i.e.≤3 or >55)