[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:

14 September 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)

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:

Octadecanoic acid, reaction products with tetraethylenepentamine

Cas Number:

71799-54-7

Molecular formula:

Not applicable - UVCB substance

IUPAC Name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Test material form:

solid

Details on test material:

- Appearance: Amber solid
- Storage: Room temperature (20 ± 5 °C); protected from light.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Species: Bos primigenius Taurus
- Source: Slaughterhouse
- Characteristics of donor animals: The cattle were between 12 and 60 months old.
- Storage, temperature and transport conditions of ocular tissue: The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1 % Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 μg/mL) in a suitable cooled container.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 673.0, 590.2 and 658.6 mg

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
- On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% foetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
- After the arrival of the corneas, they were examined and only corneas that were free from damage were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
- After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: HBSS: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10)

POSITIVE CONTROL USED: Imidazole solution: 20 %

APPLICATION DOSE AND EXPOSURE TIME
- 673.0, 590.2 or 658.6 mg, for 4 hours at 32 ± 1 °C.

TREATMENT METHOD: Open chamber
- For each treatment group (negative control solution, test material and positive control solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 μL negative and positive control solution and a defined amount of test material (673.0, 590.2 or 658.6 mg) were applied to each replicate.
- In order to apply the test material, the nut of the cornea holder was unscrewed to remove the glass disc. The test material could then be applied directly to the cornea using a weighing funnel. The test material was applied to the epithelium in such a manner that as much as possible of the cornea was covered with test material.

REMOVAL OF TEST SUBSTANCE
- Thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red was performed, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Baseline opacity was measured before treatment and the final opacity was measured after removal of the test or control material.
- Corneal permeability: After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas. For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used. The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- Calculation of Opacity:
The change of opacity value of each treated cornea with test material, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test material and positive control to obtain a corrected opacity.

Opacity = [(I0 / I) - b] / a

a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and Medium, here: Io= 1053.69
I = the measured illuminance (unit: LUX)

- Calculation of Permeability:
The corrected OD492 value of each cornea treated with test material and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea. The mean OD492 value for each treatment group (test material, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

- Calculation of IVIS (In Vitro Irritancy Score):
The IVIS of each replicate of the negative control was calculated from the following equation:

IVIS = opacity difference + (15 x corrected OD492 value)

The IVIS of each replicate of the positive control and of the test material was calculated from the following equation:

IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

DECISION CRITERIA:
- The IVIS cut-off values for identifying the test materials as inducing eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are:
In vitro score range: ≤ 3 = No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = Eye Damage Category 1

VALIDITY CRITERIA:
According to the guideline, the test is considered as valid if:
- The positive control causes an IVIS that falls within two standard deviations of the current historical mean.
- The negative control has to show an IVIS ≤ 3.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

45.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

VALIDITY
- The IVIS of the negative control (HBSS) was 0.90, this was ≤ 3 and therefore valid.
- The IVIS of the positive control (20 % imidazole solution) was 92.36, this was within the range of 70.37 – 159.65 and therefore valid.
- Values for negative and positive controls were within the range of historical data of the test facility, the test system was therefore acceptable.

ASSESSMENT
- In the negative control, no signs of eye irritation were observed.
- The positive control induced serious eye damage, which would be classified as GHS category I.
- The test material showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 45.89.
- According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category with the BCOP. Further testing may be required.
- The experiment is considered sufficient, as the resulting classification of the test material was unequivocal, because all three replicates of the test material lead to the same assessment for the test material.

Any other information on results incl. tables

Table 1: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score*
Negative control	0.49	0.0271	0.90
Positive control	72.16	1.3470	92.36
Test material	8.92	2.4641	45.89

*Calculated using the negative control mean opacity and mean permeability values for the positive control and test material.

In vitro irritancy score (IVIS) = opacity difference + (15 x corrected OD492 value)

Applicant's summary and conclusion

Interpretation of results:

other: No prediction of eye irritation can be made.

Conclusions:

Under the conditions of the study no prediction of eye irritation can be made.

Executive summary:

The eye irritation potential of the test material was determined in accordance with the standardised guidelines OECD 437 and EU method B.47, under GLP conditions. The test material was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) Test Method.

One valid experiment was performed. Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old. The test material was brought onto the cornea of a bovine eye, which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test material was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test material, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as the negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 0.90. 20 % imidazole solution was used as the positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 92.36.

Under the conditions of this study, the test material showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) was 45.89.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage with the BCOP. Further testing may be required. Under the conditions of the study no prediction of eye irritation can be made.