Poly(substitutedmethyl)amino acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2018

Materials and methods

Test guideline

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)

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Remarks:

aqueous solution

Details on test material:

Substituted amino acid (2) solution, FC-C 13587
Appearance: clear colorless solution

Specific details on test material used for the study:

No correction was made for the purity/composition of the test item.

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature, and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
750 μL directly on top of the corneas.

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ± 10 minutes
90 ± 5 minutes with Na-fluorescein cMEM solution

Number of animals or in vitro replicates:

3 replicates for the negative, positive, and treatment group.

Details on study design:

TREATMENT METHOD
The medium from the anterior compartment was removed and 750 μl of either the negative control, positive control or test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea.
Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on 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.

- POST-EXPOSURE INCUBATION: 90 ± 5 minutes in sodium-fluorescein for permeability determinations.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values
of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Other: possible pH effects of the test substance on the corneas were recorded.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score 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 upper limits of the laboratory historical range.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
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).
Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.

DECISION CRITERIA: According to the guideline (see table 1)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean of 3 replicates

Value:

110

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Mean IVIS: 0.1

Positive controls validity:

valid

Remarks:

Mean IVIS: 44

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- The corneas treated with the test item showed opacity values of 88, 90 and 80.
- Permeability values of the test item were 1.725, 1.225 and 1.867.
- Individual IVIS scores of the corneas treated with the test item were 114, 109 and 108 (n=3).

OTHER EFFECTS:
- A pH effect of the test item was observed on the rinsing medium.
- The corneas treated with the negative control item were clear after the 10 minutes of treatment.
- The corneas treated with the positive control item were turbid after the 10 minutes of treatment.

ACCEPTANCE OF RESULTS (see table 2) :
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from -0.7 to 1.3).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 30 to 52).

Any other information on results incl. tables

 Table 2: In Vitro Irritancy Score

Treatment	Final Opacity(A)	Final OD4902	In vitro Irritancy Score(B)
Negative control	-0.3	-0.002	-0.4
	1.2	0.009	1.3
	-0.8	0.006	-0.7
Positive control	22	1.869	50
	14	1.034	30
	18	2.259	52
The test item	88	1.725	114
	90	1.225	109
	80	1.867	108

(A)In vitro irritancy score (IVIS) = opacity value + (15 x OD₄₉₀value).

(B)Positive control and test item are corrected for the negative control.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed according to OECD guideline 437 and GLP principles. Substituted amino acid (2) solution FC-C 13587 induced serious eye damage through both endpoints resulting in an average IVIS > 55. Therefore Substituted amino acid (2) solution FC-C 13587 is classified as Category 1 according to GHS criteria.