[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:

12 October 2020 - 13 October 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

26 June 2020

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid

Molecular formula:

C44H86N2O4

IUPAC Name:

Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid

Test material form:

solid: particulate/powder

Remarks:

white

Details on test material:

Chemical registery number : 951-139-7
Chemical name : Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions and tested the day of arrival in the laboratory.
- 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: No
- Selection and preparation of corneas: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of Duratec Analysentechnik GmbH (Hockenheim, Germany) 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.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, Duratec GmbH). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 301.99 to 319.40 mg

NEGATIVE CONTROL: physiological saline (Merck KGaA, Darmstadt, Germany)
- Amount applied: 750 µL

POSITIVE CONTROL: 20% (w/v) Imidazole (Merck KGaA, Darmstadt, Germany)
- Amount applied: 750 µL
- Concentration: 20% (w/v) Imidazole solution

Duration of treatment / exposure:

240 ± 10 minutes at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

90 ± 5 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3 replicates

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.
The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to: The opacity value (measured with the device OP-KIT) was calculated according to: Opacity = ((I0/I) - 0.9894) / 0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS): mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA: Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints.
The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
In vitro score range - UN GHS
= 3 - No Category
> 3; = 55 - No prediction can be made
>55 - Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

-0.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

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 (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Table 1 Summary of opacity, permeability and in vitro score

1 Calculated using the negative control corrected 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).

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score 1,2
Negative control	2.5	0.011	2.7
Positive control	118	1.593	142
Test item	-1.2	0.016	-0.9

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of bovine corneal opacity and permeability test (BCOP test), performed according to OECD guideline 437 and GLP principles, the mean in vitro irritancy score (IVIS) was determined to be -0.9. As a consequence, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is not classified according to CLP criteria.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The eye damage of the test item was tested through topical application for approximately 240 minutes. The study is is based on on the most recent OECD guideline and performed according to GLP principles.
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 (20% (w/v) Imidazole) was 142 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.9 after 4 hours of treatment. As a consequence, Reaction products of 1,3-cyclohexanedimethanamine and 12-hydroxystearic acid (BA-1801) is not classified