Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

October 09, 2017

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

EC Number:

949-859-1

Molecular formula:

not applicable

IUPAC Name:

Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

Test material form:

liquid

Details on test material:

- Appearance: Clear yellow liquid
- Storage condition: At room temperature

Test animals / tissue source

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 (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- 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.

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

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three corneas were selected at random for each treatment group.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, 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.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
- Amount(s) applied (volume or weight with unit): 750 μL of physiological saline per cornea

POSITIVE CONTROL USED
- Amount(s) applied (volume or weight with unit): 750 μL of ethanol per cornea

APPLICATION DOSE AND EXPOSURE TIME
10 ± 1 minutes at 32 ± 1°C

POST-INCUBATION PERIOD: yes, 120 ± 10 minutes at 32 ± 1°C

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corp oration) and thereafter with cMEM)
- Time after start of exposure: 10 minutes

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 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.

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:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritant (UN GHS: catgeory 1);
For a test substance that induces an IVIS >3 and ≤ 55, no prediction on irritant potency can be made.

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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

single run

Value:

13

Negative controls validity:

valid

Remarks:

valid In Vitro Score 0.4

Positive controls validity:

valid

Remarks:

valid In Vitro Score 65

Remarks on result:

other: no prediction on the classification can be made.

Other effects / acceptance of results:

OTHER EFFECTS:
The corneas after the 10 minutes of treatment with the substance showed turbid spots. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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.
- Acceptance criteria met for positive control: The mean in vitro irritancy score of the positive control (Ethanol) was 65 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

Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score 1,2
Negative control	0.3	0.010	0.4
Positive control	19	3.037	65
Test substance	8.7	0.287	13

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).

Applicant's summary and conclusion

Interpretation of results:

other: no prediction on the classification can be made.

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD 437 guideline and GLP principles, it is concluded that no prediction on the classification can be made.

Executive summary:

A Bovine Corneal Opacity and Permeability test (BCOP) was performed with the substance according to OECD guideline 437 and GLP principles. The substance was tested as it is. The mean in vitro irritancy

score of the positive control (ethanol) was 65, and the mean in vitro irritancy score of the negative control (physiological saline) was 0.4. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The substance induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 13 after 10 minutes of treatment.

Since the substance induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.