Phosphoric acid, mono- and di-isotridecyl esters, potassium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Bovine Corneal Opacity and Permeability (BCOP) test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 13, 2016 to June 14, 2016

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

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

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Type:

Constituent

Type:

Constituent

Type:

Constituent

Test material form:

other: paste like

Details on test material:

- Name of test substance (as cited in study report): Phosphoric acid, mono- and di-isotridecyl esters, potassium salts
- Batch: RE 7-8
- Appearance: paste-like

Specific details on test material used for the study:

Batch no.:: Re 7-8
Purity: 95.85%
Appearance: white waxy solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

other: 20% (w/w) imidazole in physiological saline

Amount / concentration applied:

Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas

Duration of treatment / exposure:

ca. 240 minutes

Duration of post- treatment incubation (in vitro):

The opacity of the corneas was determined directly after treatment and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

Number of animals or in vitro replicates:

3

Details on study design:

The Bovine Corneal Opacity and Permeability (BCOP) test is an organic model that provides short-term maintenance of normal physiological and biological function of the bovine cornea in an isolated system. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitymeter and an ultraviolet/visible spectrophotometer, respectively.
1. Preparation of corneas
The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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.
2. Opacity reading
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. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded.
3. Treatment of corneas and opacity measurements

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Opacity and Permeability (after 240 minutes of treatment)

Value:

177

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- The individual in vitro irritancy scores for the negative controls ranged from -3.3 to 2.5, which were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- The individual positive control in vitro irritancy scores ranged from 99 to 116. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
- The corneas treated with the test substance showed opacity values ranging from 22 to 72 and permeability values ranging from 8.021 to 9.653. The corneas were turbid after the 240 minutes of treatment. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 155 to 217 after 240 minutes of treatment.

Any other information on results incl. tables

Interpretation of the results:

- In vitro irritancy score

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 substance induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

< or = 3: no category,

>3 and < or = to 55: no prediction can be made,

> 55: category 1.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the study conditions, the test substance induced an IVIS > 55 indicating serious eye damage in the bovine corneal opacity and permeability.

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance according to OECD guideline 473 (bovine corneal opacity and permeability (BCOP) test), in compliance with GLP. Bovine corneas were exposed to the test substance for a period of 240 minutes. Opacity and permeability were measured and an in vitro irritancy score was then established. Physiological saline and imidazole were used as negative and positive controls, respectively. The corneas treated with the test substance showed opacity values ranging from 22 to 72 and permeability values ranging from 8.021 to 9.653. The corneas were turbid after the 240 minutes of treatment. No pH effect of the test substance was observed on the rinsing medium. The in vitro irritancy scores ranged from 155 to 217 after 240 minutes of treatment. The negative and positive control values were within the expected range; hence the experiment was considered valid. Under the study conditions, the test substance induced serious eye damage through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score (IVIS) of 177 after 240 minutes of treatment (Eurlings, 2016). Based on the IVIS > 55, it was concluded that the test substance induced serious eye damage in the Bovine Corneal Opacity and Permeability test.