Phosphoric acid, mono- and di-C12-18-(even numbered)-alkyl esters, sodium 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 July 25, 2016 to July 26, 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

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts

Molecular formula:

C12H25O4P1Na2 (representative mono ester - mono- C12 PSE, Na+); C24H50O4P1Na1 (representative di ester - di- C12 PSE, Na+);

IUPAC Name:

Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts

Constituent 2

Reference substance name:

Alcohols, coco

EC Number:

270-351-4

EC Name:

Alcohols, coco

Cas Number:

68425-37-6

Molecular formula:

Not applicable

Constituent 3

Reference substance name:

Sodium dihydrogenorthophosphate

EC Number:

231-449-2

EC Name:

Sodium dihydrogenorthophosphate

Cas Number:

7558-80-7

Molecular formula:

H3O4P.Na

IUPAC Name:

Sodium phosphate

Constituent 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts
- Batch number: 2011-104-06-01
- ZS internal substance number: 0038
- ZS name: PHOSPHORSÄUREPARTIALESTER, NA-SALZ C12-18
- Appearance: White solid

Specific details on test material used for the study:

Batch no.: SEALS: 2011-104-06-01
Purity/Composition: 96.75%
Appearance: white 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

other: positive control: 20% (w/v) 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:

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
The medium from the anterior compartment was removed and 750 µl of the negative control and 20% (w/v) imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C. After the incubation the solutions and the test substance were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
4. Opacity measurement
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:
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).
5. Application of sodium fluorescein and Permeability determinations
Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 +/- 5 minutes at 32 +/- 1°C. 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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

corneal opacity and permeability

Value:

85

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 negative control responses (from -0.6 to 0.9) 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 121 and within two standard deviations of the current historical positive control mean. The corneas treated with the positive control were also turbid after the 240 minutes of treatment. Based on those observations, it was concluded that the test conditions were adequate and that the test system functioned properly.
- The corneas treated with the test substance showed opacity values ranging from 14 to 20 and permeability values ranging from 2.413 to 7.707. 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 55 to 129 (mean IVIS of 85) after 240 minutes of treatment. The test substance induced serious eye damage through both endpoints.

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:

other: Category 1 (causes serious eye damage) based on CLP criteria

Conclusions:

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 of 85 after 240 minutes of treatment. Since the test substance induced an IVIS > 55, it was concluded that the test substance induced serious eye damage in the Bovine Corneal Opacity and Permeability test under the reported experimental conditions.

Executive summary:

A study was conducted to determine the in vitro eye irritation potential of the test substance, mono- and di- C12 -18 PSE, Na+, according to OECD Guideline 437 (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 14 to 20 and permeability values ranging from 2.413 to 7.707. 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 55 to 129 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 of 85 after 240 minutes of treatment (Eurlings, 2016). Since the test substance induced an IVIS > 55, it was concluded that the test substance induced serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions.