Phosphoric acid C12-alkyl and C18-alkyl esters, potassium salts

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-08-06 to 2018-08-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, C12-18-alkyl esters, potassium salts

EC Number:

291-905-1

EC Name:

Phosphoric acid, C12-18-alkyl esters, potassium salts

Cas Number:

90506-43-7

Molecular formula:

C12H26KO4P - C36H74KO4P

IUPAC Name:

Phosphoric acid (C12 or C18) alkyl mono- and diester, potassium salts

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: at least 9 month old donor cattle

Test system

Controls:

yes, concurrent positive control

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the test item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.

Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.

For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).

Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).

DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:

IVIS: In vitro Irritancy Score (according to OECD 437):
≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)


Criteria for Determination of a Valid Test
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS of three runs

Value:

28.35

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Results

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	1.00	0.33	0.081	0.070	2.22	1.38	No Category
	0.00		0.061		0.92
	0.00		0.068		1.02
Positive Control	84.67*		0.364*		90.13	98.06	Category 1
	103.67*		0.357*		109.02
	88.67*		0.425*		95.04
Test Item	15.67*		0.732*		26.65	28.35	No prediction can be made
	22.67*		0.659*		32.55
	16.67*		0.613*		25.86

 

Relative to the negative control, the test item did cause a slight increase of the corneal opacity or permeability. The calculated mean IVIS was 28.35 (threshold for serious eye damage: IVIS > 55). According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results a prediction for the damage hazard cannot be made (GHS) for the test item.

Executive summary:

An in vitro study was performed to assess the corneal irritation and damage potential of the test item by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item as well as the positive and the negative controls were each applied to different corneae fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t240). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (physiological saline), neither an increase of opacity nor permeability of the corneae was observed. The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damage (EU CLP/UN GHS Category 1). Relative to the negative control, the test item did cause a slight increase of the corneal opacity and permeability. The calculated mean in vitro irritancy score was 28.35. According to OECD 437 a prediction for corneal irritation and damage potential cannot be made.