Alcohols, C16-18, ethoxylated, phosphates

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation as required by GLP regulation. This was justified in the GLP compliance statement.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom.

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C16-18, ethoxylated, phosphates

EC Number:

500-295-0

EC Name:

Alcohols, C16-18, ethoxylated, phosphates

Cas Number:

106233-09-4

Molecular formula:

UVCB

IUPAC Name:

Alcohols, C16-18 (even numbered) ethoxylated, phosphates, mono- and diesters

Details on test material:

- Name of test material (as cited in study report): Trade name (CAS 106233-09-4)
- Substance type: UVCB
- Physical state: off white solid block
- Analytical purity: >95%
- Lot/batch No.: 533935
- Expiration date of the lot/batch: 2013-09-05
- Storage condition of test material: room temperature in the dark

Test animals / tissue source

Species:

other: cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST METHOD
The bovine corneal opacity and permeability (BCOP) test is an in-vitro test method used to classify substances as “ocular corrosives and severe irritants”. The potential of a test substance to cause ocular corrosivity or severe irritancy is measured by its ability to induce opacity and increased permeability in an isolated bovine cornea. The opacity and permeability assessments of the cornea are combined to derive an in-vitro irritancy score (IVIS), which is used to classify the irritancy level of the test substance.

IDENTIFICATION OF THE SOURCE OF THE EYES, STORAGE AND TRANSPORT CONDITIONS
- Source: Local abattoir (not further specified)
- Donor animals: freshly slaughtered cattle
- Date and time of eye collection: no data
- Time interval prior to initiating testing: the corneas were prepared immediately on arrival.
- Transport medium and temperature conditions: Hanks´ Balanced Salt Solution (HBSS) on ice with penicillin/streptomycin

PREPARATION OF THE EYES (BEFORE EXPOSURE)
- Eyes free of defects (scratches, neovascularisation): yes
- Dissection of the eyes and treatment: Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders.
- Description of the cornea holder: The cornea holders consist of an anterior and a posterior compartment, which interface with the epithelial and endothelial sides of the cornea.
- Test medium and temperature conditions used in the cornea holder: complete Minimum Essential Medium (MEM); prior to use: pre-warmed to 32 ± 1 °C
- Equilibration time: 1 h at 32 ± 1 °C
- Quality check of the equilibrated corneas: free of macroscopic defects

DETERMINATION OF THE INITIAL OPACITY
- Method: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer.

Test system

Vehicle:

physiological saline

Controls:

other: number of eyes for the negative control: 3; number of eyes for the positive control: 3

Amount / concentration applied:

TEST MATERIAL
- Concentration: 20%

VEHICLE
- Substance: physiol. saline
- Concentration: Example: 0.9% NaCl solution in deionised water

POSITIVE SUBSTANCE
- Substance: imidazole
- Concentration: 20% imidazole solution in physiol. saline

Duration of treatment / exposure:

4 h at 32 ± 1 °C

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

3 corneas were used

Details on study design:

TEST CONDITIONS
- Short description of the method used:
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

POST-EXPOSURE TREATMENT
- Removal of the test substance: The test substance was removed from the anterior chamber and the epithelium washed at least three times.
- Medium for washing the corneas: complete MEM containing phenol red
- Medium for final rinsing: complete MEM without phenol red

DETERMINATION OF THE FINAL OPACITY
- Method: Corneal opacity was determined via an opacitometer.
- Time of determination: After refilling fresh MEM without phenol red in the anterior chamber the final opacity was measured.

DETERMINATION OF THE CORNEAL PERMEABILITY:
- Method: Sodium fluorescein solution was added to the anterior chamber of the cornea holder while the posterior chamber was filled with fresh medium. The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured via UV/VIS spectrophotometry at 492 nm recorded as optical density (OD492).
- Amount and concentration of the dye: 1 mL sodium fluorescein solution (5 mg/mL)
- Incubation time: no data
- Treatment for measuring: OD492 of a 360 µL aliquot was determined in a flat-bottomed 96-well plate. If the OD492 was greater than 1.500 a 1:5 dilution of the solution in complete MEM was performed.

HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: IVIS

Basis:

other: mean out of all 3 eyes

Time point:

other: 4 h

Score:

105.8

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 2

Irritation parameter:

other: opacity

Basis:

other: mean out of all 3 eyes

Time point:

other: 4 h

Score:

13

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 3

Irritation parameter:

other: permeability

Basis:

other: mean out of all 3 eyes

Time point:

other: 4 h

Score:

6.185

Reversibility:

other: not applicable

Remarks on result:

other: test substance

Results 4

Irritation parameter:

other: IVIS

Basis:

other: mean out of all 3 eyes

Time point:

other: 4 h

Score:

90.3

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Results 5

Irritation parameter:

other: opacity

Basis:

other: mean out of all 3 eyes

Time point:

other: 4 h

Score:

59

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Results 6

Irritation parameter:

other: permeability

Basis:

other: mean out of all 3 eyes

Time point:

other: 4 h

Score:

2.086

Reversibility:

other: not applicable

Remarks on result:

other: positive control

Other effects:

Corneal epithelium condition and post incubation:
The corneas of the negative control were clear, whereas the corneas of the positive control were cloudy. After treatment with the test item the corneas were slightly cloudy.

Any other information on results incl. tables

Table 1: Opacity values

Parameter	Initial opacity	Final opacity	Opacity change	Mean opacity change of NC	Corrected opacity change	Mean opacity value
Negative control	4	6	2	3.7	-	-
	4	7	3
	3	9	6
Test substance	3	19	16	-	12.3	13.0
	2	23	21		17.3
	3	16	13		9.3
Positive control	2	67	65	-	61.3	59.0
	3	65	62		58.3
	3	64	61		57.3

 

Table 2: Permeability values (optical density (OD) at 492 nm)

Parameter	OD492	Corrected OD492 change	Mean OD492 value
Negative control	0.048		0.060
	0.067
	0.065
Test substance	6.195	6.135	6.185
	6.245	6.185
	6.295	6.235
Positive control	1.449	1.389	2.086
	3.420	3.360
	1.570	1.510

 

Table 3:In-Vitro Irritancy Score (IVIS) values

	Mean IVIS
Negative Control	4.6
Test substance	105.8
Positive control	90.3

 

Applicant's summary and conclusion

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: Cat. 1, H318
DSD: Xi, R41