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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 10, 2014 to August 12, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: The OTWG of ICCVAM and the NICEATM, Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
9-Octadecenoic acid (Z)-, sulfonated, potassium salts
EC Number:
271-843-1
EC Name:
9-Octadecenoic acid (Z)-, sulfonated, potassium salts
Cas Number:
68609-93-8
Molecular formula:
A generic formula cannot be provided for this UVCB substance. The alkyl chain length of the sulfonated fatty acids range from C12-C22, however the major alkyl chain is C18.
IUPAC Name:
9-Octadecenoic acid (Z)-, sulfonated, potassium salts
Test material form:
other: powder/flakes with lumps
Details on test material:
- Name of test material (as cited in study report): 9-Octadecenoic acid (Z)-, sulfonated, potassium salts
- Physical state: Beige-yellow powder/flakes with lumps (i.e., determined at WIL Research Europe B.V.)
- Analytical purity: 100%
- Water content: 0.5%
- pH: 6 at concentration of 1g/L
- Lot/batch No.: 7495382
- Expiration date of the lot/batch: December 31, 2015
- Storage condition of test material: At room temperature in the dark
- Stability in vehicle: Water: Yes
- Solubility in vehicle: Water: Completely miscible

Test animals / tissue source

Species:
other: Bovine eyes

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750 µL 10% (w/w) solution of test substance
Duration of treatment / exposure:
10±1 minutes
Observation period (in vivo):
After the completion of the incubation period (i.e., 120±10 minutes) opacity determination was performed
Details on study design:
Preparation of corneas

- The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by submersing them in physiological saline and holding them in the light. Those exhibiting defects were discarded.

- The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen Corporation)). The isolated corneas were mounted in a corneal holder (i.e., one cornea per holder) of MC2 (Clermont, France) 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 h at 32±1°C.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
10 minutes
Value:
22
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corneas treated with the test substance showed opacity values ranging from 4 to 7 and permeability values ranging from 0.603 to 1.752. The corneas were translucent after the 10 minutes of treatment with the test substance. A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 13 to 33 after 10 minutes of treatment with test substance.

Any other information on results incl. tables

Negative and positive controls:

The individual in vitro irritancy scores for the negative controls ranged from -1.0 to 0.1. The individual positive control in vitro irritancy scores ranged from 164 to 186. The corneas treated with the positive control were turbid after the 10 minutes of treatment.

In Vitro irritancy score

Eye

Negative control correctedFinal Opacity

Negative control correctedFinal OD490

In vitro Irritancy Score1

Negative control

1

-1

-0.002

-1.0

2

0

-0.005

-0.1

3

0

0.008

0.1

Positive control

4

115

4.596

183.9

5

114

4.806

186.1

6

82

5.430

163.5

Test substance

7

7

1.752

33.3

8

5

1.073

21.1

9

4

0.603

13.0

 

1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

Summary of opacity, permeability and in vitroscores (10% solution, 10 minutes treatment)

Treatment

Mean

Opacity

Mean

Permeability

Mean In vitro Irritation Score1, 2

Negative control

0

0.000

0

Positive control

104

4.944

178

Test substnace

5

1.143

22

1 Calculated using the negative control mean opacity and mean permeability values.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).

Applicant's summary and conclusion

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the study conditions, mean in vitro irritancy score for the test substance was established at 22, and hence no prediction can be made regarding its eye irritation potential.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 437 (Bovine Corneal Opacity and Permeability test), in compliance with GLP. The test substance (750 µL of a 10% w/w solution) was applied topically onto the epithelium of the bovine cornea for 10±1 minutes at 32±1°C. After exposure, the corneas were washed with MEM (Minimum Essential Medium), phenol red and cMEM (Eagle's Minimum Essential Medium), then incubated for 120±10 minutes at 32±1°C. Opacity and permeability were determined. The negative control responses for opacity and permeability were within laboratory historical control ranges. The mean in vitro irritancy score of the positive control (10% w/v benzalkonium chloride) was 178, which was also within the historical control range. Under the study conditions, mean in vitro irritancy score for the test substance was established at 22, and hence no prediction can be made regarding its eye irritation potential (Verspeek, 2014).