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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 Sep 2017 to 28 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
additive
Type:
impurity
Test material form:
liquid: viscous
Details on test material:
- Other: R-9817

Test animals / tissue source

Species:
cattle
Strain:
not specified
Remarks:
bovine cornea
Details on test animals or tissues and environmental conditions:
Test System
The bovine eyes, in a Hanks’ Balance Salt Solution (HBSS) with penicillin-streptomycin, were received from Spear Products on 28 Sep 2017 and transported to MB Research in a refrigerated container.

Pretest Procedures
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar of MEM powder (sufficient to make one liter of solution), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 ml of fetal bovine serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution was kept in a 32 (±1)°C incubator for the duration of testing. HBSS was prepared by stirring together HBSS powder (sufficient to make one liter) and 0.35 g of sodium bicarbonate and brought to a final volume of 1000 ml with distilled water. HBSS was maintained at room temperature. In addition, MEM solution with phenol red was prepared by stirring together 9.3 g of MEM with phenol red (sufficient to make one liter), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 ml of fetal bovine serum (FBS) and brought to a final volume of 1000 ml with distilled water. The MEM solution with phenol red was kept in a 32 (±1)°C incubator for the duration of testing. The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim
of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects. The entire holder was incubated at 32 (±1)°C and allowed to equilibrate for at least one hour, but not longer than two hours. A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied by the opacitometer. Any cornea with a value greater than 7 units was discarded.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 ml of test article, Minimal Essential Media (MEM, negative control), or 100% ethanol (positive control)
Duration of treatment / exposure:
10-minute exposure for each group
Duration of post- treatment incubation (in vitro):
All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. After 10 (±1) minutes), the test article, ethanol or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.
Number of animals or in vitro replicates:
Thre replicates per sample
Details on study design:
Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 ml of test article, ethanol, or MEM solution was applied to the epithelium of each of the three test article corneas, three positive control corneas and three negative control corneas in a manner which ensured that the entire cornea was covered. Test article corneas were dosed via the open-chamber method. The negative and positive controls were dosed via the closed-chamber method. All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C incubator. After 10 (±1) minutes), the test article, ethanol or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill.

All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations. Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphatebuffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea. After 90 (±5) minutes, the fluid from the posterior chamber was removed and the amount of dye that passed through the cornea (permeability) was measured as the optical density at 490 nm by a spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Value:
4.67
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Minimal Essential Media; value= 0.021
Positive controls validity:
valid
Remarks:
100% Ethanol; value 25.34
Remarks on result:
no indication of irritation
Remarks:
Non-Corrosive
Irritation parameter:
in vitro irritation score
Remarks:
IVIS
Value:
4.87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS=32.84
Positive controls validity:
valid
Remarks:
IVIS=-0.36
Remarks on result:
no indication of irritation
Remarks:
Non-Corrosive

Any other information on results incl. tables

The ethanol positive control IVIS was 32.84, which fell within the acceptance range of 16.96 - 37.00 (± 2 standard deviations of the historical mean).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
The test material is not considered an ocular corrosive or severe irritant (Category 1).
Conclusions:
The test material is not consider an ocular corrosive or severe irritant (Category 1).
Executive summary:

To determine the potential for ocular irritation using an alternative to the Draize methodology, a study based on the methodology described in the current OECD Guideline for the Testing of Chemicals No. 437 was conducted. Three bovine corneas per group were dosed with 0.75 ml of Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts, Minimal Essential Media (MEM, negative control), or 100% ethanol (positive control). Following a 10-minute exposure for each group of dosed corneas, opacity measurements and sodium fluorescein permeability were determined. The MEM solution is then removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution. After 90 min the fluid from the posterior chamber is removed and the amount of dye that passed through the cornea is measured as the optical density at 490 nm by a plate reader. For the test substance, the in vitro irritancy score (IVIS) is 4.87, with a corneal opacity score of 4.67. Based on these results, benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts cannot be consider an ocular corrosive or severe irritant.