1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.06.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to EC and OECD guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals; Guideline no. 437: "Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants" (adopted September 07, 2009).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

EC Number:

700-464-0

Cas Number:

99591-74-9

Molecular formula:

C2H4O6S2

IUPAC Name:

1,5,2,4-dioxadithiane 2,2,4,4-tetraoxide

Details on test material:

- Name of test material (as cited in study report): MMDS
- Substance type: organic
- Physical state: White powder
- Analytical purity: Not indicated by the sponsor; treated as 100% pure
- Purity test date: no data
- Lot/batch No.: 100910
- Expiration date of the lot/batch: 20 April 2012
- Stability under test conditions: hydrolyses rapidly, handle in nitrogen environment in glovebox
- Storage condition of test material: In refrigerator (2-8°C) in the dark under nitrogen

Test system

Details on study design:

Test System: Bovine eyes were used as soon as possible after slaughter on the same day.

Test substance preparation
Due to the physical properties of the test substance, MMDS should be applied as soon as possible after weighing in a nitrogen environment to minimize exposure to air. Since MMDS was not soluble in water, an excessive test substance was applied directly on top of the corneas (967 to 1222 mg).

Reference substances
Negative control: A negative control, physiological saline (Merck, Darmstadt, Germany) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Preparation of corneas: All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.
The isolated corneas were stored at 32 +/- 1 degreeC 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 (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 degreeC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 degreeC.

Cornea selection and 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 (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 microl of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. MMDS was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (967 to 1222 mg). 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 degreeC. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) 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 degreeC.

Permeability determinations
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 microl 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 (Multiskan spectrum, Thermo labsystems, Breda, The Netherlands). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Results and discussion

In vivo

Irritant / corrosive response data:

The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 0.1. The individual positive control in vitro irritancy scores ranged from 99 to 109. The corneas treated with the positive control were turbid after the 240 minutes of treatment.

The corneas treated with MMDS showed opacity values ranging from 168 to 174 and permeability values ranging from -0.007 to 0.000. The corneas were white and turbid after the 240 minutes of treatment with MMDS. 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 168 to 174 after 240 minutes of treatment with MMDS.

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	0	0.000	0.0
Positive control	72	2.076	103.1
MMDS	172	-0.003	172.0

 

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:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The negative control responses of the opacity and permeability values 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 103 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

MMDS induced severe ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 172 after 240 minutes of treatment.

Since MMDS induced an IVIS ≥ 55.1, it is concluded that MMDS is corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report