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

Endpoint summary

Description of key information

Two in vitro irritation/corrosion studies are available to determine the potential irritancy of the test substance. The EPISKIN Standard Model was used for the skin irritation study according to OECD Guideline 439 and GLP principles. The Bovine Corneal Opacity Test was used for the eye irritation study according to OECD Guideline 437 and GLP principles. 
The in vivo skin irritation/corrosion study was performed according to OECD Guideline 404 and GLP principles.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December - 24 December, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

(2002)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Version / remarks:

(1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, April 2011, including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: at least 6 weeks old
- Weight at study initiation: at least 1.0 kg
- Housing: Animals were individually housed in labeled cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) and shelters (Ebeco, Germany, dimensions 40 x 32 x 23 cm).
- Diet: Free access to pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad, Italy). Hay and wooden sticks were abailable during the study period.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

other: dehydrated corn oil

Controls:

other: Adjacent areas of the untreated skin of each animal served as controls.

Amount / concentration applied:

TEST MATERIAL - Amount(s) applied: 0.5 grams

VEHICLE - The test substance was mixed with 0.3 mL dehydrated corn oil

Duration of treatment / exposure:

Single application.

Observation period:

14 days.

Number of animals:

3 males.

Details on study design:

STUDY DESIGN
The study was performed in a stepwise manner and started with the treatment of one animal (sentinel) with a stepwise exposure regime. The two other animals were exposed to the substance for four hours only and in a similar manner 6 days later, after considering the degree of skin irritation observed in the first animal.

TEST SUBSTANCE PREPARATION
The powdery test substance was mixed with dehydrated corn oil, specific gravity 0.92 (Fagron, Nieuwerkerk a/d IJssel, The Netherlands), immediately before application, to ensure close contact with the animal's skin.

TEST SITE
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations.

The study was initiated by treatment of one rabbit.
The test substance was applied to the skin of one flank, using a Metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. The dressing was removed 3 minutes after application.
Since no signs of severe skin reactions (i.e. necrosis or corrosion) were observed and it was considered that exposure could be continued humanely, two samples of 0.5 grams of the test substance mixed with 0.3 mL of the vehicle were then applied to separate skin-sites on the intact, clipped skin of the same animal, using an identical procedure and one sample per dressing. One of the dressings was removed after a 1-hour exposure.
After similar considerations (i.e. no severe skin reactions, necrosis or corrosion), the other dressing was removed after a 4-hour exposure.

Since no signs of severe skin reactions (i.e. necrosis or corrosion) were observed after 4 hours exposure and the irritation was considered reversible in the first animal, two further animals were treated at a later stage. These animals received single samples of 0.5 grams of the test substance mixed with 0.3 mL of the vehicle on the intact, clipped skin of one flank applied in a similar manner as in the first animal. Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using vehicle.

REMOVAL OF TEST SUBSTANCE
After each removal of a dressing, the treated skin was cleaned of residual test substance using dehydrated corn oil.

OBSERVATIONS
- Mortality/Viability: Twice daily.
- Toxicity: At least once daily.
- Body Weight: Day of treatment (prior to application) and after the final observation.
- Necropsy: No necropsy was performed.
- Irritation:
In the initially treated animal, the skin reactions of all visible treated sites were assessed immediately after removal of a dressing and approximately 1, 24, 48, 72 hours after the removal of the last dressing and test substance. After the 4 hours exposure in two further animals, the skin reactions were assessed approximately 1, 24, 48, 72 hours after the removal of the dressing and test substance.
The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of untreated skin of each animal serve as controls.

SCORING SYSTEM:
The irritation was assessed according to the numerical scoring system according to OECD 404.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1, #2 and #3

Time point:

other: 24, 48 and 72 hours.

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1, #2 and #3

Time point:

other: 24, 48 and 72 hours.

Score:

0

Max. score:

4

Irritant / corrosive response data:

No skin irritation was caused by 0.5 g of MMDS after exposure periods of 3 minutes, 1 hour and 4 hours.
There was no evidence of a corrosive effect on the skin.

Other effects:

No staining of the treated skin by the test substance was observed and no test substance remnants were seen.
No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an skin irritation study with rabbits, performed according to OECD/EC test guidelines, no irritation was observed.

Executive summary:

Primary skin irritation/corrosion study with MMDS in the rabbit (semi-occlusive application).

Initially, one rabbit was exposed to three samples of 0.5 grams of MMDS mixed with dehydrated corn oil applied to separate skin-sites on intact, clipped skin using a semi-occlusive dressing. The exposure periods were 3 minutes, 1 hour and 4 hours, respectively. Based on the absence of severe skin reactions, two further animals exposed for 4 hours to MMDS at a later stage.

No skin irritation was caused by MMDS after exposure periods of 3 minutes, 1 hour and 4 hours.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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.

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

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)

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.

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.

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).

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro Skin Irritation/Corrosion:

In vitro skin irritation test with MMDS using a human skin model.

This report describes the ability of MMDS to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SM^TM)). The possible skin irritation potential of MMDS was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 100910 of MMDS was a white powder. Skin tissue was moistened with 5 µl of Milli-Q water and MMDS was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with MMDS compared to the negative control tissues was 105%. Since the mean relative tissue viability for MMDS was above 50% after 15 minutes treatment MMDS is considered to be non-irritant.

 

The positive control had a mean cell viability of 5% after 15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 3%, indicating that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that MMDS is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

In vivo Skin Irritation/Corrosion:

Primary skin irritation/corrosion study with MMDS in the rabbit (semi-occlusive application).

Initially, one rabbit was exposed to three samples of 0.5 grams of MMDS mixed with dehydrated corn oil applied to separate skin-sites on intact, clipped skin using a semi-occlusive dressing. The exposure periods were 3 minutes, 1 hour and 4 hours. Based on the absence of severe skin reactions, two further animals were exposed for 4 hours to MMDS at a later stage.

No skin irritation was caused by MMDS after exposure periods of 3 minutes, 1 hour and 4 hours.

Eye irritation/Corrosion:

Screening for the eye irritancy potential of MMDS using the Bovine Corneal Opacity and Permeability test (BCOP test). This report describes the ocular irritation properties of MMDS on an isolated bovine cornea.

The possible ocular irritancy of MMDS was tested through topical application for 240 ± 10 minutes. The study procedures described in this report were based on the most recent OECD and EC guideline. Batch 100910 of MMDS was a white powder. MMDS was applied directly on top of the corneas (967 to 1222 mg). 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.

Justification for selection of skin irritation / corrosion endpoint:
One key study available.

Justification for selection of eye irritation endpoint:
One key study available.

Effects on eye irritation: corrosive

Justification for classification or non-classification

Skin Irritation/Corrosion:

Based on the results of the in vitro and in vivo studies, the test substance, MMDS, is not considered to be a skin irritant in accordance with Regulation EC No. 1272/2008 and Directive 67/548/EEC

Eye irritation/Corrosion:

Based on the results of the key study, the test substance,MMDS, should be considered to be a Category 1 corrosion in accordance with Regulation EC No. 1272/2008 and should have the signal word Danger and the Hazard statement H318: Cause serious eye damage associated with it. According to Directive 67/548/EEC, the test substance should be classified as an Irritant (Xi) and have the risk phrase R41: Risk of serious damage to eye associated with it.