Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
adopted 09. Oct. 2017
Deviations:
not specified
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 14. Feb. 2017
Bovine Corneal Opacity and Permeability Test Method for Identifying (i) Chemicals Inducing Serious Eye Damage and (ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage
Deviations:
not specified
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160
Version / remarks:
25. Oct. 2011 / “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PER-MEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COL-LECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”
Deviations:
not specified
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: 1,3,2-dioxathiane 2,2-dioxide
- IUPAC name: 1,3,2-Dioxathiane 2,2-dioxide
- Molecular formula: C3H6O4S
- Molecular weight: 138.14 g/mol
- Substance type: Organic
- Physical state: Solid

Test animals / tissue source

Species:
other: bovine
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
Test item: Replicate 1: 1.0655g, Replicate 2: 1.0282 g, Replicate 3:1.0712 g
Positive control: 750 µL
Negative control: 750 µL

- Concentration (if solution):
Test item: The test item was given on the epithelium in such a manner that the cornea was covered with test item.
Positive control: Imidazole solution: 20% C3H4N2 (CAS-No. 288-32-4), dissolved in HBSS, batch no.: 20180829.
Negative control: Hank’s Balanced Salt Solution (HBSS) 10-fold concentrated, diluted in demin. water (1:10), batch no.: 20180829
Duration of treatment / exposure:
4 hours at 32 ± 1 °C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1 °C
Number of animals or in vitro replicates:
3
Details on study design:
-SELECTION AND PREPARATION OF CORNEAS:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container within 1 hour.
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C.
The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

-QUALITY CHECK OF THE ISOLATED CORNEAS:
The corneas were examined and only corneas which were free from damages were used

-NUMBER OF REPLICATES: 3

-NEGATIVE CONTROL USED: Yes (HBSS)

-POSITIVE CONTROL USED: Yes (Imidazole, 20 % solution in HBSS)

-APPLICATION DOSE AND EXPOSURE TIME:
Test item: Replicate 1: 1.0655g, Replicate 2: 1.0282 g, Replicate 3:1.0712 g
Positive control: 750 µL
Negative control: 750 µL.
EXPOSURE TIME: 4 hours at 32 ± 1 °C

-TREATMENT METHOD: Open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
Rinsing the anterior chamber with cMEM with phenol red and final rinsing with cMEM without phenol red.
- POST-EXPOSURE INCUBATION:
90 minutes at 32 ± 1 °C.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
106.38
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The calculated IVIS for each replicate and the corresponding means are presented in the following table:

Test Group

IVIS

Mean IVIS

Relative Standard Deviation IVIS

Negative Control
HBSS

0.84

1.54

88.21%

0.67

3.10

Test Item
1,3,2-Dioxathiane, 2,2-dioxide

154.87

106.38

50.33%

115.36

48.92

Positive Control

20% imidazole

solution

105.22

103.28

7.59%

94.66

109.97

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS (The calculated mean IVIS was 106.38.) > 55 induces serious eye damage, that should be classified as UN GHS Category I.
Executive summary:

Thisin vitrostudy was performed to assess corneal damage potential of1,3,2-Dioxathiane, 2,2-dioxideby quantitative measurements of changes in opacity and permeability in a bovine cornea.

 

Two experiments were performed. The first experiment was not valid, because the mean IVIS value of the negative control was too high. Therefore the experiment was declared invalid and repeated. This experiment is not reported, but the raw data are kept in the GLP-archive of the test facility. The second performed experiment was valid and reported.

 

The test item1,3,2-Dioxathiane, 2,2-dioxidewas brought onto the cornea of a bovine eye which previously had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hoursat32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

 

The test item was tested neat. Under the conditions of this test, the test item1,3,2-Dioxathiane, 2,2-dioxideinduced serious eye damage on the cornea of the bovine eye. The calculated mean IVIS was 106.38.

According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.

 

The negative control (HBSS) and the positive control (20% imidazole solution) have met the validity criteria.

 

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.