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EC number: 250-001-7 | CAS number: 30007-47-7
Toxicological information
Eye irritation
Currently viewing:
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- 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)
- Qualifier:
- according to guideline
- Guideline:
- EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 5-bromo-5-nitro-1,3-dioxane
- EC Number:
- 250-001-7
- EC Name:
- 5-bromo-5-nitro-1,3-dioxane
- Cas Number:
- 30007-47-7
- Molecular formula:
- C4H6BrNO4
- IUPAC Name:
- 5-bromo-5-nitro-1,3-dioxane
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- batch No.of test material: A14-Q0201
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solid test substance was ground with pestle and mortar before application
Test animals / tissue source
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: The test system is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle.
-Supplier: Schlachthof Mannheim, Schlachthofstr. 21, 68165 Mannheim, Germany
- Number of animals:
- Characteristics of donor animals: age of the animals: minimum 12 months, maximum 60 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
- Time interval prior to initiating testing:
- indication of any existing defects or lesions in ocular tissue samples: only corneas free of defects (opacity, scratches, pigmentation etc.) were used
- Indication of any antibiotics used:
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 200 mg of undiluted solid test substance
CONTROLS
- 750 µL of NC or PC were applied using a pipette
Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method. - Duration of treatment / exposure:
- 4 hours at 32°C in horizontal position
- Number of animals or in vitro replicates:
- 3 corneas per treatment group
- Details on study design:
- SELECTION AND PREPARATION OF CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour
QUALITY CHECK OF THE ISOLATED CORNEAS: Yes
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: Yes, de-ionized water
POSITIVE CONTROL USED: Yes, Imidazole as 20% solution in de-ionized water
APPLICATION DOSE AND EXPOSURE TIME: 200 mg for 4 hours
TREATMENT METHOD: closed chamber
POST-INCUBATION PERIOD: No
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: corneas exposed to the NC and PC were removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red); both chambers were then refilled with fresh Eagle’s MEM (without phenol red).
Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry; (OD490)
- Others (histopathology): After determination of opacity and permeability, corneas were fixed in 4% formaldeyhde for further histotechnical
processing and examination by light microscopy
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA:
IVIS Prediction
< 1.5 No classification for eye irritation1
1.5 – 4.5 Borderline
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another
suitable method is required1,2
45 - 65 Borderline
> 65 Ocular corrosive
Results and discussion
In vitro
Resultsopen allclose all
- Irritation parameter:
- cornea opacity score
- Run / experiment:
- 1st test run
- Value:
- 44.7
- Negative controls validity:
- valid
- Remarks:
- mean 10.6
- Positive controls validity:
- valid
- Remarks:
- mean 84.5
- Irritation parameter:
- fluorescein leakage
- Run / experiment:
- 1st test run
- Value:
- 1.247
- Negative controls validity:
- valid
- Remarks:
- mean 0.003
- Positive controls validity:
- not valid
- Remarks:
- mean 2.259
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 1st test run
- Value:
- 63.4
- Negative controls validity:
- valid
- Remarks:
- mean 10.6
- Positive controls validity:
- valid
- Remarks:
- mean 118.4
- Irritation parameter:
- cornea opacity score
- Run / experiment:
- 2nd test run
- Value:
- 14.6
- Negative controls validity:
- valid
- Remarks:
- mean 10.5
- Positive controls validity:
- valid
- Remarks:
- mean 79.6
- Irritation parameter:
- fluorescein leakage
- Run / experiment:
- 2nd test run
- Value:
- 4.1
- Negative controls validity:
- valid
- Remarks:
- mean 0.002
- Positive controls validity:
- valid
- Remarks:
- mean 3.413
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 2nd test run
- Value:
- 76
- Negative controls validity:
- valid
- Remarks:
- mean 10.5
- Positive controls validity:
- valid
- Remarks:
- mean 130.8
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
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