5-bromo-5-nitro-1,3-dioxane

Endpoint summary

Description of key information

In a GLP study conducted in accordance with OECD guideline 431 (in vitro skin corrosion), exposure of reconstructed human skin to the test substance redcued tissue viability to 16.2% and 3.8% of controls at 3 minutes and 1 hr respectively, when measured by MTT reduction. Three supporting studies are available, none of whcich are suitable for assessment given the lack of experimental details reported and the low concentrations of test substance (0.1 -2.5 % w/v) used.

Eye damage potential was assessed in a GLP, guideline study using Bovine Cornea Opacity and Permeability (OECD 437). Exposure of bovine cornea to the test substance for 4 hours resulted in In Vitro Irritation Scores of 63.4 and 76 in duplicate studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 28 July 2015

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/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 system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM 200 kit; 24 EPI-200 tissues (reconstructed epidermis): surface 0.6 cm²
cultured in Millicells, diameter: 1 cm
- Tissue batch number(s): EPI-200, EPI-212,EPI-218
- Date of initiation of testing: 04 August 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: once with PBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT: 1.0 mg / mL MTT diluent
- Incubation time: 3 hrs
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.850 ± 0.053
- Barrier function: 6.18 hrs
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freezing
- N. of replicates : 2

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%.]

Acceptance criteria for tissue variability:
For every treatment 2 tissues are treated in parallel. In the range of 20% and 100% viability, variability between the
tissues is considered to be acceptable if the coefficient of variation (CV) of %-viability is ≤ 30%.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL de-ionized water was applied first; thereafter, a bulk volume of ca. 25 μL of the solid
ground test material was applied with a sharp spoon and homogeneously distributed with the water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of de-ionized water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8 N potassium hydroxide

Duration of treatment / exposure:

3 min or 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

3 min incubation

Value:

16.2

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1 hour incubation

Value:

3.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

The CV of %-viability of the 3-minute exposure of the test-substance treated tissues, which was 37.4%, is out of the acceptance range, which was ≤ 30%. Since all other quality criteria of the study were met and both viability values are well below the cut off for skin corrosion, this deviation is not considered to adversely affect the result of this study.

Interpretation of results:

Category 1A (corrosive) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

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

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)

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

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:

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

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

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the results obtained in the in vitro reconstructed human skin assay for skin corrosion, the test substance fulfils the classificaiotn criteria for Skin Corrosion and Severe eye Damage Category 1. The results obtained in the Bovine Corneal Opacity and Permeability study confirm the hazard classification for Severe Eye Damage category 1.