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Diss Factsheets

Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
31 March 2017 to 28 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
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)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-bis(hydroxymethyl)urea
EC Number:
205-444-0
EC Name:
1,3-bis(hydroxymethyl)urea
Cas Number:
140-95-4
Molecular formula:
C3H8N2O3
IUPAC Name:
1,3-bis(hydroxymethyl)urea
Test material form:
solid

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Number of animals: not specified, nine corneas
- Characteristics of donor animals: age 15 - 42 months (cornea diameter 25-27 mm)
- Storage, temperature and transport conditions of ocular tissue: The eyes were kept and transported in transport medium cooled on ice.
- Time interval prior to initiating testing: not specified
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS).

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount applied : 150 mg
- Concentration: 20% (w/v), 150 mg/750 µL

VEHICLE
- Amount applied : 750 µL
- Concentration: 0.9 % sodium chloride solution
Duration of treatment / exposure:
240 min
Duration of post- treatment incubation (in vitro):
90 min with fluorescein
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

SOLVENT/NEGATIVE CONTROL USED: 0.9 % sodium chloride solution

POSITIVE CONTROL USED: 20 % (w/v) Imidazole in 0.9 % sodium chloride solution

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: three times with wash medium, once with incubation medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others: Each cornea was observed visually and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: <= 3: No category, >3 - <= 55: no prediction, > 55: Category 1

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Tissue 1
Value:
5.885
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Tissue 2
Value:
9.115
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
Tissue 3
Value:
3.55
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: A study with reference substances (Feb. 2017) confirmed the accuracy and reliability of the test method used in the laboratory.

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

Applicant's summary and conclusion

Interpretation of results:
other: Not Category 1 (irreversible effects on the eye) based on GHS
Conclusions:
The test substance should not be considered for classification as Category 1 (severely eye damaging) due to the observed IVIS of 6.2 according to UN GHS classification.
Executive summary:

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20 % (w/v) suspension in a 0.9 % sodium chloride solution according to OECD 437. As negative control 0.9 % sodium chloride solution and as positive control 20 % (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS). After treatment with the negative control the calculated IVIS was 0.9 (study acceptance criteria range: -1.4 - 3.3). Treatment with the positive control revealed an IVIS of 105.5 (study acceptance criteria range: 79.8 - 133.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 6.2 and, thus higher than 3 and lower than 55. Therefore according to UN GHS classification the test substance should not be considered for classificaion as UN GHS Category 1 (severely eye damaging).