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EC number: 209-247-0 | CAS number: 563-41-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- This study was conducted on 19 September 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
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:
- 26 July 2013
- 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:
- EC No. 440/2008
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Semicarbazide hydrochloride
- EC Number:
- 209-247-0
- EC Name:
- Semicarbazide hydrochloride
- Cas Number:
- 563-41-7
- Molecular formula:
- CH5N3O.ClH
- IUPAC Name:
- semicarbazide hydrochloride
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- Identification: Semicarbazide hydrochloride
CAS Number: 563-41-7
Batch: 55507861
Purity: 99.36%
Physical state/Appearance: white solid
Expiry Date: 28 April 2019
Storage Conditions: room temperature in the dark
Test animals / tissue source
- Species:
- cattle
- Details on test animals or tissues and environmental conditions:
- Source of Bovine Eyes
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival
Test system
- Vehicle:
- physiological saline
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- For the purpose of this study the test item was prepared as a 20% w/v solution in sodium chloride 0.9% w/v.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
0.75 mLof the test item was added to each cornea. - Duration of treatment / exposure:
- 240 Mins
- Duration of post- treatment incubation (in vitro):
- 90 Minutes
- Number of animals or in vitro replicates:
- triplicates
- Details on study design:
- Study Design
Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.
Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Data Evaluation
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
Results and discussion
In vitro
Results
- Irritation parameter:
- in vitro irritation score
- Value:
- 73.5
- Negative controls validity:
- valid
- Remarks:
- 1.4
- Positive controls validity:
- valid
- Remarks:
- 128.8
- Remarks on result:
- positive indication of irritation
- Other effects / acceptance of results:
- Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was above the range of 65.1 to 123.3. The positive control acceptance criterion was therefore not satisfied. This is reported as a deviation.
The negative control gave opacity of ≤2.4 and permeability ≤0.072. The negative control acceptance criteria were therefore satisfied
DEVIATIONS FROM STUDY PLAN
The following deviations from the Study Plan occurred:
Deviation 1
The positive control group had an overall IVIS of 128.8, which was marginally higher than the criteria range set for an acceptable test. As the score was only marginally exceeded, it was decided that the result was acceptable as the positive control group still provided its intended function, which was to show the sensitivity of the test system to a known ocular irritant.
This deviation was considered to have not affected the integrity or validity of the study.
Any other information on results incl. tables
Corneal Opacity and Permeability Measurement
Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1.
Corneal Epithelium Condition
The condition of each cornea is given in Appendix 2.
The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.
In VitroIrritancy Score
TheIn Vitroirritancy scores are summarized as follows:
Treatment |
In VitroIrritancy Score |
Test Item |
73.5 |
Negative Control |
1.4 |
Positive Control |
128.8 |
Appendix 1 Individual and Mean Corneal Opacity and Permeability Measurements
Treatment |
Cornea Number |
|
Opacity |
|
Permeability (OD) |
In Vitro Irritancy Score |
||
Pre-Treatment |
Post-Treatment |
Post-Treatment Pre-Treatment |
Corrected Value |
|
Corrected Value |
|||
Negative Control |
1 |
4 |
6 |
2 |
|
0.022 |
|
|
2 |
6 |
6 |
0 |
|
0.030 |
|
|
|
3 |
4 |
5 |
1 |
|
0.022 |
|
|
|
|
|
|
1.0* |
|
0.025 |
|
1.4 |
|
Positive Control |
4 |
4 |
93 |
89 |
88.0 |
3.195 |
3.170 |
|
5 |
6 |
104 |
98 |
97.0 |
2.525 |
2.500 |
|
|
6 |
6 |
102 |
96 |
95.0 |
1.440 |
1.415 |
|
|
|
|
|
|
93.3 |
|
2.362 |
128.8 |
|
Test Item |
7 |
4 |
81 |
77 |
76.0 |
0.076 |
0.051 |
|
8 |
4 |
77 |
73 |
72.0 |
0.051 |
0.026 |
|
|
9 |
6 |
78 |
72 |
71.0 |
0.052 |
0.027 |
|
|
|
|
|
|
73.0 |
|
0.035 |
73.5 |
OD = Optical density * = Mean of the post-treatment pre-treatment values = Mean permeability = Mean corrected value
Appendix 2 Corneal Epithelium Condition Post Treatment
Treatment |
Cornea Number |
Observation Post Treatment |
Negative Control |
1 |
Clear |
2 |
Clear |
|
3 |
Clear |
|
Positive Control |
4 |
Cloudy |
5 |
Cloudy |
|
6 |
Cloudy |
|
Test Item |
7 |
Cloudy |
8 |
Cloudy |
|
9 |
Cloudy |
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Conclusions:
- Category 1. UN GHS or EU CLP Causes serious eye damage.
- Executive summary:
The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.
The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.
Method
The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS).
Data Interpretation
The test item is classified according to the prediction model as follows:
IVIS
Classification
≤ 3
No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55
No prediction of eye irritation can be made
> 55
Category 1. UN GHS or EU CLP Causes serious eye damage
Results
TheIn Vitroirritancy scores are summarized as follows:
Treatment
In VitroIrritancy Score
Test Item
73.5
Negative Control
1.4
Positive Control
128.8
Conclusion
Category 1. UN GHS or EU CLP Causes serious eye damage.
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