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EC number: 690-651-2 | CAS number: 1227296-24-3
- 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
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Remarks:
- .
- Adequacy of study:
- key study
- Study period:
- 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- No deviations from the study plan were observed.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
Materials and methods
Test guideline
- 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:
- OECD Guideline for the Testing of Chemicals, Method No. 437, adopted 09. Oct. 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:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- GLP-Conformity All procedures according to the principles of GLP (Chemikaliengesetz §19a and §19b and annexes 1 and 2 from 28. Aug. 2013, published in Federal Law Gazette, Germany (BGBl) No. 55/2013 as of 06. Sep. 2013, and further revisions).
Test material
- Test material form:
- liquid
- Details on test material:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance, and no significant differences in the purity profile exist
- Expiration date of the lot/batch: not relevant (stable)
- Purity test date: not relevant, the test material is representative of the registered substance
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
- Reactivity of the test substance with the solvent: none (contains water)
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: The test material is representative of the registered substance
- Expiration date of the lot/batch: not relevant
- Purity test date: not relevant
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- Species Bos primigenius Taurus (fresh bovine corneas)
Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old:
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 15 minutes.
The following Guideline was applied: OECD Guideline for the Testing of Chemicals, Series on Testing and Assessment No. 160: “GUIDANCE DOCUMENT ON “THE BOVINE CORNEAL OPACITY AND PERMEABILITY (BCOP) AND ISOLATED CHICKEN EYE (ICE) TEST METHODS: COLLECTION OF TISSUES FOR HISTOLOGICAL EVALUATION AND COLLECTION OF DATA ON NON-SEVERE IRRITANTS”; 25. Oct. 2011
Additional information was taken from:
• “Bovine Corneal Opacity and Permeability (BCOP) Assay”, SOP of Microbiological Associates Ltd., UK, Invittox (UK) protocol no. 124, Procedure Details, April 1997, last update Aug. 1999; based on Gautheron et al. (1992), refined by Vanparys et al. (1994)
• “The Bovine Corneal Opacity and Permeability Assay – Method of Gautheron”; Invit-tox (UK) protocol no. 98, April 1996
• Commission Regulation (EU) 2017/735 amending Regulation (EC) No. 440/2008, EU Method B.47 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, adopted 14. Feb. 2017
Test system
- Vehicle:
- unchanged (no vehicle)
- Remarks:
- The test item is a liquid substance. It was tested directly, without dilution or preparation of a solution. The test item was shaken before use.
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- Chemicals
• Minimum Essential Medium (MEM) without phenol red
• Minimum Essential Medium (MEM) with phenol red
• L-glutamine
• Sodium bicarbonate NaHCO3
• 1% fetal calf serum
• cMEM without phenol red:
495 mL MEM without phenol red are supplemented with 5 mL FCS, 146 mg L-Glutamine and 0.55 g NaHCO3
248 mL MEM without phenol red are supplemented with 2.5 mL FCS, 73 mg L-Glutamine and 0.28 g NaHCO3
(as soon as the MEM without phenol red is supplemented with FCS and L-Glutamine and NaHCO3, it is designated in the following as cMEM (= complete MEM) without phenol red)
• cMEM with phenol red:
495 mL MEM with phenol red are supplemented with 5 mL FCS and 146 mg L-Glutamine (as soon as the MEM with phenol red is supplemented with FCS and L-Glutamine, it is designated in the following as cMEM (= complete MEM) with phenol red)
• Sodium fluorescein (C20H11O5Na)
• Hank’s Balanced Salt Solution (HBSS) with Penicillin-Streptomycin solution (transport medium)
• Hank’s Balanced Salt Solution (HBSS) without Penicillin-Streptomycin solution (negative control)
• Penicillin-Streptomycin solution - Duration of treatment / exposure:
- 1 h + 10 min
- Duration of post- treatment incubation (in vitro):
- 90 min
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- Purpose and Principle of the Study
Thisin vitro study was performed to assess the corneal damage potential of test item by quantitative measurements of changes in opacity and permeability in a bovine cornea. The study was performed for regulatory purposes.
The 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. Both measurements are used to calculate an “In Vitro Irritancy Score (IVIS)”, which is used to classify the test item in the UN Globally Harmonised System (GHS).
The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. Test item is applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation parameter:
- cornea opacity score
- Remarks:
- Mean Opacity Difference corrected
- Run / experiment:
- Opacity Values Test Item and Positive Control: Mean Opacity Difference corrected
- Value:
- ca. 95.92
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- Hank’s Balanced Salt Solution (HBSS) 0.96 Mean Opacity Difference
- Positive controls validity:
- valid
- Remarks:
- Dimethylformamide (DMF) 71.35 Mean Opacity Difference corrected
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- other: Optical density
- Run / experiment:
- Optical density at 492 nm
- Value:
- ca. 3.256
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 0.0052
- Positive controls validity:
- valid
- Remarks:
- 1.3221
- Remarks on result:
- positive indication of irritation
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Mean IVIS +/- Relative Standard Deviation IVIS
- Value:
- ca. 144.77
- Negative controls validity:
- valid
- Remarks:
- Mean IVIS 1.03 +/- 8.73 %
- Positive controls validity:
- valid
- Remarks:
- Mean IVIS 91.18 +/- 15%
- Other effects / acceptance of results:
- For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:
Table 9.1 d Optical density at 492 nm of Blank
Parameter cMEM without phenol red
1. Measurement 0.035
2. Measurement 0.033
3. Measurement 0.032
Mean 0.033
Validity
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The negative control has to show an IVIS ≤ 3.
The validity criteria and findings are the following
Parameter Criterion Found Assessment
IVIS of negative control
HBSS ≤ 3 1.03 ok
IVIS of positive control
DMF undiluted 36.93 – 150.79 91.18 ok
Values for negative and positive controls were within the range of historical data of the test facility. Therefore, the test system was acceptable.
Any other information on results incl. tables
Findings and Results
Opacity and Permeability Values
Theilluminance(unit: LUX) values which were measured before and after exposure are given in the following table:
Table9.1‑a Illuminance Values
Parameter |
Negative Control |
Test Item |
Positive Control |
||||||
1. Rep. |
2. Rep. |
3. Rep. |
1. Rep. |
2. Rep. |
3. Rep. |
1. Rep. |
2. Rep. |
3. Rep. |
|
(I) Measured values before exposure |
938 |
952 |
994 |
958 |
978 |
1010 |
993 |
989 |
957 |
(I) Measured values after exposure |
917 |
934 |
972 |
292 |
282 |
342 |
377 |
342 |
386 |
Rep. = Replicate
The values in the following tables present the calculated opacity values, according to evaluation:
Opacity Values Negative Control
Parameter |
Negative Control |
||
1. Rep. |
2. Rep. |
3. Rep. |
|
Opacity before exposure |
5.89 |
5.22 |
3.34 |
Opacity after exposure |
6.93 |
6.08 |
4.31 |
Opacity Difference |
1.04 |
0.86 |
0.97 |
Mean Opacity Difference |
0.96 |
Rep. = Replicate
Opacity Values Test Item and Positive Control
Parameter |
Test Item |
Positive Control |
||||
1. Rep. |
2. Rep. |
3. Rep. |
1. Rep. |
2. Rep. |
3. Rep. |
|
Opacity before exposure |
4.94 |
4.04 |
2.66 |
3.38 |
3.55 |
4.99 |
Opacity |
106.13 |
111.29 |
84.85 |
73.31 |
84.85 |
70.68 |
Opacity |
101.18 |
107.25 |
82.19 |
69.93 |
81.29 |
65.69 |
Opacity corrected |
100.23 |
106.30 |
81.23 |
68.98 |
80.34 |
64.74 |
Mean Opacity corrected |
95.92 |
71.35 |
Rep. = Replicate
For the permeability measurement, three replicates for each treatment group were measured three times. cMEM without phenol red was measured as blank value as well. The optical density values at 492 nm are given in the following tables:
Optical density at 492 nm of Blank
Parameter |
cMEM without phenol red |
1. Measurement |
0.035 |
2. Measurement |
0.033 |
3. Measurement |
0.032 |
Mean |
0.033 |
Optical density at 492 nm of Negative Control, Test Item and Positive Control
Parameter |
Negative Control |
Test Item |
Positive Control |
||||||
1. Rep. |
2. Rep. |
3. Rep. |
1. Rep. * |
2. Rep. * |
3. Rep. * |
1. Rep. |
2. Rep. |
3. Rep. |
|
1. Measurement |
0.040 |
0.044 |
0.032 |
0.711 |
0.684 |
0.670 |
1.789 |
1.484 |
0.777 |
2. Measurement |
0.039 |
0.039 |
0.034 |
0.701 |
0.680 |
0.671 |
1.837 |
1.475 |
0.780 |
3. Measurement |
0.041 |
0.042 |
0.036 |
0.705 |
0.679 |
0.670 |
1.842 |
1.489 |
0.773 |
|
|||||||||
1. Measurement – blank |
0.0067 |
0.0107 |
-0.0013 |
0.6777 |
0.6507 |
0.6367 |
1.7557 |
1.4507 |
0.7437 |
2. Measurement – blank |
0.0057 |
0.0057 |
0.0007 |
0.6677 |
0.6467 |
0.6377 |
1.8037 |
1.4417 |
0.7467 |
3. Measurement – blank |
0.0077 |
0.0087 |
0.0027 |
0.6717 |
0.6457 |
0.6367 |
1.8087 |
1.4557 |
0.7397 |
Mean of each replicate |
0.0067 |
0.0083 |
0.0007 |
0.6723 |
0.6477 |
0.6370 |
1.7893 |
1.4493 |
0.7433 |
Mean of the 3 replicates |
0.0052 |
-- |
-- |
||||||
Corrected |
-- |
-- |
-- |
3.3564 |
3.2331 |
3.1798 |
1.7841 |
1.4441 |
0.7381 |
Corrected mean of the 3 replicates |
-- |
3.2564 |
1.3221 |
Rep. = Replicate
* Note: All values of the three replicates of the test item were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.
IVIS Values
The calculated IVIS for each replicate and the corresponding means are presented in the following table:
Table9.2‑a IVIS
Test Group |
IVIS |
Mean IVIS |
Relative Standard Deviation IVIS |
Negative Control |
1.14 |
1.03 |
8.73% |
0.99 |
|||
0.98 |
|||
Test Item |
150.57 |
144.77 |
9.58% |
154.79 |
|||
128.93 |
|||
Positive Control |
95.74 |
91.18 |
15.00% |
102.00 |
|||
75.81 |
Assessment
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.
Table9.4‑a Classification Scheme
IVIS |
UN GHS Category Eye Damage |
≤ 3 |
No category |
> 3 and≤ 55 |
No prediction can be made |
> 55 |
Eye damage Category I |
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS category I.
The test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 144.77.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Conclusions:
- Under the conditions of this test, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS (In Vitro Irritancy Score) is 144.77.
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. - Executive summary:
In a Study following OECD Guideline 437, cornea of bovine eyes were incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and exposed to the test item for 10 minutes at 32 ± 1 °C. After removal of the test item and 2 hours post-incubation, opacity and permeability values were measured.
Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In VitroIrritancy Score) is 1.03.
Dimethylformamide (DMF) undiluted was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 91.18.
Under the conditions of this study, the test item induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 144.77.
According to OECD Guideline 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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