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EC number: 813-152-5 | CAS number: 152261-44-4
- 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
Endpoint summary
Administrative data
Description of key information
Skin Irritation
REACH_not corrosive | Epiderm | OECD 431 | #key study#
REACH_not irritating | Epiderm | OECD 439 | #key study#
Eye Irritation
REACH_no prediction | BCOP | OECD 437 | #key study#
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin corrosion: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-01-16 to 2018-01-18
- 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:
- 29 July 2016
- Qualifier:
- according to guideline
- Guideline:
- other: Council Regulation 440/2008, Method B.40 BIS: “In Vitro Skin Corrosion: Human Skin Model Test”, May 30, 2008
- Qualifier:
- according to guideline
- Guideline:
- other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Corrosion Test (EPI-200-SCT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm; Version 07/11/2014
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
- Test system:
- human skin model
- Remarks:
- EpiDerm
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal (non-cancerous), human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm skin model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous, granular and cornified layers analogous to those found in vivo.
- Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 50 µL
- Duration of treatment / exposure:
- 3 minutes and 60 minutes
- Duration of post-treatment incubation (if applicable):
- 3 h MTT incubation
- Number of replicates:
- 2
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 60 min. treatment
- Value:
- 97.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100.0
- Positive controls validity:
- valid
- Remarks:
- 1.5
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 min. treatment
- Value:
- 106.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100.0
- Positive controls validity:
- valid
- Remarks:
- 5.2
- Other effects / acceptance of results:
- Value Cut off pass/fail
Mean Absolute OD570 nm NK
(3 min Experiment) 1.609 0.8 ≤ NK ≤ 2.8 pass
Mean Absolute OD570 nm NK
(60 min Experiment) 1.670 0.8 ≤ NK ≤ 2.8 pass
Mean Relative Tissue Viability [%] of
PC (60 min experiment) 1.5 < 15% pass
CV [%]
(in the range of 20 – 100% viability) 7.8% - 20.1% ≤ 30% pass - Interpretation of results:
- other: not corrosive
- Conclusions:
- In this study under the given conditions the test item showed no corrosive effects. The test item is classified as “non-corrosive“.
- Executive summary:
In the present study the skin corrosivity potential of the test item was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm, a reconstituted three-dimensional human epidermis model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 3 min and 60 min exposure period and compared to those of the concurrent negative controls.
The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TODTT). The test item showed no water-colouring potential.
The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (110.7%, NSMTT-corrected) after 3 min treatment and ≥ 15% (97.6%, NSMTT-corrected) after 60 min treatment.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-02-21 to 2018-02-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 28 July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Commission Regulation (EC) No. 640/2012, L 193, Part B.46. “In vitro Skin Irritation: Reconstructed Human Epidermis Test Method” 06-Jul-2012
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: MatTek Corporation Protocol for: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT) For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDerm (EPI-200-SIT)
- Version / remarks:
- Version 07-Nov-2014
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
- Test system:
- human skin model
- Remarks:
- EpiDerm
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- This in vitro method is designed to predict and classify the skin irritation potential of a chemical by assessment of its effect on EpiDermTM, a reconstituted three-dimensional human epidermis model. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure period.
The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have
been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- Dose Groups
1. Negative control 30 µL Dulbecco’s phosphate buffered saline
2. Positive control 30 µL 5% sodium dodecyl sulfate solution
3. Test Item 30 µL (undiluted) - Duration of treatment / exposure:
- 60 ± 1 min
- Duration of post-treatment incubation (if applicable):
- 24 ± 2 h + 18 ± 2 h (42 ± 2 h)
- Number of replicates:
- The test was performed on a total of 3 tissues per dose group.
- Details on study design:
- The test was performed on EpiDerm, an organotypic reconstructed three-dimensional model of the human epidermis. 3 replicate tissues are dosed with the test item, the negative control (30 µL DPBS) and the positive control (30 µL 5% SDS), respectively. After 60 ± 1 min treatment period at 37°C (35 min) and room temperature (25 min) the test item and the controls are rinsed off with DPBS and the tissues are post-incubated for 42 +/- 1 h. Then the tissues are stained via MTT for 3 hours. The extracts are measured photometrically at 570 nm.
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1
- Value:
- 94.9
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100.0
- Positive controls validity:
- valid
- Remarks:
- 3.0
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
- Executive summary:
In the present study the skin irritant potential of the test item was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.
The mixture of 30 µL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 30 µL of the test item per 300 µL aqua dest. and/ per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.9%) after 60 min treatment and 42 h post-incubation.
Referenceopen allclose all
The test item showed non-specific MTT-reducing potential. Therefore, additional killed tissue controls were treated with the test item to determine the non-specific reduction of MTT (NSMTT) and the results were corrected to the true MTT metabolic conversion (TODTT). The test item showed no water-colouring potential.
The test item showed no corrosive effects. The mean relative tissue viability (% negative control) was ≥ 50% (110.7%, NSMTT-corrected) after 3 min treatment and ≥ 15% (97.6%, NSMTTcorrected) after 60 min treatment.
The controls confirmed the validity of the study. The mean OD570nm of the two negative control tissues was ≥ 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (1.5%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was ≤ 30% (7.8% - 20.1%).
Results of 3 minExperiment
Name |
Negative Control |
Test Item |
Positive Control |
|||
Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
Absolute OD570 |
1.818 |
1.381 |
1.687 |
1.694 |
0.130 |
0.122 |
1.854 |
1.399 |
1.702 |
1.704 |
0.129 |
0.125 |
|
1.823 |
1.380 |
1.712 |
1.789 |
0.129 |
0.125 |
|
OD570- |
1.773 |
1.335 |
1.642 |
1.648 |
0.085 |
0.077 |
1.808 |
1.354 |
1.656 |
1.659 |
0.083 |
0.079 |
|
1.777 |
1.334 |
1.666 |
1.744 |
0.084 |
0.079 |
|
Mean OD570of 3 Aliquots (Blank Corrected) |
1.786 |
1.341 |
1.655 |
1.684 |
0.084 |
0.079 |
SD OD570 of 3 Aliquots |
0.019 |
0.011 |
0.012 |
0.052 |
0.001 |
0.001 |
Total Mean OD570of 2 Replicate Tissues (Blank Corrected) |
1.564* |
1.669 |
0.081 |
|||
TODTT |
- |
1.731 |
- |
|||
SD OD570 of 2 Replicate Tissues |
0.315 |
0.021 |
0.004 |
|||
Mean Relative Tissue |
100.0 |
106.8 |
5.2 |
|||
Mean Relative Tissue Viability [%] |
- |
110.7 |
- |
|||
Coefficient Of Variation [%]*** |
20.1 |
1.2 |
4.8 |
* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.
*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.
Results of the NSMTT control of 3 min Experiment
NSMTT |
KU |
KT |
Negative Control |
||||
Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
|
absolute OD570 -values |
0.576 |
0.495 |
0.449 |
0.501 |
1.818 |
1.381 |
|
0.590 |
0.490 |
0.460 |
0.497 |
1.854 |
1.399 |
||
0.597 |
0.493 |
0.445 |
0.497 |
1.823 |
1.380 |
||
OD570(Blank Corrected) |
0.531 |
0.450 |
0.404 |
0.455 |
1.773 |
1.335 |
|
0.545 |
0.445 |
0.415 |
0.451 |
1.808 |
1.354 |
||
|
0.552 |
0.448 |
0.399 |
0.452 |
1.777 |
1.334 |
|
mean OD570 |
0.538 |
0.448 |
0.409 |
0.453 |
1.791 |
1.344 |
|
total mean OD570 |
0.493 |
0.431 |
1.568* |
||||
SD OD570(of the replicate tissues) |
0.064 |
0.031 |
0.315 |
||||
NSMTT [%] |
-3.92 |
- |
|||||
Relative Tissue Viability [%] |
- |
114.2 |
85.8 |
||||
Mean Relative Tissue Viability [%] |
- |
100.0 |
|||||
SD Tissue Viability [%] |
- |
20.1 |
|||||
CV [% Viabilities] |
- |
20.1 |
Results of 60 min Experiment
Name |
Negative Control |
Test Item |
Positive Control |
|||
Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
Absolute OD570 |
1.576 |
1.710 |
1.415 |
1.817 |
0.070 |
0.069 |
1.579 |
1.783 |
1.419 |
1.816 |
0.072 |
0.069 |
|
1.587 |
1.787 |
1.494 |
1.822 |
0.072 |
0.069 |
|
OD570- |
1.531 |
1.665 |
1.370 |
1.771 |
0.025 |
0.024 |
1.533 |
1.738 |
1.374 |
1.771 |
0.027 |
0.024 |
|
1.542 |
1.742 |
1.449 |
1.776 |
0.027 |
0.024 |
|
Mean OD570of 3 Aliquots (Blank Corrected) |
1.535 |
1.715 |
1.397 |
1.773 |
0.026 |
0.024 |
SD OD570 of 3 Aliquots |
0.006 |
0.043 |
0.044 |
0.003 |
0.001 |
0.000 |
Total Mean OD570of 2 Replicate Tissues (Blank Corrected) |
1.625* |
1.585 |
0.025 |
|||
TODTT |
- |
1.586 |
- |
|||
SD OD570 of 2 Replicate Tissues |
0.127 |
0.266 |
0.002 |
|||
Mean Relative Tissue |
100.0 |
97.5 |
1.5** |
|||
Mean Relative Tissue Viability [%] |
- |
97.6 |
- |
|||
Coefficient Of Variation [%]*** |
7.8 |
16.8 |
6.3 |
* corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability.
** mean relative tissue viability of the 60 min positive control < 15%.
*** coefficient of variation (CV) (in the range of 20 – 100% viability) between two tissues treated identically is ≤ 30%.
Results of the NSMTT control of 60 min Experiment
NSMTT |
KU |
KT |
Negative Control |
||||
Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
|
absolute OD570 -values |
0.106 |
0.095 |
0.087 |
0.113 |
1.576 |
1.710 |
|
0.107 |
0.095 |
0.088 |
0.112 |
1.579 |
1.783 |
||
0.106 |
0.094 |
0.087 |
0.112 |
1.587 |
1.787 |
||
OD570(Blank Corrected) |
0.061 |
0.049 |
0.041 |
0.068 |
1.531 |
1.665 |
|
0.062 |
0.049 |
0.043 |
0.067 |
1.533 |
1.738 |
||
|
0.061 |
0.049 |
0.042 |
0.066 |
1.542 |
1.742 |
|
mean OD570 |
0.061 |
0.049 |
0.042 |
0.067 |
1.532 |
1.702 |
|
total mean OD570 |
0.055 |
0.055 |
1.617* |
||||
SD OD570(of the replicate tissues) |
0.008 |
0.018 |
0.120 |
||||
NSMTT [%] |
-0.04 |
- |
|||||
Relative Tissue Viability [%] |
- |
97.7 |
108.6 |
||||
Mean Relative Tissue Viability [%] |
- |
103.1 |
|||||
SD Tissue Viability [%] |
- |
7.6 |
|||||
CV [% Viabilities] |
- |
7.4 |
The mixture of 30 μL test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 30 μL of the test item per 300 μL aqua dest. and per 300 μL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (94.9%) after 60 min treatment and 42 h post-incubation.
The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (2.002). The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.0%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.1% - 9.8%).
Result of the Test Item
Name |
Negative Control |
Positive Control |
Test Item |
||||||
Tissue |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
Absolute OD570 |
2.070 |
1.785 |
2.187 |
0.100 |
0.101 |
0.100 |
1.948 |
1.996 |
1.686 |
2.045 |
1.789 |
2.133 |
0.103 |
0.107 |
0.100 |
1.975 |
2.028 |
1.775 |
|
OD570(Blank Corrected) |
2.027 |
1.741 |
2.144 |
0.057 |
0.057 |
0.056 |
1.905 |
1.953 |
1.643 |
2.002 |
1.746 |
2.090 |
0.060 |
0.063 |
0.057 |
1.931 |
1.985 |
1.731 |
|
Mean OD570of the Duplicates (Blank Corrected) |
2.014 |
1.743 |
2.117 |
0.058 |
0.060 |
0.056 |
1.918 |
1.969 |
1.687 |
Total Mean OD570of 3 Replicate Tissues (Blank Corrected) |
1.958* |
0.058 |
1.858 |
||||||
SD OD570 |
0.193 |
0.002 |
0.150 |
||||||
Relative Tissue Viability [%] |
102.9 |
89.0 |
108.1 |
3.0 |
3.1 |
2.9 |
97.9 |
100.5 |
86.1 |
Mean Relative Tissue Viability [%] |
100.0 |
3.0** |
94.9 |
||||||
SD Tissue Viability [%]*** |
9.8 |
0.1 |
7.7 |
||||||
CV [% Viabilities] |
9.8 |
3.2 |
8.1 |
* Blank-corrected mean OD570 nm of the negative control corresponds to 100% absolute tissue viability.
** Mean relative tissue viability of the three positive control tissues is ≤ 20%.
*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2018-02-13 to 2018-05-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline for the Testing of Chemicals, no. 437 (adopted: 09 October 2017)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Munich, Germany
- Species:
- other: bovine cornea
- Strain:
- not specified
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- 750 μL
- Duration of treatment / exposure:
- After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red).
- Duration of post- treatment incubation (in vitro):
- 2 h before illuminance measurement followed by 90 min before determination of optical density
- Number of animals or in vitro replicates:
- 3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100% - Details on study design:
- Preparation of the Corneas
The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
The eyes were carefully examined for defects and any defective eyes were discarded.
The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.
Calibration of the Opacitometer
The opacitometer (BASF-OP3.0, Duratec) was switched on 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout should lie in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer should display values between 300-310 lux and between 95-105 lux. If this is not the case, the calibration procedure has to be repeated. The calibration procedure is performed before each test and is documented in the raw data.
Treatment of the Corneas
After the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI. An initial measurement was performed on each of the corneas using the opacitometer. Three corneas with illuminance readings approximately equivalent to the median illuminance of all corneas were selected as negative-control corneas. The illuminance of each cornea was read and recorded. Only corneas that had an initial illuminance reading I > I0/1.1651 lux were used for the assay. The medium was removed from the anterior chamber and replaced with the test item or control.
750 µL of the test substance or the control substance was introduced into the anterior chamber. After 10 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed after 2 hours incubation at 32 ± 1 °C. Also, each cornea was observed visually and pertinent observations were recorded.
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 mL of a 4 mg/mL sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).
Test Groups
3 corneas for the test item
3 corneas as negative controls treated with physiological saline 0.9% NaCl
3 corneas as positive controls treated with ethanol 100%
The BCOP assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Evaluation of Results
The following formula was used to calculate the opacity, whereas the values a and b are equipment-specific variables empirically determined by the manufacturer:
Opacity= ( I0/I-b)/a
with a = 0.025 and b = 0.9894
The value I0 (=I zero) is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment.
The mean OD490 for the blank cuvettes was calculated. The mean blank OD490 was subtracted from the OD490 of each cuvette (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average-corrected OD490 of the negative-control corneas from the corrected OD490 value of each treated cornea:
Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
The following formula was used to determine the in vitro irritation score (IVIS):
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The IVIS cut-off values for identifying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given below:
Evaluation of the BCOP Assay
IVIS UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
An identification of test substances that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances that should be classified as mildly irritating to eyes (UN GHS Category 2B) cannot be made.
For this purpose further testing with another suitable method is required. - Irritation parameter:
- in vitro irritation score
- Remarks:
- mean
- Run / experiment:
- test item
- Value:
- 46.32
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 0.81
- Positive controls validity:
- valid
- Remarks:
- 63.38
- Other effects / acceptance of results:
- No prediction can be made regarding the classification of the test substance according to the evaluation criteria.
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- No prediction can be made regarding the classification of the test substance Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine according to the evaluation criteria.
- Executive summary:
Summary Results
The eye irritancy potential of Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine was investigated in the bovine corneal opacity and permeability assay.
Preparation of the test item: tested as provided by the sponsor
Visual Observation after treatment: All 3 corneas treated with Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine showed opacity of the tissue.
Mean in vitro irritation score: 46.32
UN GHS No Category
x
No prediction can be made
UN GHS Category 1
Classification
The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid.
The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.
Conclusion
No prediction can be made regarding the classification of the test substanceEthanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine according to the evaluation criteria.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irreversible damage)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Skin Irritation
The in vitro testing of Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine according to OECD 431 and OECD 439 showed no irritational potential on the skin.
Eye Irritation
The eye irritancy potential of Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine was investigated in the bovine corneal opacity and permeability assay.
All 3 corneas treated with Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine showed opacity of the tissue. Furthermore, the permeability score of the tissues was also significantly increased. An IVIS of 46.32 was determined which is close to the cut-off for classification as severely eye damaging of 55.1.
Due to the severe corneal effects observed, Ethanol, 2,2’[1,2-ethanediylbis(oxy)] bis-, reaction products with 3-(triethoxysilyl)-1-propanamine is classified as severely damaging to eyes.
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