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EC number: 846-828-3 | CAS number: 1872341-39-3
- 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
Endpoint summary
Administrative data
Description of key information
A GLP-compliant in vitro skin irritation turnkey strategy was used to assess the skin irritating potential of the test substance. Both, the skin corrosion test (according to OECD Guideline 431) and the skin irritation test (according to OECD Guideline 439), were negative. Therefore, the substance is not considered to be classified for skin irritation.
A GLP-compliant in vitro eye irritation turnkey strategy was used to assess the eye irritating potential of the test substance. According to the results of the BCOP (OECD Guideline 437) the substance is not classified in UN GHS Category 1. Based on the combined assessment together with the EpiOcular (according to OECD Guideline 492) the substance is considered to be classified for eye irritation UN GHS Category 2.
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:
- weight of evidence
- Study period:
- Feb 2019
- 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
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
FORM AS APPLIED IN THE TEST (if different from that of starting material) : solid - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 28684
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: one washing step with PBS
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: measurement without reference filter
NUMBER OF REPLICATE TISSUES: two tissues per exposure time and test group (12 tissues per test)
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed at - 20 °C
- N. of replicates: two killed control tissues per exposure time were treated with the test substance and the negative control, respectively
- Method of calculation used: In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean corrected OD570 KC). Since killed tissues might still have a residual enzyme activity that is able to produce some formazan net, OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero, it is subtracted from the respective mean OD570 to result in the mean corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 % - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N - Duration of treatment / exposure:
- 3 min and 1 h
- Number of replicates:
- 2
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 min exposure period
- Value:
- 95.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 h exposure period
- Value:
- 105.4
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Interpretation of results:
- GHS criteria not met
- Remarks:
- no indication of skin corrosion
- Conclusions:
- Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.
- Executive summary:
The potential of the test substance to cause dermal corrosion was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion test:
The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 95.5%, and it was 105.4% after an exposure period of 1 hour.
Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Feb 2019
- 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
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
FORM AS APPLIED IN THE TEST (if different from that of starting material): solid - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 28684
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C and room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: two washing steps with PBS
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: measurement without using a reference filter
NUMBER OF REPLICATE TISSUES: three tissues were treated with the test substance, the PC and the NC, respectively
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed at - 20 °C
- N. of replicates: three
- Method of calculation used: Since killed tissues might still have a residual enzyme activity that is able to produce some formazan net, OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero, it is subtracted from the respective mean OD570 to result in the mean corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal to 50 %. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % - Duration of treatment / exposure:
- 1 hour
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 hour exposure period
- Value:
- 98.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Interpretation of results:
- GHS criteria not met
- Remarks:
- no indication of skin irritation
- Conclusions:
- Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.
- Executive summary:
The potential of the test substance to cause dermal irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).
The skin irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour postincubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin irritation test:
The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 98.1%.
Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation test under the test conditions chosen.
Referenceopen allclose all
Table 2: Summary of results after 3 minutes exposure period
Test substance identification |
|
|
Tissue 1 |
Tissue 2 |
Mean |
SD |
CV [%] |
NC |
Viable tissues |
Mean OD570 |
2.016 |
1.996 |
2.006 |
|
|
Viability [% of NC] |
100.5 |
99.5 |
100.0 |
0.7 |
0.7 |
||
KC tissues |
Mean OD570 |
0.114 |
0.092 |
0.103 |
|
|
|
Viability [% of NC] |
5.7 |
4.6 |
5.1 |
0.8 |
15.4 |
||
Test substance |
Viable tissues |
Mean OD570 |
1.995 |
1.849 |
1.922 |
|
|
Viability [% of NC] |
99.5 |
92.2 |
95.8 |
5.1 |
5.4 |
||
KC tissues * |
Mean OD570 KC NC corrected |
0.011 |
0.000 |
0.006 |
|
|
|
Viability [% of NC] |
0.56 |
0.00 |
0.28 |
|
|
||
Final relative mean viability of tissues after KC correction [% of NC] |
95.5 |
||||||
PC |
Viable tissues |
Mean OD570 |
0.254 |
0.216 |
0.235 |
|
|
Viability [% of NC] |
12.7 |
10.8 |
11.7 |
1.3 |
11.4 |
*negative values are set to zero for further calculation
Table 3: Summary of results after 1 hour exposure period
Test substance identification |
|
|
Tissue 1 |
Tissue 2 |
Mean |
SD |
CV [%] |
NC |
Viable tissues |
Mean OD570 |
1.854 |
1.875 |
1.864 |
|
|
Viability [% of NC] |
99.4 |
100.6 |
100.0 |
0.8 |
0.8 |
||
KC tissues |
Mean OD570 |
0.085 |
0.092 |
0.088 |
|
|
|
Viability [% of NC] |
4.5 |
4.9 |
4.7 |
0.3 |
5.6 |
||
Test substance |
Viable tissues |
Mean OD570 |
1.941 |
2.030 |
1.986 |
|
|
Viability [% of NC] |
104.1 |
108.9 |
106.5 |
3.4 |
3.2 |
||
KC tissues |
Mean OD570 KC NC corrected |
0.023 |
0.019 |
0.021 |
|
|
|
Viability [% of NC] |
1.2 |
1.0 |
1.1 |
0.1 |
11.9 |
||
Final relative mean viability of tissues after KC correction [% of NC] |
105.4 |
||||||
PC |
Viable tissues |
Mean OD570 |
0.097 |
0.106 |
0.101 |
|
|
Viability [% of NC] |
5.2 |
5.7 |
5.4 |
0.3 |
6.3 |
Table 4: Historic control data of NC and PC of skin corrosion test
|
|
Exposure Time |
Period |
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
NC |
OD570 |
3 minutes |
Jan 2017 – Jan 2019 |
1.728 |
0.216 |
2.160 |
1.295 |
60 minutes |
1.724 |
0.252 |
2.227 |
1.220 |
|||
PC |
3 minutes |
0.222 |
0.071 |
0.363 |
0.081 |
||
60 minutes |
0.097 |
0.031 |
0.159 |
0.036 |
|||
Relative viability [%] |
3 minutes |
13.0 |
4.2 |
21.3 |
4.6 |
||
60 minutes |
5.7 |
1.8 |
9.3 |
2.2 |
Table 2: Individual and mean OD570 values, individual and mean viability and standard deviations
Test substance identification |
|
|
Tissue 1 |
Tissue 2 |
Tissue 3 |
Mean |
SD |
NC |
Viable tissues |
Mean OD570 |
2.142 |
2.053 |
2.180 |
2.125 |
|
Viability [% of NC] |
100.8 |
96.6 |
102.6 |
100.0 |
3.1 |
||
KC tissues |
Mean OD570 |
0.046 |
0.042 |
0.042 |
0.043 |
|
|
Viability [% of NC] |
2.2 |
2.0 |
2.0 |
2.0 |
0.1 |
||
Test substance |
Viable tissues |
Mean OD570 |
2.057 |
2.072 |
2.138 |
2.089 |
|
Viability [% of NC] |
96.8 |
97.5 |
100.6 |
98.3 |
2.0 |
||
KC tissues |
Mean OD570 KC NC corrected |
0.0045 |
0.0045 |
0.0015 |
0.0035 |
|
|
Viability [% of NC] |
0.21 |
0.21 |
0.07 |
0.16 |
0.08 |
||
Final relative mean viability of tissues after KC correction [% of NC] |
98.1 |
||||||
PC |
Viable tissues |
Mean OD570 |
0.045 |
0.046 |
0.045 |
0.045 |
|
Viability [% of NC] |
2.1 |
2.2 |
2.1 |
2.1 |
0.0 |
Table 3: Historic control data of NC and PC of skin irritation test
|
|
Period |
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
NC |
OD570 |
Jan 2017 – Jan 2019 |
1.816 |
0.190 |
2.195 |
1.437 |
PC |
0.048 |
0.005 |
0.059 |
0.037 |
||
Relative viability [%] |
2.7 |
0.4 |
3.4 |
1.9 |
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:
- weight of evidence
- Study period:
- Feb - Mar 2019
- 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)
- Version / remarks:
- 09 Oct 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study); ca. 3.5 (20 % aqueous preparation, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparation was produced on a weight per volume (w/v) basis shortly before application by stirring with a high-speed homogenizer (Ultra-Turrax) and a magnetic stirrer. The homogeneity of the test substance preparation during application was provided by stirring with a magnetic stirrer.
FORM AS APPLIED IN THE TEST (if different from that of starting material) : 20 % (w/v) suspension in deionized water - Species:
- cattle
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Schlachthof Alzey, Emil Färber GmbH & Co. KG, Robert-Bosch-Straße 23, 55232 Alzey, Germany
- Characteristics of donor animals (e.g. age, sex, weight): minimum 12 months and maximum 60 months of age
- indication of any existing defects or lesions in ocular tissue samples: corneas free of defects (opacity, scratches, pigmentation etc.) were used - Vehicle:
- water
- Remarks:
- deionized
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 20 %
VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL - Duration of treatment / exposure:
- 4 hours
- Number of animals or in vitro replicates:
- 3
- 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.
NUMBER OF REPLICATES : 3
NEGATIVE CONTROL USED : deionized water
POSITIVE CONTROL USED : Imidazole 20 % (w/v) in deionized water
APPLICATION DOSE AND EXPOSURE TIME : 750 µL for 4 hours
TREATMENT METHOD: open chamber
POST-INCUBATION PERIOD: no
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: one
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Before measurement, each cornea was visually observed 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)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA:
- IVIS ≤ 3: no UN GHS Category
- IVIS > 3; ≤ 55: No prediction can be made
- IVIS > 55: UN GHS Category 1 - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 1st test run
- Value:
- 39.5
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 2nd test run
- Value:
- 36.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- other: not identified as corrosive or severe irritant
- Conclusions:
- Based on the results of the BCOP and by applying the evaluation criteria, it was concluded that the test substance was not identified as corrosive or severe irritant under the test conditions chosen. No further assumptions can be made based on this in vitro study alone.
- Executive summary:
The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL 20% test-substance preparation to the epithelial surface of isolated bovine corneas.
Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the
cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test:
Two test runs were performed. The results obtained in the 1st test run did not indicate a severe eye irritation potential of the
test substance. However, high variability between the individual corneas treated with the negative control (NC) and positive control (PC) were noted. Therefore, the study was repeated to verify the result and to meet the acceptance criteria of the test.
The obtained results of the test substance in the 2nd test run of the BCOP test verified the findings of the 1st experiment. All acceptance criteria of the test were fulfilled.
Based on the results of the BCOP and by applying the evaluation criteria, it was concluded that the test substance was not identified as corrosive or severe irritant under the test conditions chosen. No further assumptions can be made based on this in vitro study alone.
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Jan 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
- Version / remarks:
- 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Landesamt für Umwelt Rheinland-Pfalz
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study); ca. 3.5 (20 % aqueous preparation, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
FORM AS APPLIED IN THE TEST (if different from that of starting material): The test substance is applied undiluted. - Species:
- human
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method and considerations regarding applicability
: The test is based on the experience that irritant chemicals produce cytotoxicity in human reconstructed cornea after a short term topical exposure. The test is designed to predict an eye irritation potential of a chemical by using the three-dimensional, human cornea model EpiOcularTM. After application of the test material to the surface of the EpiOcularTM tissue, the
induced cytotoxicity (= loss of viability) is measured by a colorimetric assay. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity. The mitochondrial dehydrogenase reduces the yellow colored water-soluble 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) to the insoluble blue colored formazan. After isopropanol extraction of the formazan from the tissues, the optical density of the extract is spectrophotometrically determined. The optical density of the extracts of the tissues treated with the test substance is compared to values from negative control tissues and expressed as relative tissue viability.
- Description of the cell system used: The EpiOcular model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived, epidermal keratinocytes used to model the human corneal epithelium. The EpiOcular tissues (surface 0.6 cm²) are cultured on cell culture inserts and are commercially available as kits (EpiOcular 200) containing 24 tissues on shipping agarose. - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- other: MTT reduction control (KC): sterile deionized water or test substance
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): ca. 50 µL - Duration of treatment / exposure:
- 6 hours
- Duration of post- treatment incubation (in vitro):
- 18 hours
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- - RhCE tissue construct used, including batch number
: OCL-200, tissue lot no. 27089
- Doses of test chemical and control substances used : 50 µL
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable) : OCL-200 tissue that is killed by freezing at - 20 °C
- Number of tissue replicates used per test chemical and controls : 2
- Wavelength used for quantifying MTT formazan: 570 nm
- The irritation potential of the test material is predicted from the mean relative tissue viabilities compared to the negative control tissues treated concurrently with sterile water. A chemical is considered as "non-irritant" (no UN GHS Category) if the mean relative tissue viability with a test material is greater than 60 %. - Irritation parameter:
- other: tissue viability [%]
- Run / experiment:
- mean
- Value:
- 40
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Interpretation of results:
- other: irritant
- Conclusions:
- The final relative mean viability of the tissues treated with the test substance was 40 %. The test substance is therefore considered to be classified for eye irritation. No prediction can be made on the UN GHS Category based on this in vitro assay alone.
- Executive summary:
The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 12 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).
Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by a 18-hour post-incubation period.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation assay:
The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.
The final relative mean viability of the tissues treated with the test substance was 40 %.
Referenceopen allclose all
Table 1: 1st test run: IVIS of the test substance, the NC and the PC
Test substance identification |
Cornea-No. |
Opacity per cornea |
Permeability per cornea |
IVIS |
||
Per cornea |
Per group |
|||||
Mean |
SD |
|||||
Test substance |
19 20 21 |
30.2 41.5 46.9 |
0.003 0.003 0.000 |
30.2 41.5 46.9 |
39.5 |
8.5 |
NC |
13 14 15 |
4.3 12.0 24.2 |
0.000 0.001 0.003 |
4.3 12.0 24.3 |
13.5 |
10.1 |
PC |
16 17 18 |
51.5 256.0 61.6 |
3.670 1.982 2.385 |
106.6 285.8 97.4 |
163.2 |
106.2 |
Table 2: 2nd test run: IVIS of the test substance, the NC and the PC
Test substance identification |
Cornea-No. |
Opacity per cornea |
Permeability per cornea |
IVIS |
||
Per cornea |
Per group |
|||||
Mean |
SD |
|||||
Test substance |
7 8 9 |
37.9 37.7 32.5 |
0.004 0.005 0.000 |
38.0 37.7 32.5 |
36.1 |
3.1 |
NC |
1 2 3 |
2.3 6.7 2.9 |
0.002 0.004 0.003 |
2.3 6.7 2.9 |
4.0 |
2.4 |
PC |
4 5 6 |
96.0 96.5 104.3 |
1.360 2.012 1.569 |
116.4 126.7 127.8 |
123.6 |
6.3 |
Table 3: Historic control data of the BCOP Test: Negative control (protocol for solids), Jan 2017 - Jul 2018 (No. of tests performed: 8)
|
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
Opacity |
9.6 |
3.7 |
17.0 |
2.1 |
Permeability [OD570] |
0.003 |
0.001 |
0.005 |
0.001 |
Table 4: Historic control data of the BCOP Test: Positive control (20 % Imidazole), Jan 2017 - Jul 2018 (No. of tests performed: 8)
|
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
Opacity |
92.9 |
10.4 |
113.7 |
72.1 |
Permeability [OD570] |
2.307 |
0.571 |
3.449 |
1.165 |
In Vitro Irritation Score (IVIS) |
127.5 |
14.4 |
156.4 |
98.7 |
Table 1: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability
Test substance identification |
|
|
Tissue 1 |
Tissue 2 |
Mean |
Inter-tissue variability [%] |
NC |
Viable tissues |
Mean OD570 |
2.284 |
2.280 |
2.282 |
|
Viability [% of NC] |
100.1 |
99.9 |
100.0 |
0.2 |
||
KC tissues |
Mean OD570 |
0.029 |
0.031 |
0.030 |
|
|
Viability [% of NC] |
1.3 |
1.4 |
1.3 |
0.1 |
||
Test substance |
Viable tissues |
Mean OD570 |
0.915 |
0.929 |
0.922 |
|
Viability [% of NC] |
40.1 |
40.7 |
40.4 |
0.6 |
||
KC tissues |
Mean OD570 KC NC corrected |
0.008 |
0.013 |
0.010 |
|
|
Viability [% of NC] |
0.3 |
0.6 |
0.4 |
0.2 |
||
Final relative mean viability of tissues after KC correction [% of NC] |
40.0 |
|||||
PC |
Viable tissues |
Mean OD570 |
0.396 |
0.337 |
0.367 |
|
Viability [% of NC] |
17.3 |
14.8 |
16.1 |
2.6 |
Table 2: Historic control data of the EpiOcular Test
|
Period |
Protocol |
Mean |
SD |
Mean + 2 SD |
Mean – 2 SD |
NC [OD570] |
Jan 2017 – Dec 2018 |
Protocol for liquids |
1.901 |
0.497 |
2.896 |
0.906 |
Jan 2017 – Oct 2018 |
Protocol for solids |
1.822 |
0.400 |
2.623 |
1.021 |
|
PC [OD570] |
Jan 2017 – Dec 2018 |
Protocol for liquids |
0.536 |
0.166 |
0.869 |
0.203 |
Jan 2017 – Oct 2018 |
Protocol for solids |
0.326 |
0.113 |
0.553 |
0.100 |
|
Relative viability [%] |
Jan 2017 – Dec 2018 |
Protocol for liquids |
28.7 |
7.4 |
43.5 |
13.8 |
Jan 2017 – Oct 2018 |
Protocol for solids |
17.9 |
4.9 |
27.7 |
8.0 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (irritating)
Additional information
In Vitro Skin Irritation Turnkey Testing Strategy
The objective was to assess the skin irritation and corrosion potential of the test substance. Using the methods currently available, a single in vitro assay is not sufficient to cover the full range of skin irritating/corrosion potential.
Therefore, two in vitro assays were part of this in vitro skin irritation and corrosion test strategy: The Skin Corrosion Test (SCT) and Skin Irritation Test (SIT). The potential of the test substance to cause dermal corrosion/irritation was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The skin irritation test was performed with three EpiDerm™ tissues, which were incubated with the test substance for 1 hour followed by a 42-hour postincubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion/irritation test:
The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.
Results of the Skin Corrosion Test (SCT):
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 95.5%, and it was 105.4% after an exposure period of 1 hour.
Results of the Skin Irritation Test (SIT):
The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 1 hour with an about 42-hour post-incubation was 98.1%.
Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm™ in vitro skin irritation and corrosion test strategy under the test conditions chosen.
In Vitro Eye Irritation Turnkey Testing Strategy
The objective was to assess the eye irritating potential of 550Acid. By using the methods currently available a single in vitro assay is not sufficient to cover the full range of eye irritating potential. Therefore, two in vitro assays were part of this in vitro eye irritation test strategy: The Bovine Corneal Opacity and Permeability Test (BCOP Test) and EpiOcular Eye Irritation Test.
BCOP
The potential of the test substance to cause ocular irritation or serious damage to the eyes was assessed by a single topical application of 750 μL 20% test-substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test-substance preparation for an exposure period of 4 hours. In addition to the test substance, a negative control (NC; deionized water) and a positive control (PC; 20% imidazole in deionized water) were applied to three corneas each. Corneal opacity was quantitatively measured as the amount of light transmitted through the cornea. Permeability was quantitatively measured as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance. The following results were obtained in the BCOP Test: Two test runs were performed. The results obtained in the 1st test run did not indicate a severe eye irritation potential of the test substance. However, high variability between the individual corneas treated with the negative control (NC) and positive control (PC) were noted. Therefore, the study was repeated to verify the result and to meet the acceptance criteria of the test. The obtained results of the test substance in the 2nd test run of the BCOP test verified the findings of the 1st experiment. All acceptance criteria of the test were fulfilled.
EpiOcular
The potential of the test substance to cause ocular irritation was assessed by a single topical application of ca. 50 μL bulk volume (about 12 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™).
Two EpiOcular™ tissues were incubated with the test substance for 6 hours followed by a 18-hour post-incubation period.
Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The ratio of the values indicates the relative tissue viability. The following results were obtained in the EpiOcular™ eye irritation assay:
The test substance is able to directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. The final relative mean viability of the tissues treated with the test substance was 40%.
Based on the results of the BCOP and EpiOcular Tests and by applying the evaluation criteria, it was concluded that the test substance shows an eye irritation potential in the in vitro eye irritation test strategy under the test conditions chosen.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) 1272/2008. A GLP-compliant turnkey testing strategy including the EpiDerm skin corrosion test (SCT, OECD Guideline 431) and the EpiDerm skin irritation test (SIT, OECD Guideline 439) is available for skin irritation. Both tests indicate that the substance is negative and therefore not considered to be classified for skin irritation.
A GLP-compliant turnkey testing strategy including the BCOP (OECD Guideline 437) and the EpiOcular (OECD Guideline 492) is available for eye irritation. In the BCOP, the IVIS for the treated tissues indicates that the test substance does not cause irreversible damage to the eyes. The result of the EpiOcular demonstrates that the test substance has to be classified for eye irritation. Therefore, it can be concluded that the test item is considered to be classified for eye irritation Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.
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