Registration Dossier
Registration Dossier
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Reaction mass of Chromate(1-), [2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)][methyl [7-hydroxy-8-[[2-hydroxy-5-(methylsulfonyl)phenyl]azo]-1-naphthalenyl]carbamato(2-)]-, sodium and sodium bis[2-(3-chlorophenyl)-2,4-dihydro-4-[[2-hydroxy-5-mesylphenyl]azo]-5-methyl-3H-pyrazol-3-onato(2-)]chromate(1-) and sodium bis[methyl [7-hydroxy-8-[[2-hydroxy-5-mesylphenyl]azo]-1-naphthyl]carbamato(2-)]chromate(1-)
EC number: 915-704-1 | CAS number: -
Endpoint summary
Administrative data
Description of key information
Skin:
In an in vitro GLP-skin corrosion study according to OECD Guideline 431, the test substance showed no skin corrosive properties in an reconstructed human epidermis model (BASF Colors&Effects, 2017).
In an in vitro GLP-skin irritation study according to OECD Guideline 439, the test substance showed no skin irritating properties in an reconstructed human epidermis (RhE) model (BASF Colors&Effects, 2017).
Eye:
In an ex vivo GLP-eye irritation study according to OECD Guideline 437, an IVIS score of 14.6 was determined in the BCOP-model (BASF Colors&Effects, 2017).
In an in vitro GLP-eye irritation study according to OECD Guideline 492, no cell viability could be determined in an reconstructed human corneal epidermis (RhCE) model (BASF Colors&Effects, 2017).
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:
- 2016-10-06 to 2017-08-15
- 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:
- 2015-07-28
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No. of test material: 001-140902
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
OTHER SPECIFICS: Solid / brown, pH ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study) - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- Demanded by the Guideline.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM model, EPI-200, MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
- Tissue lot number: 23362
- Date of initiation of testing: 2016-10-11
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature, 1 min or 37 °C, 1 h
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.
- Observable damage in the tissue due to washing: No
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1 %) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
NUMBER OF REPLICATE TISSUES: 2
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- No of replicates: 2
- Method of calculation used: see "Any other information on materials and methods"
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%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount applied: 25 µL deionized water + 25 µL test substance (bulk volume)
NEGATIVE CONTROL
- Amount applied: 50 µL
POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration: 8 N - Duration of treatment / exposure:
- 3 min or 1 h
- Number of replicates:
- 2
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 3 min
- Value:
- 113.3
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1 h
- Value:
- 95.6
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: Mechanical damage of KC tissue No. 2 (1 h incubation) occurred during the washing procedure
- Direct-MTT reduction: Due to the intense color of the test substance, it was not possible to evaluate whether the test substance is able to directly reduce MTT. Therefore, freeze-killed control tissues (KC) were treated with the test article and the negative control in the same way as described above.
- Colour interference with MTT: Due to the color of the test substance, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed as follows: the test substance was applied to a KC tissue, incubated for 1 hour and removed by washing. Thereafter, extraction in isopropanol was performed and the OD570 of the extract was spectrophotometrically determined. Based on the result of the pretest, it was judged that application of color control tissues is not necessary.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes - Interpretation of results:
- GHS criteria not met
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2016-10-06 to 2017-08-15
- 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:
- 2015-07-28
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No. of test material: 001-140902
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
OTHER SPECIFICS: Solid / brown, pH ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study) - Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- Demanded by the Guideline.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM model, EPI-200
- Tissue lot number: 23362
- Date of initiation of testing: 2016-10-11
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 25 min at room temperature, 35 min at 37 °C (Incubator)
- Temperature of post-treatment incubation: 37 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates pre-filled with 0.9 mL fresh medium. When all tissues were rinsed the surface of each tissue was carefully dried with a sterile cotton swab.
- Observable damage in the tissue due to washing: No
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: SunriseTM Absorbance Reader
- Wavelength: 570 nm
- Filter: without reference filter
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 value following exposure to Triton X-100 (1 %) for each EpiDermTM batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD guidelines. Lower acceptance limit: ET50 = 4.0 hours; Upper acceptance limit: ET50 = 8.7 hours
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues
- Procedure used to prepare the killed tissues: freezing
- N. of replicates : 3
- Method of calculation used: Application of measurements using killed control tissues: 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 OD570 KC corrected). Since killed tissue 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 OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the mean relative tissue viability with a test material 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 applied: 25 µL deionized water + 25 µL test substance (bulk volume)
NEGATIVE CONTROL
- Amount applied: 30 µL
POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5 % (w/v) in water - Duration of treatment / exposure:
- 1 h
- Duration of post-treatment incubation (if applicable):
- 42 ± 4 h
- Number of replicates:
- 3
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 111
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Due to the color of the test substance, it could not be determined whether the test substance can directly reduce MTT. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced.
- Colour interference with MTT: Brownish discoloration and/or minimal compound residues were observed on all test-substance treated tissues after the washing procedure. However, this did not interfere with the colorimetric test as was demonstrated in the pretest prior to start of the study.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes - Interpretation of results:
- GHS criteria not met
Referenceopen allclose all
Table 3: Individual and ean OD570 values, individual and mean viability values, standard deviations and coefficient of variation
Exposue Time: 3 min |
Tissue 1 |
Tissue 2 |
Mean |
SD |
CV (%) |
||
NC |
Viable Tissue |
Mean OD570 |
1.971 |
1.899 |
1.935 |
|
|
Viability (% of NC) |
101.8 |
98.2 |
100.0 |
2.6 |
2.6 |
||
KC Tissue |
Mean OD570 |
0.120 |
0.102 |
0.111 |
|
|
|
Viability (% of NC) |
6.2 |
5.3 |
5.7 |
0.7 |
11.4 |
||
Test Substance |
Viable Tissue |
Mean OD570 |
2.141 |
2.273 |
2.207 |
|
|
Viability (% of NC) |
110.6 |
117.5 |
114.0 |
4.8 |
4.2 |
||
KC Tissue * |
Mean OD570 |
0.000 |
0.029 |
0.015 |
|
|
|
Viability (% of NC) |
0.0 |
1.5 |
0.7 |
1.1 |
141.4 |
||
Final mean viability of tissues after KC correction (% of NC) |
113.3 |
|
|
||||
PC |
Viable Tissue |
Mean OD570 |
0.370 |
0.462 |
0.416 |
|
|
Viability (% of NC) |
19.1 |
23.9 |
21.5 |
3.3 |
15.6 |
||
* Negative values are set to zero for further calculation.
Brownish discoloration and minimal compound residues were observed on all test-substance treated tissues after the washing procedure.
Due to the intense color of the test substance, the ability of the test substance to directly reduce MTT could not be determined in a pretest. Therefore, KC tissues were applied in parallel. The results of the KC tissues indicate an increased MTT reduction (mean viability 0.7 % of NC). Thus for the test substance, the final mean viability is given after KC correction.
Table 4: Individual and ean OD570 values, individual and mean viability values, standard deviations and coefficient of variation
Exposue Time: 1 h |
Tissue 1 |
Tissue 2 |
Mean |
SD |
CV (%) |
||
NC |
Viable Tissue |
Mean OD570 |
1.814 |
1.984 |
1.899 |
|
|
Viability (% of NC) |
95.5 |
104.5 |
100.0 |
6.3 |
6.3 |
||
KC Tissue |
Mean OD570 |
0.0889 |
0.089 |
0.089 |
|
|
|
Viability (% of NC) |
4.7 |
4.7 |
4.7 |
0.0 |
0.0 |
||
Test Substance |
Viable Tissue |
Mean OD570 |
1.832 |
1.838 |
1.835 |
|
|
Viability (% of NC) |
96.5 |
96.8 |
96.6 |
0.2 |
0.3 |
||
KC Tissue** |
Mean OD570 |
0.020 |
0.233 |
0.020 |
|
|
|
Viability (% of NC) |
1.1 |
12.3 |
1.1 |
|
|
||
**Final mean viability of tissues after KC correction (% of NC) |
95.6 |
|
|
||||
PC |
Viable Tissue |
Mean OD570 |
0.078 |
0.087 |
0.082 |
|
|
Viability (% of NC) |
4.1 |
4.6 |
4.3 |
0.3 |
7.3 |
||
** Mechanical damage of KC tissue No. 2 occurred during the washing procedure, thus, the result of KC tissue 2 is excluded from calculation of the mean value. Due to the unambiguous results, this deviation did not adversely affect the evaluation of the study.
Brownish discoloration and minimal compound residues were observed on all test-substance treated tissues after the washing procedure.
Due to the intense color of the test substance, the ability of the test substance to directly reduce MTT could not be determined in a pretest. Therefore, KC tissues were applied in parallel. The result of KC tissue 1 indicate an increased MTT reduction (viability 1.1 % of NC). Thus for the test substance, the final mean viability is given after KC correction.
Table 3: Individual and mean OD570 values, individual and mean viability values and standard deviations
Tissue 1 |
Tissue 2 |
Tissue 3 |
Mean |
SD |
|||
NC |
Viable Tissue |
Mean OD570 |
1.973 |
1.838 |
1.654 |
1.821 |
|
Viability (% of NC) |
108.3 |
100.9 |
90.8 |
100.0 |
8.8 |
||
KC Tissue |
Mean OD570 |
0.063 |
0.076 |
0.057 |
0.065 |
|
|
Viability (% of NC) |
3.5 |
4.2 |
3.1 |
3.6 |
0.5 |
||
Test Substance |
Viable Tissue |
Mean OD570 |
2.091 |
2.060 |
1.930 |
2.027 |
|
Viability (% of NC) |
114.8 |
113.1 |
106.0 |
111.3 |
4.7 |
||
KC Tissue * |
Mean OD570 |
0.000 |
0.001 |
0.013 |
0.005 |
|
|
Viability (% of NC) |
0.0 |
0.1 |
0.7 |
0.3 |
0.4 |
||
Final mean viability of tissues after KC correction (% of NC) |
111.0 |
|
|||||
PC |
Viable Tissue |
Mean OD570 |
0.054 |
0.063 |
0.065 |
0.060 |
|
Viability (% of NC) |
3.0 |
3.4 |
3.5 |
3.3 |
0.3 |
||
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:
- 2016-10-12 to 2017-08-15
- 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:
- 2013-07-26
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
- Version / remarks:
- 2010-12-08
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No. of test material: 001-140902
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
OTHER SPECIFICS: Solid / brown, pH ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study) - Species:
- cattle
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Schlachthof Mannheim, 68165 Mannheim, Germany
- Characteristics of donor animals: > 12 months, < 60 months of age - Vehicle:
- water
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount applied: 750 µL
- Concentration: 20 % (w/v) - Duration of treatment / exposure:
- 4 h
- 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. Isolated corneas were mounted in cornea holders that consists of anterior and posterior chambers. Both chambers were filled to excess with pre-warmed Eagle’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
QUALITY CHECK OF THE ISOLATED CORNEAS: After the equilibration period, the medium in both chambers was replaced by fresh pre-warmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that showed macroscopic tissue damage or an opacity value < 550 opacity units were discarded.
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: Yes
POSITIVE CONTROL USED: Yes
APPLICATION DOSE AND EXPOSURE TIME: 750 µL, 4 h
TREATMENT METHOD: open chamber
POST-INCUBATION PERIOD: no
REMOVAL OF TEST SUBSTANCE:
- Number of washing steps after exposure period: The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids). The NC and the PC were then removed from the anterior chamber by using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed by using a syringe the epithelium was rinsed with the open chamber method.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
Table 2: Decision criteria of BCOP
IVIS Prediction
< 1.5 No classification for eye irritation*
1.5 – 4.5 Borderline *, ***
> 4.5; < 45 No prediction can be made for eye irritation, further testing with another suitable method is required *, **
45 - 65 Borderline *, ***
> 65 Ocular corrosive or severe irritant
* According to OECD Guideline 437 (adopted July 2013), this prediction is possible. However, due to limited accuracy of the BCOP test to correctly identify substances that do not require classification for eye irritation or serious eye damage, this test method should not be the first choice to initiate a bottom-up approach. Based on this statement and the experience of the test facility, test substances not leading to the prediction “ocular corrosive or severe irritant” are generally examined in the EpiOcular test as well.
** The test method according to the OECD Guideline 437 revised and adopted in 2013 does not allow for the evaluation of eye irritation, i. e., the result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study are needed.
*** The “borderline“ evaluation (IVIS 3.0 ± 1.5 and 55.0 ± 10.0) was statistically determined by using historic BASF data and takes the test facility specific variance of the test method into account. This evaluation is an amendment to the evaluation provided in OECD Guideline 437. - Irritation parameter:
- in vitro irritation score
- Run / experiment:
- Mean
- Value:
- 14.6
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: yes - Interpretation of results:
- GHS criteria not met
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2016-10-12 to 2017-08-15
- 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:
- 2015-07-28
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No. of test material: 001-140902
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
OTHER SPECIFICS: Solid / brown, pH ca. 6 (undiluted test substance, moistened with de-ionized water; determined in the lab prior to start of the GLP study) - Species:
- human
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method and considerations regarding applicability
The EpiOcularTM 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 (compare Figure 1). The EpiOcularTM tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm diameter) and are commercially available as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
Tissue lot No 23740, Tissue lot No 23747, Tissue lot No 23760; for details, see "any other information on materials and methods". - Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount applied: 50 µL (bulk volume) - Duration of treatment / exposure:
- 6 h
- Duration of post- treatment incubation (in vitro):
- ~ 18 h
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- - Details of the test procedure used
- RhCE tissue construct used, including lot number. EpiOcularTM model (OCL-200); Tissue lot No: 23740 (test run 1), 23747 (test run 2), 23760 (test run 4)
- Doses of test chemical and control substances used: 50 µL (bulk volume) test substance, 50 µL positive or negative control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods:
Preincubation: 16 - 24 h at 37 °C (Incubator)
Test: 6 h at 37 °C (Incubator)
Postincubation: 18 h at 37 °C (Incubator)
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: In order to assess the ability of the test material to directly reduce MTT, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. The test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37 °C for 3 hours. A negative control (deionized water) was tested concurrently. If the MTT solution color or in case of water-insoluble test substances the border to the water-phase turned blue / purple the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case of direct MTT reduction, two freeze-killed control tissues were treated with the test article and the negative control each in the same way as described in the protocol.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan: spectrophotometrically
Data Evaluation
Principle: The OD570 values determined for the various tissues are measures of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material was an irritant.
Calculation of individual and mean optical densities: The OD570 value measured and corrected for each individual tissue was calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way was calculated.
Application of measurements using killed control tissues: 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 OD570 KC-corrected). 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 OD570 KC corrected. The mean OD570 KC-corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.
Application of measurements using color control tissues: The OD570 values measured in the color control tissues (CC) was used to correct the mean OD570 of the tissues treated with the test substance (mean OD570 CC-corrected). The mean net OD570 CC was subtracted from the respective mean OD570 to result in the mean OD570 CC only corrected when interference of the test substance in the colorimetric test is noticed. The mean OD570 CC-corrected represents the formazan production without the absorbance of the colored test substance.
Tissue viability: The quantification of tissue viability is presented as the ratio of the mean OD570 (or mean OD570 KC-/CC-corrected, if applicable) divided by the respective OD570 NC value in percent.
Acceptance Criteria
Barrier function and Quality control (QC): The supplier demonstrates that each batch of the model used meets the defined production release criteria. MatTek determines the ET50 (min) value following exposure to 100 µL of 0.3 % Triton X-100 for each EpiOcular™ EIT (OCL-200) batch. The ET50 must fall within a range established based on a historical database of results. The following acceptability range (upper and lower limit) for the ET50 is established by the supplier as described in the cited OECD Guideline.
Lower acceptance limit: ET50 = 12.2 min
Upper acceptance limit: ET50 = 37.5 min
Acceptance criteria for the NC: The absolute OD570 of the NC tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is >0.8. The mean OD570 of the NC should not exceed 2.5.
Acceptance criteria for the PC: Methyl acetate used as PC usually leads to a tissue viability of approx. 25 %. A viability of < 50 % is acceptable.
Acceptance criteria for tissue variability: Two tissues were treated under the same conditions.A variability between the two tissues is considered to be acceptable if the relative difference of the viability is < 20 %.
Acceptance criteria for the KC: The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT reduction of a test substance should be ≤ 30 % of the NC.
Acceptance criteria for the CC: The OD570 value for the color control of a test substance should be ≤ 30 % of the OD570 of the NC. If the OD570 value for direct MTT reduction or the color control tissues of a test substance is > 30 % of the OD570 of the NC expert judgment must be performed to determine if the test substance must be considered as incompatible with the test. - Irritation parameter:
- other: viability (%)
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes - Interpretation of results:
- study cannot be used for classification
Referenceopen allclose all
Table 4: Opacity score of the test substance, the NC and the PC
Test substance identification |
Cornea-No. |
Initial opacity |
Final opacity |
Opacity Change |
Corrected Opacity Change |
Mean |
SD |
Test substance |
7 8 9 |
5.2 6.3 4.5 |
24.6 28.0 25.2 |
19.4 21.7 20.7 |
13.2 15.5 14.5 |
14.4 |
1.1 |
NC |
1 2 3 |
6.8 5.2 5.0 |
8.7 14.8 12.3 |
1.8 9.6 7.3 |
NA NA NA |
6.2 |
4.0 |
PC |
4 5 6 |
4.6 5.2 3.7 |
100.9 99.4 100.9 |
96.3 94.1 97.2 |
90.1 87.9 91.0 |
89.6 |
1.6 |
Table 5: Permeability score of the test substance, the NC and the PC
Test substance identification |
Cornea-No. |
Mean OD490 |
Dilution Factor |
Mean Corrected OD490 |
Mean |
SD |
Test substance |
7 8 9 |
0.032 0.016 0.013 |
1 1 1 |
0.029 0.014 0.010 |
0.018 |
0.010 |
NC |
1 2 3 |
0.002 0.002 0.004 |
1 1 1 |
NA NA NA |
0.003 |
0.002 |
PC |
4 5 6 |
0.544 0.454 0.494 |
5 5 5 |
2.719 2.266 2.467 |
2.484 |
0.227 |
Table 6: In Vitro Irritancy score (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 |
13.2 15.5 14.5 |
0.029 0.014 0.010 |
13.6 15.7 14.6 |
14.6 |
1.0 |
NC |
1 2 3 |
1.8 9.6 7.3 |
0.002 0.002 0.004 |
1.9 9.6 7.3 |
6.3 |
4.0 |
PC |
4 5 6 |
90.1 87.9 91.0 |
2.719 2.266 2.467 |
130.8 121.9 128.0 |
126.9 |
4.6 |
Due to the intense color of the test substance, it could not be determined whether the test substance can reduce MTT directly. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. In a pretest, it was demonstrated that the color of the test substance interferes with the colorimetric test.Therefore, color controls (viable tissues CC) and freeze-killed control tissues (KC CC) were performed to differentiate formazan produced by the cells in the MTT test from color residues of the test substance in the colorimetric test. In all test runs of the main study compound residues remained on all test-substance treated tissues after the washing procedure and these tissues were brown discolored.
The values of the color control (CC) tissues indicated considerable interference due to the color of the test substance. Thus, for the test substance the final mean viability is given after CC correction. However, no comparable results for CC correction could be obtained within three test runs. In addition, the values of the KC and KC CC tissues were partly massively increased, because in this case the killed tissues absorb and bind the colored test substance in a higher amount as viable tissues.
Finally, the assessment of several test runs of the EpiOcular™ eye irritation assay showed that the intense dye interfered with the measurements and influenced the determination of the viability values and thus no valid results could be derived for the EpiOcular™ eye irritation assay.
Table 6: 1st test run: 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 |
2.429 |
2.158 |
2.293 |
|
viability |
105.9 |
94.1 |
100.0 |
11.8 |
||
KC tissues |
mean OD570 |
0.030 |
0.030 |
0.030 |
|
|
viability |
1.3 |
1.3 |
1.3 |
0.0 |
||
16/0083-1 |
viable tissues |
mean OD570 |
1.330 |
2.621 |
1.975 |
|
viability |
58.0 |
114.3 |
86.1 |
56.3 |
||
CC tissues |
mean OD570 |
0.536 |
0.328 |
0.432 |
|
|
viability |
23.4 |
14.3 |
18.8 |
|
||
Final mean viability of tissues after CC correction [% of NC]: |
67.3 |
|
||||
KC tissues |
mean OD570 KC NC corrected |
0.954 |
0.782 |
0.868 |
|
|
viability |
41.6 |
34.1 |
37.8 |
7.5 |
||
KC CC tissues |
mean OD570 |
4.405 |
1.688 |
3.046 |
|
|
viability |
192.1 |
73.6 |
73.6** |
|
||
*Mean viability of KC-tissues after KC CC correction [% of NC]: |
0.0 |
|
||||
Final mean viability of tissues after KC and CC correction |
67.3 |
|
||||
PC |
viable tissues |
mean OD570 |
0.375 |
0.473 |
0.424 |
|
viability |
16.3 |
20.6 |
18.5 |
4.3 |
||
* Negative values are set to zero for further calculation
** Mechanical damage of KC CC tissue 1 occurred during the washing period; thus, the result of KC CC tissue 1 is excluded from calculation of the mean value
Table 7: 2nd test run: 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 |
1.506 |
1.469 |
1.487 |
|
viability |
101.2 |
98.8 |
100.0 |
2.5 |
||
KC tissues |
mean OD570 |
0.033 |
0.025 |
0.029 |
|
|
viability |
2.2 |
1.7 |
2.0 |
0.5 |
||
16/0083-1 |
viable tissues |
mean OD570 |
1.083 |
1.193 |
1.138 |
|
viability |
72.8 |
80.2 |
76.5 |
7.4 |
||
CC tissues |
mean OD570 |
0.685 |
0.624 |
0.654 |
|
|
viability |
46.1 |
41.9 |
44.0 |
4.1 |
||
Mean viability of tissues after CC correction [% of NC]: |
32.5 |
|
||||
KC tissues |
mean OD570 KC NC corrected |
0.721 |
0.850 |
0.786 |
|
|
viability |
48.5 |
57.2 |
52.8 |
8.7 |
||
KC CC tissues |
mean OD570 |
2.661 |
0.708 |
1.684 |
|
|
viability |
178.9 |
47.6 |
47.6** |
|
||
Mean viability of KC-tissues after CC correction [% of NC]: |
5.2 |
|
||||
Final mean viability of tissues after KC and CC correction |
27.3 |
|
||||
PC |
viable tissues |
mean OD570 |
0.152 |
0.209 |
0.181 |
|
viability |
10.2 |
14.1 |
12.2 |
3.8 |
||
** mechanical damage of KC CC tissue 1 occurred during extraction period; thus, the result of KC CC tissue 1 is excluded from calculation of the mean value
Table 8: 4th test run: 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 |
1.490 |
1.523 |
1.506 |
|
viability |
98.9 |
101.1 |
100.0 |
2.2 |
||
KC tissues |
mean OD570 |
0.026 |
0.029 |
0.027 |
|
|
viability |
1.7 |
1.9 |
1.8 |
0.2 |
||
16/0083-1 |
viable tissues |
mean OD570 |
0.582 |
0.265 |
0.424 |
|
viability |
38.6 |
17.6 |
28.1 |
21.0 |
||
CC tissues |
mean OD570 |
1.146 |
1.602 |
1.374 |
|
|
viability |
76.1 |
106.3 |
91.2 |
30.2 |
||
*Mean viability of tissues after CC correction [% of NC]: |
0.0 |
|
||||
KC tissues |
mean OD570 KC NC corrected |
1.228 |
2.540 |
1.884 |
|
|
viability |
81.5 |
168.6 |
125.1 |
87.1 |
||
KC CC tissues |
mean OD570 |
2.465 |
2.469 |
2.467 |
|
|
viability |
163.6 |
163.9 |
163.8 |
0.3 |
||
*Mean viability of KC-tissues after CC correction [% of NC]: |
0.0 |
|
||||
*Final mean viability of tissues after KC and CC correction |
0.0 |
|
||||
PC |
viable tissues |
mean OD570 |
0.345 |
0.222 |
0.283 |
|
viability |
22.9 |
14.7 |
18.8 |
8.2 |
||
* Negative values are set to zero for further calculation
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin:
The objective was to assess the skin irritation and corrosion potential of the test substance. By using the currently available methods 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) (BASF Colors&Effects, 2017).
The SCT was conducted according to OECD Guideline 431, the SIT was conducted according to OECD Guideline 439. Both tests followed the GLP. 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 13 mg) of the undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™). For the corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each. The irritation test was performed with three EpiDerm™ tissues which were incubated with the test substance for 1 hour followed by a 42-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 test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. Positive and negative controls were valid in both assays.
Results of the Corrosion Test (SCT): The final mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was ca. 113 % and it was ca. 96 % after an exposure period of 1 hour.
Based on the decision criteria for evaluation of results of corrosion test (see below), the test substance does not show skin corrosive properties.
Mean tissue viability |
Prediction |
3 min: < 45 |
Corrosive |
3 min: 45 - 55 |
Borderline |
3 min: > 55 and1 hour: 10 - 20 |
Borderline |
3 min: > 55 and1 hour: < 10 |
Corrosive |
3 min: > 55 and1 hour: > 20 |
Non-corrosive |
Results of the Irritation Test (SIT): The final 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 ca. 111 %. Based on the decision criteria for evaluation of results of the irritation test (see below), the test substance does not show skin irritating properties.
Mean tissue viability |
Prediction |
<45 |
Irritant |
45- 55 |
Borderline |
> 55 |
Non-irritant |
Conclusion
Based on the results of the skin corrosion and skin irritation study, the test substance is considered to be not skin irritating.
Eye:
The objective was to determine a possible eye irritating potential of the test substance by in vitro studies. A single in vitro assay may not always be sufficient, using the currently available methods, to cover the full range of eye irritating potential. Consequently, two in vitro assays were performed: The Bovine Corneal Opacity and Permeability Test (BCOP Test) according to OECD guideline 437 and the EpiOcular Eye Irritation Test according to OECD guideline 492.
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 of a 20 % test-substance preparation in deionized water to the epithelial surface of isolated bovine corneas (BASF Colors&Effects, 2017). Three corneas were treated with the test-substance for an exposure period of 4 h. 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 measured quantitatively as the amount of light transmitted through the cornea. Permeability was measured quantitatively 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 (IVIS) of the test substance. The following results were obtained in the BCOP Test:
|
Mean Opacity Value |
Mean Permeability Value |
Mean In Vitro Irritany Score |
Test Substance |
14.4 |
0.018 |
14.6 |
NC |
6.2 |
0.003 |
6.3 |
PC |
89.6 |
2.484 |
126.9 |
Minimal discoloration (brown) of the test-substance treated corneas was observed after the washing procedure.
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 26 mg) undiluted test substance to a reconstructed three-dimensional, human cornea model (EpiOcular™). Four test runs of the EpiOcular™ eye irritation assay were performed. Two EpiOcular™ tissues per test run were incubated with the test substance for 6 hours followed by an18-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:
Due to the intense color of the test substance, it could not be determined whether the test substance can reduce MTT directly. Therefore, an additional MTT reduction control (freeze-killed control tissues (KC)) was introduced. In a pretest, it was demonstrated that the color of the test substance interferes with the colorimetric test.Therefore, color controls (viable tissues CC) and freeze-killed control tissues (KC CC) were performed to differentiate formazan produced by the cells in the MTT test from color residues of the test substance in the colorimetric test. In all test runs of the main study compound residues remained on all test-substance treated tissues after the washing procedure and these tissues were brown discolored. The values of the color control (CC) tissues indicated considerable interference due to the color of the test substance. Thus, for the test substance the final mean viability is given after CC correction. However, no comparable results for CC correction could be obtained within three test runs. In addition, the values of the KC and KC CC tissues were partly massively increased, because in this case the killed tissues absorb and bind the colored test substance in a higher amount as viable tissues. Finally, the assessment of several test runs of the EpiOcular™ eye irritation assay showed that the intense dye interfered with the measurements and influenced the determination of the viability values and thus no valid results could be derived for the EpiOcular™ eye irritation assay.
Conclusion
The result of the BCOP Test was negative and the test substance did not cause ocular corrosion or severe irritation. However, the result the BCOP Test alone is not sufficient for the evaluation of eye irritation. On basis of the result of this study, a potential of the test substance to bear a risk of eye irritation cannot be excluded. Thus, a combined assessment of the eye irritating potential is not possible, because no unambiguous result could be derived for the EpiOcular™ eye irritation assay.
In summary, it must be concluded that the results of the test substance should be considered as “inconclusive” in the in vitro eye irritation test strategy under the test conditions chosen for final assignment of a risk phrase at present.
Justification for classification or non-classification
Skin:
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.
No skin irritating properties were determined. As a result the substance should not be considered for skin irritation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.
Eye:
The available experimental test data derived from the BCOP assay are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. No corrosive properties were determined. The EpiOcular however yielded inconclusive results due to intensive, permanent staining of the tissues and interferences of the test compound with the viability measurements. The results obtained in the EpiOcular are therefore not suitable for classification and are considered as inconclusive.
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