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EC number: 227-492-1 | CAS number: 5858-51-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Boiling point
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- Particle size distribution (Granulometry)
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- Aquatic toxicity
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- Short-term toxicity to fish
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- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
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- Carcinogenicity
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- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- other: read across from similar substance
- Adequacy of study:
- key study
- Study period:
- June to July 2016
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- 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:
- July 2015
- Qualifier:
- according to guideline
- Guideline:
- other: MatTek Corporation Protocol: EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation's Reconstructed Human EpiOcularTM Model; 29 June 2015
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
- EC Number:
- 228-589-1
- EC Name:
- Disodium 7-amino-4-hydroxy-3-[[4-[(4-sulphonatophenyl)azo]phenyl]azo]naphthalene-2-sulphonate
- Cas Number:
- 6300-50-1
- Molecular formula:
- C22H17N5O7S2.2Na
- IUPAC Name:
- disodium 7-amino-4-hydroxy-3-({4-[(4-sulfonatophenyl)diazenyl]phenyl}diazenyl)naphthalene-2-sulfonate
Constituent 1
Test animals / tissue source
- Species:
- other: Human Cornea Model
- Details on test animals or tissues and environmental conditions:
- - Justification of the test method and considerations regarding applicability:
1. Assessment of Direct Test Item Reduction by MTT
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a 6-well plate and the mixture was incubated in the dark at 37+-1.5ºC in a humidified atmosphere of 5+-0.5% CO2 in air for three hours. A control (50 µL of dionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Since the MTT solution color did not turn blue/purple, the test item was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.
2. Assessment of Coloured or staining materials
Coloured test items or test items which become coloured after application to the tissues may interfere with the quantitative photometric MTT measurement, if the colorant binds to the tissue and is extracted together with MTT. Therefore, each test item has to be checked for its colorant properties.
For this purpose each approximately 50 mg of the test item was aded to 1.0 ml of water and to 2 ml isopropanol in 6-well plates. the water mixture was incubated in the dark at 37 +-1.5ºC in a humidified atmosphere of 5+-0.5% CO2 in air for at least 1 hour, the isopropanol mixture or for 2 to 3 hours at room temperature.
Since the test item stained water and isopropanol, it had to be considered as possibly interacting with the MTT measurement and an additional test on colorant controls had to be performed.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia) received under sterile conditions.
The certificate of analysis is included in the report.
Test system
- Controls:
- yes
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- The test material was crushed and ground in a mortar with pestle for improving its consistency. Approximately 50 mg of the test item was tested topically on duplicate EpiOcularTM tisses.
- Duration of treatment / exposure:
- 6 hours (37+-1.5ºC, 5+-0.5% CO2, 95% RH)
- Duration of post- treatment incubation (in vitro):
- 18 hours
- Number of animals or in vitro replicates:
- 2
- Details on study design:
- Test system: Lot No.: 23719
1. EpiOcular Kit Components Needed for the Assay
Sealed 24-well plate: Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium: DMEM-Medium
Positive control: Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTI viability assay
6-well plates For storing inserts, or for topically applying test agents
Serum-free assay medium DMEM-based medium
2. MTT-I00 Assay Kit Components
1 vial, 2 mL: MTT concentrate
1 vial, 8 mL: MTI diluent (supplemented DMEM), For diluting MTT concentrate prior to use in the MTT assay
60mL: Extractant solution (Isopropanol), For extraction of formazan crystals
3. MTT -Solution
For the pre-test for "Assessment of Direct Test Item Reduction by MTT" a 1.0 mg/mL MTT solution in DMEM was prepared directly prior to the pre-test.
MTT concentrate and MTT diluent together with the extractant solution was ordered as MTT test kit (MTT-100, MatTek) and was used for the main experiment. A MTT solution was prepared. MTT concentrate was diluted in MTT diluent to produce a 1.0 mg/mL solution.
3.1. Cell culture
EpiOcular™ kits and MTT -100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm2) are cultured on specially prepared cell culture inserts (MILLICELL ®, 10 mm 0).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24- well plate on Tuesday. On day of receipt of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. An appropriate volume of EpiOcular™ Assay Medium was warmed to approximately 3 7 CC. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-Iabeled 6-well plates.
Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper.
The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6- well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37°C and the EpiOcular™ tissues were incubated at standard culture conditions overnight (about 16.5 hours).
4 Experimental Performance
After the overnight incubation, the tissues were pre-wetted with 20 µL ofCa++Mg++free DPBS. The tissues were incubated at standard culture conditions for 30 minutes.
Test item exposure: After the 30 minute Ca ++Mg++free-DPBS pre-treatment, the test and control item was tested by applying approximately 50 mg (test item) or 50 µL (controls) topically on the EpiOcular™ tissues. The tissues were incubated at standard culture conditions (37 ± 1.5 QC, 5 ± 0.5% C02, 95% RH) for 6 hours.
At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues with Ca++Mg++-free DPBS (brought to room temperature).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously-warmed assay medium (room temperature) in a pre-Iabeled 12-well plate for a 25 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium was intended to remove any test item or control absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material and transferred to the appropriate well of the pre-Iabeled 6-well plate containing 1 mL of warm assay medium. The tissues were incubated for 18 hours at standard culture conditions (posttreatment incubation).
5 MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 ml of MTT solution. Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions and rinsed 3 times with DPBS afterwards.
Inserts were removed from the 24-well plate after 180 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-Iabelled 6-well plate containing 2 ml isopropanol in each well so that no isopropanol was flowing into the insert. The corresponding negative and positive controls had to be treated identically without piercing. For this procedure it was necessary to seal the plates particularly thorough since a higher evaporation rate had to be expected due to the larger surface of wells in 6-well plates.
Extraction was performed for 20 hours in the refrigerator.
The extract solution was mixed and two 200 ilL aliquots were transferred to the appropriate wells of a pre-Iabeled 96-well plate(s).
The absorbance at 570 nm (OD570) of each well was measured with a plate reader reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro, version 4.7.1). No reference wavelength measurement was used.
6 Colorant Controls for Assessment of Colored or Staining Test Items
Since the test item showed to have or to develop relevant colour, which could interact with the MTT measurement, an additional test had to be performed to determine the amount of colour bound to and extracted from the tissues. For this purpose the coloured test item was applied to two additional tissues (= colorant controls (CC», and were treated in the same way as described in 4 and 5. In contrast to the normal viability test, no MTT assay was performed. The bound colour was extracted and the absorbance of the isopropanol extracts was measured identically as in the MTT assay for coloured test items (according to 4, as described for the MTT assay with 2 ml extraction solution in 6-well plates without piercing the tissue, and starting with a 180 min incubation in medium instead of MTT solution addition). The amount of extracted colour was subtracted from the results of the viability assay.
7 Data evaluation
1) The mean OD value of the blank control wells (ODBlk) for each experiment was calculated.
2) The mean value of the two aliquots for each tissue (= corrected test item OD) was calculated.
3) The mean ODBlk from the mean OD value of the same experiment (blank corrected values) was subtracted.
4) The mean value of the two relating tissues for each control and test item (= corrected mean OD) was calculated. For further calculations only the corrected mean negative control OD value was needed.
5) The corrected OD value of the negative control corresponds to 100% viability.
Corrected negative control OD = Negative Control OD - ODBlk = 100% Viability
7.1 Calculations for Viability Tests only
1) The percent viability of each ofthe two relating tissues for each control and test item relative to the negative control (100% control) was calculated:
Viability [%] = 100 * ( corrected test item OD / corrected mean negative control OD)
2) The difference of the viability between duplicate tissues was calculated. If the difference was >20% the test is considered as non-qualified.
3) The mean test item viability (TI viability) was calculated and the test item is classified according to the prediction model.
7.2. Calculation for Viability plus Colorant Control Tests
1) The OD values of the colorant control test (blank corrected) were calculated as described at the beginning of chapter 7.
2) The colorant control OD for the two relating tissues was transferred to a percentage value relative to the viability scale (colorant control "viability") by comparison to the corrected viability negative control of the same experiment.
Colorant control viability [%] = 100 * (OD colorant control / corrected mean negative control OD)
3) The difference of the viability of the two tissues was calculated. If the difference is >20%, the colorant control test is considered as non-qualified.
4) The mean viability for the colorant control was calculated and this mean colorant control "viability" value (CC "viability") was subtracted from the relating mean viability of the same test item (TI viability) to determine the colorant control corrected viability (CC corrected viability).
CC corrected viability = TI viability - CC viability
5) The test item was classified regarding the colorant control corrected viability according to the prediction model.
7.3 Prediction Model
If the test item-treated tissue viability is > 60% relative to the negative control treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ~ 60% relative to negative control treated tissue viability, the test item is labeled irritant.
7.4 Acceptability of the Assay
The results are acceptable according to MatTek Protocol, if:
1) The negative control OD is> 0.8 and < 2.5,
2) The mean relative viability of the positive control is below 50% of the negative control viability.
3) The difference of viability between the two relating tissues of a single test item is < 20% in the same run (for positive and negative control tissues and tissues of test items). This applies also to the killed controls (items and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.
Results and discussion
In vitro
Results
- Irritation parameter:
- in vitro irritation score
- Remarks:
- The indicated value is the mean relative absorbance value, corresponding to the cell viability (%)
- Run / experiment:
- test item
- Value:
- 89.9
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- The optical pre-experiment (colour interference pre-experiment) to investigate the test item's colour change potential in water or isopropanol did lead to a change in colour. Therefore, an additional test with viable tissues without MTT addition was necessary.
Optical evaluation of the MTT-reducing capacity of the test item with MTT-reagent did not show blue colour. Therefore, an additional test with freeze-killed tissues was not necessary.
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 89.9% (threshold for irritancy ≤ 60%), consequently the test item was not
irritant to eye.
Concerning acceptance criteria:
• The negative control OD is > 0.8 and < 2.5 (1.248).
• The mean relative viability of the positive control is below 50% of the negative control viability (42.3%).
• The difference of viability between the two relating tissues of a single item is < 20% (values between 1.3% to 8.2%) in the same run (for positive and negative control tissues and tissues of single test items). This applied also to the colorant controls which were calculated as percent values related to the viability of the relating negative control.
Any other information on results incl. tables
Table: Results after treatment for 6 hours with the tested substance and the controls
Dose group |
absorbance well 1 (tissue 1/2) |
absorbance well 2 (tissue 1/2) |
mean absorbance (tissue 1/2) |
mean absorbance * |
mean absorbance of 2 tissues * |
rel. absorbance [%] tissue 1 and 2** |
absolute value of the difference of the Rel. absorbances [%] tissue 1 and 2 |
mean rel. absorbance [% of negative control]** |
corrected viability |
|
negative control |
1.267 1.297 |
1.282 1.284 |
1.275 1.290 |
1.240 1.256 |
1.248 |
99.4 100.6 |
1.3 |
100.0 |
|
|
positive control |
0.531 0.594 |
0.532 0.595 |
0.531 0.594 |
0.497 0.560 |
0.528 |
39.8 44.9 |
5.0 |
42.3 |
|
|
test item | 1.128 1.161 |
1.170 1.179 |
1.149 1.170 |
1.114 1.135 |
1.125 | 89.3 91.0 |
1.7 | 90.1 | 89.9 | |
negative control viable tissue | 0.039 0.039 |
0.038 0.038 |
0.038 0.038 |
0.004 0.004 |
0.004 | 95.9 104.1 |
8.2 | 100.0 | ||
test item viable tissue | 0.037 0.038 |
0.038 0.037 |
0.037 0.037 |
0.003 0.003 |
0.003 | 71.2 75.3 |
4.1 | 73.3 |
* Mean of two replicate wells after blank correction
** Relative absorbance [rounded values]: 100 x (absorbance test item/positive control) / (absorbance negative control)
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, it can be stated that in this study and under the experimental conditions reported, the test substance does not possess any eye irritating potential.
- Executive summary:
This in vitro study was performed to assess the eye irritation potential of the substance by means of the Human Cornea Model Test.
Additional tests with viable tissues were performed, since the test item dyed water or isopropanol. Additional tests with freeze killed tissues were not necessary, since the test item did not prove to be a MTT reducer.
About 50 mg of the test item and each 50 µL of he controls, respectively, were applied to each of duplicate EpiOcular TM tissue for 6 hours.
Treatment with the positive control induced a decrease in the mean relative absorbance compared with the negative control to 42.3%, thus the validity of the test system is ensured.
The acceptance criteria were met.
Since the viability value of the test item exposed tissues did not decrease below 60%, the test item is not considered to possess an eye irritating potential.
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