Registration Dossier

Administrative data

Description of key information

Skin irritation:

The dermal irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 105.2 %.  Thus, Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was considered to be not irritating to the human skin.

Eye irritation:

The ocular irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3)  was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance Tris[5-amino -4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 79.2%. Thus, the substance Tris[5-amino-4-hydroxy -3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) is considered to be "not irritating" to the human eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 05, 2017 to July 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test articles to be dermal irritants. The dermal irritation potential of test article Tris[5- amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) may be predicted by measurement of their cytotoxic effect, as reflected in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiDerm™ model (MatTek Corp., Ashland, MA).
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Test material identity: Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (D & C Red 33, Al Lake/Lavanya Lilliput)- Source: Neelikon Food Dyes & Chemicals Ltd.- Lot No.of test material: FG/16-17/1055- Date of manufacture: 16/07/2016- Expiration date of the lot/batch: January 2020- Purity: 19.7%RADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A- Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature - Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test articles is tested as provided (neat).- Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data availableFORM AS APPLIED IN THE TEST: SolidOTHER SPECIFICS: No data available
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EpiDerm™ 3-dimensional human tissues used in this study
Source strain:
other: Not applicable
Details on animal used as source of test system:
- Description of the cell system used:The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.- Test System IdentificationAll of the EpiDerm™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues are included in this report. Tissue plates were appropriately labeled with study information.
Justification for test system used:
The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD). Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
The tissues were exposed to the test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium neat (undiluted) on June 28, 2017 (Run 1 of 1). EpiDerm™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately 1 hour, with 35 minutes in an approximately 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature. Following the exposure time, the tissues were rinsed and placed in fresh media for approximately 24 hours. The media was then changed again and the tissues were incubated in fresh media for another ~18 hours for a total of approximately 42 hour post-exposure recovery period. The tissue viability was then assessed by MTT assay. The tissue CoA was used instead of verification of barrier properties of the tissue.MTT and Color Pre-testsPretesting for MTT auto-reduction and coloring was not performed for this study but was based on the results obtained from another study (CYP1690_R1b).MTT AssayFollowing the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours, 57 minute and 25 second MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was approximately 2 hours 04 minutes and 11 seconds with gentle shaking. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability is calculated for each tissue as % of the mean negative control tissues.Evaluation of Test Article in the Cell Models:1. Cell system: Upon receipt, the MatTek EpiDerm™ tissue cultures were placed in 0.9 mL of fresh Maintenance medium (in a 6-well plate). The culture inserts are incubated for ~one hour. The tissues were then transferred to 6-well plates containing 0.9 mL fresh Maintenance medium and they were incubated overnight at ~37°C, 5% CO2 in a humidified incubator.2. Control and Test Article Exposures: On the day of dosing, the tissues are then removed from the incubator and the controls and the test articles are applied topically to tissues by pipette. Tissues were exposed to controls and the test articles for one hour, with ~35 minutes in a 37°C, 5% CO2 humidified incubator and the remaining 25 minutes at room temperature.a) Controls30 µL of negative control DPBS, positive control 5% SDS was applied topically to the tissue and gently spread by placing a nylon mesh on the apical surface of each tissue, if necessary.b)Test ArticleFor Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium, the tissues were moistened with 25 μL of ultrapure water to improve contact of the tissue surface with the test article. Approximately 25 mg of Tris[5-amino-4-hydroxy-3-(phenylazo)naphtha lene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was evenly applied to the apical surface of each tissue (n=3). All the tissues were placed into the ~37°C incubator with 5% CO2. The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature. 3.Post-exposure treatmentAfter the 1 hour exposure, the tissues were rinsed 20 to 25 times with 1 mL of DPBS. The apical surface was gently blotted with a cotton swab. The tissues were placed in 0.9 mL of fresh Maintenance medium (6-well plate) for either 25 hours, 38 minutes and 23 seconds or for 24 hours, 10 minutes and 09 seconds (as there were numerous tissues, they had to be broken down into 2 sets to complete dosing in a timely manner). After this initial ~24 hour incubation, the tissues were placed in 6-well plates containing 0.9 mL fresh Maintenance medium and incubated for another 17 hours, 03 minutes and 34 seconds prior to performing the MTT assay, for a total of an approximately 42 hour post-exposure incubation.RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: The EpiDerm™ 3 dimensional human tissue model- Tissue Lot number(s): 26459- Date of initiation of testing: 6/08/2017TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37°C- Temperature of post-treatment incubation (if applicable): 37°CREMOVAL OF TEST MATERIAL AND CONTROLS-Volume and number of washing steps: TwiceMTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE- MTT concentration: 300 µL MTT medium (1.0 mg/mL).- Incubation time: After 2 hours, 57 minute and 25 second MTT incubation- Spectrophotometer: Synergy H4 spectrophotometer - Wavelength: 570 nm- Filter: No data- Filter bandwidth: No data- Linear OD range of spectrophotometer: No dataNUMBER OF REPLICATE TISSUES: 3CALCULATIONS and STATISTICAL METHODSAll data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows: MTT AssayBlanks:·        The optical density (OD) mean from all replicates for each plate (ODblank). Negative Controls (NC):·        The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·        The OD mean per NC tissue was calculated. ·        The mean OD for all tissues corresponds to 100% viability. ·        The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Positive Control (PC):·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Tested compound :·        Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·        The OD mean per tissue was calculated. ·        The viability per tissue was calculated: %TT = [ODTT/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean TT = Σ %TT / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. Data Correction Procedure for MTT Interfering Compounds (if applicable)True viability = Viability of treated tissue – Interference from test article = ODtvt– ODktwhere ODkt= (mean ODtkt– mean ODukt).ODtvt= optical density of treated viable tissueODkt= optical density of killed tissuesODtkt= optical density of treated killed tissueODukt= optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored Compounds (if applicable)True viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt– ODvt.ODtvt= optical density of treated viable tissue incubated in MTT mediaODvt= optical density of viable tissues incubated in media alone- Evaluation of data The results of the assay was evaluated and compared to negative control.  Table: Criteria for in vitro Interpretation: In VitroResults In VivoPredictionMean tissue viability ≤50% Irritant (I), R38Mean tissue viability >50% Non-irritant (NI)- Assay quality controls- Negative Controls (NC)The Dulbecco’s phosphate buffered saline (DPBS) was used as a NC. The assay passed all acceptance criteria if the ODs of the negative control exposed tissues were between ≥0.8 and ≤2.8.  - Positive Controls (PC)5% solution of sodium dodecyl sulfate was used as a PC. The assay is meeting the acceptance criteria if the viability of the PC is ≤20% of the negative control.   - Standard Deviation (SD)The standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was ≤18.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied (volume or weight with unit): 25 mg- Concentration (if solution): neat (undiluted)VEHICLE (Not used)- Amount(s) applied (volume or weight with unit): none- Concentration (if solution): none- Lot/batch no. (if required): none- Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 30 µL- Concentration (if solution): 5% solution of sodium dodecyl sulfate
Duration of treatment / exposure:
The exposure times were approximately 1 hour, with ~35 minutes exposure in the incubator and ~25 minutes at room temperature.
Duration of post-treatment incubation (if applicable):
For a total of an approximately 42 hour post-exposure incubation.
Number of replicates:
3 tissues will be used per test compound and control.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
105.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 7.3 passing the acceptance criteria. The Standard Deviation, initially caused the test article to not pass acceptance criteria (reported as 32.2 which is >18%) but after removing a single outlier, it has passed the acceptance criteria.
Interpretation of results:
other: not irritating
Conclusions:
The dermal irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 439 test guideline followed for this study. The Mean % tissue viability compared to negative control (n=3) of the test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 105.2 %. Thus, Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was considered to be not irritating to the human skin.
Executive summary:

The dermal irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 439 test guideline followed for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article. Tissues were exposed to test articls and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 7.3 passing the acceptance criteria.

 

The Mean % tissue viability compared to negative control (n=3) of the test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 105.2 %.

 

Hence, under the experimental test conditions it was concluded that test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 05, 2017 to July 12, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to
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)
Principles of method if other than guideline:
The purpose of this study was to assess potential for the test article to be ocular irritants. The ocular irritation potential of a test article Tris[5-am ino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) may be predicted by measurement of its cytotoxic effect, as reflected in the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in the MatTek EpiOcular™ model (MatTek Corp., Ashland, MA).
GLP compliance:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL- Test material identity: Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (D & C Red 33, Al Lake/Lavanya Lilliput)- Source: Neelikon Food Dyes & Chemicals Ltd.- Lot No.of test material: FG/16-17/1055- Date of manufacture: 16/07/2016- Expiration date of the lot/batch: January 2020- Purity: 19.7%RADIOLABELLING INFORMATION (Not applicable)- Radiochemical purity: N/A- Specific activity: N/A - Locations of the label: N/A- Expiration date of radiochemical substance: N/ASTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Room temperature- Stability under test conditions: No data available- Solubility and stability of the test substance in the solvent/vehicle: No data available- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No data availableTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: The test article tested as provided neat (undiluted).- Preliminary purification step (if any): No data available- Final dilution of a dissolved solid, stock liquid or gel: No data available- Final preparation of a solid: No data available FORM AS APPLIED IN THE TEST: SolidOTHER SPECIFICS: No data available
Species:
human
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
- Description of the cell system usedThe normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native corneal mucosa. Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness.- Test System IdentificationAll of the EpiOcular™ 3-dimensional human tissues used in this study were identified by the date of arrival and the lot number. Certificate of Analysis for the tissues is included in this report. Tissue plates were appropriately labeled with study information. Bias was not a factor in this test system.- Justification of the test method and considerations regarding applicabilityHuman Corneal Epithelia (HCE) by MatTek, Inc.:The test article and controls were evaluated for potential ocular irritancy using the EpiOcular™ 3 dimensional human tissue model purchased from MatTek Corporation (Ashland, MA). This model consists of normal human keratinocytes cultured on a permeable synthetic membrane at the air-liquid interface in a chemically defined medium. The cells form a stratified, squamous corneal epithelium resembling the corneal mucosa of the human eye. This model has been used with several common tests of cytotoxicity including MTT and interleukin 1-alpha (IL-1α). A growing body of evidence indicates that EpiOcular™ effectively provides a non-animal means to assess potential irritancy. The EpiOcular™ model closely mimics the human corneal mucosa and thus provides an important in vitro approach in the evaluation of ocular irritancy and toxicity. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).
Vehicle:
unchanged (no vehicle)
Controls:
other: See ''Remark" for Control Samples used in the study
Amount / concentration applied:
TEST MATERIAL - Amount(s) applied (volume or weight with unit): 50 mg - Concentration (if solution): neat (undiluted) VEHICLE (no vehicle) - Amount(s) applied (volume or weight with unit): none - Concentration (if solution): none - Lot/batch no. (if required): none - Purity: noneNEGATIVE CONTROL- Amount(s) applied (volume or weight): 50 µL- Concentration (if solution): neatPOSITIVE CONTROL- Amount(s) applied (volume or weight): 50 µL- Concentration (if solution): neat
Duration of treatment / exposure:
Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~18 hours for test article and controls.
Number of animals or in vitro replicates:
3 tissues were used for test compound and control.
Details on study design:
- Details of the test procedure usedThe tissues were exposed to the test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium neat (undiluted). EpiOcular™ tissues were purchased from MatTek. Quality control of the tissues was performed by MatTek and the Certificate of Analysis (CoA) for the tissues is provided and is kept in the study binder. Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator. After the exposure, the test article was rinsed off the tissues and the tissues were soaked in media for ~25 minutes for test article and controls. Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~18 hours for solid test articles and controls. Tissue viability was assessed by 3-(4,5-dimethylthiazol -2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. - MTT Auto reduction and colouring assessment- MTT Pre-test~50 mg of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was added to 1 mL of MTT media (~1 mg/mL) and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 55 minutes and 02 seconds. 50 µL of ultrapure water was used as a negative control. - Test Article Color TestApproximately 50 mg of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was added to 1.0 mL of ultrapure water and 2.0 mL isopropanol and incubated in a humidified incubator at approximately 37°C and approximately 5% CO2 for 2 hours, 04 minutes and 35 seconds. Samples were then added to the wells of a clear 96-well plate and the plate was read on a BioTek Synergy H4 (or equivalent) plate reader set to 570 nm. Test article that tested positive for excessive coloration (OD >0.08) were assessed on living-tissue controls that were incubated in both culture media and MTT media as well (n=3 for both conditions). - MTT Assay After the recovery period, the MTT assay was performed on run 1 tissues by transferring the tissues to 24-well plates containing 300 µL MTT medium (1.0 mg/mL). After 2 hours 55 minutes and 00 seconds (solids) of MTT incubation at approximately 37°C, approximately 5% CO2 in a humidified incubator, the tissues were rinsed twice with DPBS. The blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissues.- Evaluation of Test Article in the cell Models1. Cell System: Upon receipt, the MatTek EpiOcular™ tissue cultures were placed in 1.0 mL of fresh Maintenance medium (in a 6-well plate) for 59 minutes and 00 seconds. The tissues were not incubated overnight. Control and Test Article Exposures:20 µL of calcium and magnesium free DPBS was added to each tissue and the tissues placed back into the incubator for 31 minutes 00 seconds. The controls and the test article will be applied topically to tissues by pipette. Three tissues will be used per test compound and control.a)Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time. b)Test Article: Approximately 50 mg of the test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time. 3. Post exposure treatmentAfter the exposure, the tissues were rinsed 20 to 25 times with ~1 mL of DPBS to remove test material. The apical surface was gently blotted with a cotton swab and cultures were immediately transferred to a 12-well plate containing 5 mL of media per well. Tissues exposed to solid test article (and the respective control) were incubated, submerged in the media for ~25 minutes at room temperature. Tissuses were then transferred to 6-well plates containing 1.0 mL fresh Maintenance medium per well and incubated for a post-exposure recovery period for 17 hours 48 minutes 01 seconds at approximately 37 degC, 5% CO2 in a humidified incubator.- Doses of test chemical and control substances useda)Test Article: Approximately 50 mg of Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) were added to the tissues as a fine powder. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time. b)Controls: 50 µL of negative control sterile ultrapure water, positive control methyl acetate were added to the tissues. The tissues were placed into the ~37°C humidified incubator with 5% CO2 for the approximately 6 hour exposure time. - Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: Tissues were exposed for approximately ~6 hours for test article and controls, at approximately 37°C, 5% CO2 in a humidified incubator.Following the post soak, the tissues were rinsed and incubated at approximately 37°C, 5% CO2 in a humidified incubator for a post-exposure recovery time totaling ~18 hours for test article and controls.- Justification for the use of a different negative control than ultrapure H2O (Not applicable)- Justification for the use of a different positive control than neat methyl acetate (Not applicable)- Number of tissue replicates used per test chemical and controls: 3 tissues will be used per test compound and control.- Description of the method used to quantify MTT formazanThe blue formazan salt was extracted by submerging tissues in 2 mL isopropanol in a 24-well plate. The extraction time was 2 hours 00 minutes with gentle shaking for solid exposed tissues. The optical density of the extracted formazan (200 µL/well of a 96-well plate) was determined using a Synergy H4 spectrophotometer at 570 nm.- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction modelCalculations and Statistical MethodsMTT AssayBlanks: ·  The OD mean from all replicates for each plate (ODblank). Negative Controls (NC): ·  The blank corrected value was calculated: ODNC= ODNCraw– ODblank. ·  The OD mean per NC tissue was calculated. ·  The mean OD for all tissues corresponds to 100% viability. ·  The mean, standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. ODblank= optical density of blank samples (isopropanol alone). ODNCraw= optical density negative control samples. ODNC= optical density of negative control samples after background subtraction. Positive Control (PC): ·        Calculate the blank corrected value: ODPC= ODPCraw– ODblank. ·        The OD mean per PC tissue was calculated. ·        The viability per tissue was calculated: %PC = [ODPC/ mean ODNC] x 100. ·        The mean viability for all tissues was calculated: Mean PC = Σ %PC / number of tissues. ·        The standard deviation (SD), standard error of the mean (SEM) and the percent coefficient of variation (% CV) was calculated. ODPCraw= optical density positive control samples. ODPC= optical density of positive control samples after background subtraction. Tested Articles: ·  Calculate the blank corrected value ODTT= ODTTraw– ODblank. ·  The OD mean per tissue is calculated. ·  The viability per tissue is calculated: %TT = [ODTT/ mean ODNC] x 100. ·  The mean viability for all tissues is calculated: Mean TT = Σ %TT / number of tissues. ·  The standard deviation (SD) and the percent coefficient of variation (% CV)for the controls and the test articles will be calculated. ODTTraw= optical density test article samples. ODPC= optical density of test article samples after background subtraction. Data Correction Procedure for MTT Interfering CompoundsTrue viability = Viability of treated tissue – Interference from test article = ODtvt – ODkt where ODkt = (mean ODtkt – mean ODukt). ODtvt = optical density of treated viable tissue ODkt = optical density of killed tissues ODtkt = optical density of treated killed tissue ODukt = optical density of untreated killed tissue (NC treated tissue) Data Correction Procedure for Colored CompoundsTrue viability = Viability of treated tissue incubated in MTT media – Viability of treated tissue incubated in media without MTT = ODtvt – ODvt. ODtvt = optical density of treated viable tissue incubated in MTT media ODvt = optical density of viable tissues incubated in media alone. Proposed Statistical methods The mean, standard deviation (SD) and the percent coefficient of variation (% CV) for the controls and the test article will be calculated. - Evaluation of data The results of the assay was evaluated and compared to negative control. Table: Irritancy Prediction In VitroResults In VivoPredictionMean tissue viability ≤60% Irritant (I) – Category 1 or 2Mean tissue viability >60% Non-irritant (NI) – No Category- Assay quality controls- Negative Controls (NC)The assay is meeting the acceptance criterion if the mean viability of the NC in terms of Optical Density (OD570) of the NC tissues (treated with sterile ultrapure water) in the MTT assay are >0.8 to <2.5. This is an indicator of tissue viability following shipping and conditions under use.   - Positive Controls (PC)Methyl acetate was used as a PC and tested concurrently with the test article. The assay is meeting the acceptance criteria if the viability of the PC is <50% of the negative control.   - Standard Deviation (SD)Each test of ocular irritancy potential is predicted from the mean viability determined on 3 single tissues. The assay meets the acceptance criteria if SD calculated from individual percent tissue viabilities of the replicates is <18% for three replicate tissues.  
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
79.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 4.2 passing the acceptance criteria.
Interpretation of results:
other: not irritating
Conclusions:
The ocular irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 492 test guideline followed for this study. The mean % tissue viability of test substance Tris[5-amino -4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 79.2%. Thus, the substance Tris[5-amino-4-hydroxy -3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) is considered to be "not irritating" to the human eye.
Executive summary:

The ocular irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to solid test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 4.2 passing the acceptance criteria.

The mean % tissue viability of test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 79.2%.

Hence, under the experimental test conditions it was concluded that test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) is considered to be not irritating to the human eye and being classified as “Not classified’’ as per CLP Regulation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

Various studies have been investigated for assessing the dermal irritation potential of Aluminum, tris(5-amino-4-hydroxy-3-(2-phenyldiazenyl)-2,7-naphthalenedisulfonato(2-))di - (CAS No. 68475-50-3) to a greater or lesser extent. The studies are based on in vitro experiment for target chemical along with its Read across substances for in vivo data

The dermal irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 439 test guideline followed for this study. The MatTek EpiDerm™ model was used to assess the potential dermal irritation of the test article by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the dermal irritation potential of test article. Tissues were exposed to test articls and controls for ~one hour, followed by a 42 hour post-exposure recovery period. The viability of each tissue was determined by MTT assay. 

 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 7.3 passing the acceptance criteria.

 

The Mean % tissue viability compared to negative control (n=3) of the test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 105.2 %.

 

Hence, under the experimental test conditions it was concluded that test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

In other study by (JAMES E. FULTON, JR, 1989) with similar substance (3567-66-6) was observed in rabbit. The test uses a single rabbit ear, to indicate the Comedogenicity and irritancy of group of skin care products that may contain follicular and surface epithelial irritating ingredients. A dose of 1 ml of the test material is applied and spread once daily to the entire inner surface of once for five days per week for two weeks. The opposite untreated ear of each animal served as an untreated control. The irritancy produced by repeated application of a chemical or skin care product on the surface epidermis in the rabbit ear is evaluated on a scale of 0 to 5. The test chemical falls under Grade 2 (diffuse scaling, no erythema) Hence it can be concluded that the test chemical is non-irritating to rabbit ears. Based on the above result the test chemical D&C Red 33 not classified as skin irritant.

 

In other study by (Scientific Committee on Consumer Safety, 2007) with similar substance (3567-66-6) was observed in rabbit.Test substance Acid Red 33 of dose concentration 0.5 g/kg bw of a 0, 0.1 and 1% formulation in white petrolatum or a hydrophilic cream was applied on intact skin of rabbits for 65 applications or on damaged skin for 15 applications. Under the conditions of the study, the test material was considered not be irritant to rabbit skin.

In other study by (EUROPEAN COMMISSION – European Chemicals Bureau, 2000) with similar substance (5460-09-3) was observed in rabbit.The skin irritation study of Monosodium 8-amino-1-naphthol-3,6-disulfonate(CAS No:5460–09–3)was performed in rabbits to determine its irritation potential for observation period of 7 days. The test was applied on 2 rabbits at a concentration of 500 mg/animal on ventral site of one ear and exposed for 24 hours. The test site was covered by a gauze patch and held in place by a tape. Since there was no evidence of any skin reaction, the test material Monosodium 8-amino-1-naphthol-3,6-disulfonate(CAS No:5460–09–3) was found to be non irritant.

 

In other study by (EUROPEAN COMMISSION – European Chemicals Bureau, 2000) with similar substance (5460-09-3) was observed in rabbit.The skin irritation study of Monosodium 8-amino-1-naphthol-3,6-disulfonate (CAS No: 5460–09–3) was performed in rabbits to determine its irritation potential. Since no irritation was noted the test material Monosodium 8-amino-1-naphthol-3,6-disulfonatewas found to be non irritant on rabbits’ skin.

 

In other study by (Commission of the European Communities, 1988) with similar substance (1064-48-8) was observed in rabbit. The skin irritation studyof D+C Black No. 1(CAS No: -1064-48-8)wascarried out inrabbitsto determine its irritation potency.0.5ml of 10% aqueous solution on a patch of test sample was applied on rabbits for 2 hours. No known skin irritation effects were observed. Hence the test chemical D+C Black No. 1 (CAS No: -1064-48-8) was found to be non irritant on rabbits’ skin.

 

In other study by (Scientific Committee on Consumer Safety (SCCS), 2010) with similar substance (1064-48-8) was observed in rabbit. The skin irritation studyof D+C Black No. 1wascarried out in 6 malerabbitsto determine its irritation potency. The rabbits were clipped on the back 24 hours prior to the test. 0.5 ml of 10% aqueous solution/suspension (pH adjusted with ammonia to pH 8-10) of the dye was applied on the skin and covered with occlusive patches, which were secured to the skin for 2 hours exposure period. Observations were made immediately and after 24, 48 and 72 hours of patch removal. None of the 6 animals showed signs of irritation, neither after immediate reading nor after 72 hours. Therefore D+C Black No. 1 (CAS No: -1064-48-8) was found to be non irritant on New Zealand albino rabbits’ skin.

 

On the basis of available information for the target as well as read across substance and applying weight of evidence approach, the test substance can be considered as not irritating to the skin.

Eye Irritation:

Various studies have been investigated for assessing the occular irritation potential of Aluminum, tris(5-amino-4-hydroxy-3-(2-phenyldiazenyl)-2,7-naphthalenedisulfonato(2-))di - (CAS No. 68475-50-3) to a greater or lesser extent. The studies are based on in vitro experiment for target chemical along with its Read across substances for in vivo data.

The ocular irritation potential of test article Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) was determined according to the OECD 492 test guideline followed for this study. The MatTek EpiOcular™ model was used to assess the potential ocular irritation of the test articles by determining the viability of the tissues following exposure to the test article via MTT. The objective of this study was to assess the ocular irritation potential of test article. Tissues were exposed to solid test article and controls for ~6 hours, followed by a ~25 minute post-soak and approximately 18 hour recovery after the post-soak. The viability of each tissue was determined by MTT assay. 

 

The MTT data show the assay quality controls were met, as the OD of the negative control tissues was between 0.981 and 1.284 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be 11.3% of negative control (for 6 hour exposures with solids) in run one and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was 4.2 passing the acceptance criteria.

The mean % tissue viability of test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium was determined to be 79.2%.

Hence, under the experimental test conditions it was concluded that test substance Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminium (CAS No.- 68475-50-3) is considered to be not irritating to the human eye and being classified as “Not classified’’ as per CLP Regulation.

The ocular irritation potential of similar substance (3567-66-6) was assessed (Scientific Committee on Cosmetic Products, adopted by the SCCNFP adopted on18 December 2007) on rabbits.0.2 ml of a 10% aqueous solution (20 mg) or suspension of the test material was applied twice daily, five times weekly, for four weeks to the conjunctival sac of one eye of each of a group of six or more albino rabbits (40 applications). One hour after each application, the eyes were examined for evidence of staining and the irritation was scored according to Draize. The study concluded that Acid Red 33 was not irritating to the rabbit eye.

Reports from Committee for Hazardous Substances - AGS Management - BAuA - 2006 for similar substance (81-16-3), suggests that the test substance is not an eye irritant Tobias acid was evaluated for ocular irritation in rabbits according to current relevant guidelines. No irritation reactions of cornea, iris and conjunctiva were observed. Hence it was reported that Tobias acid was not irritating to rabbit eyes.

The ocular irritation potential of similar substance (1064-48-8) was assessed (COLIPA n° B15, Scientific Committee on Consumer Safety, SCCS adopted on23 March 2010) on New Zealand White rabbits.On test day 1, an aliquot of 0.1ml of 5% aqueous solution/suspension (pH adjusted with ammonia to pH 8 - 10) was applied in the conjunctival sac of the right eye of each animal. The left eye served as reference control. Rinsing of the eyes was not performed. Scoring of irritation based on the Draize method was performed 1, 24, 48 and 72 hours after single application. No irritation reactions of cornea, iris and conjunctiva were observed. Hence it was reported that Acid Black 1 was not irritating to rabbit eyes.

 

Therefore, on the basis of available information for the target chemical as well as its various read across, and applying the weight of evidence approach, Aluminum, tris(5-amino-4-hydroxy-3-(2-phenyldiazenyl)-2,7-naphthalenedisulfonato(2-))di- was considered to be not irritating to rabbit eyes.


Justification for classification or non-classification

Skin and Eye irritation properties ofAluminum, tris(5-amino-4-hydroxy-3-(2-phenyldiazenyl)-2,7-naphthalenedisulfonato(2-))di- was determined .The substance Aluminum, tris(5-amino-4-hydroxy-3-(2-phenyldiazenyl)-2,7-naphthalenedisulfonato(2-))di-is determined not  to be irritating to both skin and eye.