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Toxicological information

Eye irritation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name of the test chemical:Tris[5-amino-4-hydroxy-3-(phenylazo)naphthalene-2,7-disulphonato(2-)]dialuminiumMolecular Formula: C48H33Al2N9O21S6Molecular Weight: 1318.19 g/molSubstance type: OrganicPhysical State: Solid
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

Test animals / tissue source

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).

Test system

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.  

Results and discussion

In vitro

Results
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
79.2
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
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.

Applicant's summary and conclusion

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.