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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:
December 06, 2017 to March 24, 2018
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 is to provide classification of chemicals concerning their eye irritation potential using an alternative to the Draize Rabbit Eye Test, according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”. The EpiOcular™ EIT is intended to differentiate those materials that are UN GHS No Category (i.e., do not meet the requirements for UN GHS classification) from those that would require labeling as either UN GHS Category 1 or 2. This assay is not intended to differentiate between UN GHS Category 1 / HazardCode 318 and UN GHS Category 2 / Hazard Codes 319 and 320.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
IUPAC name : 2-methyl-9,10-anthraquinone
Molecular formula : C15H10O2
Molecular weight : 222.242 g/mol
Substance type: Organic
Physical property: Orange powder
InChI: 1S/C15H10O2/c1-9-6-7-12-13(8-9)15(17)11-5-3-2-4-10(11)14(12)16/h2-8H,1H3
Smiles: c12c(C(c3ccccc3C1=O)=O)ccc(c2)C

Test animals / tissue source

Species:
other: MatTek EpiOcular Tisssue Model OCL-200
Strain:
other: Not applicable
Details on test animals or tissues and environmental conditions:
-Test System: MatTek EpiOcular™ Tissue Model OCL-200Storage:EpiOcular™ tissues and assay medium will be refrigerated at approximately 2-8°C upon arrival and until use.Supplier:MatTek Corporation, Ashland, MA- Justification of the test method and considerations regarding applicabilityThe EpiOcular™ Tissue Model closely parallels human ocular tissue, thus providing a useful in vitro means to assess ocular irritancy and toxicology

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Duration of treatment / exposure:
Tissues will be topically exposed to the test article and control articles for 6 hours ± 15 minutes.
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
Following the post soak,Tissues will be incubated in 1 ml fresh assay medium in a humidified 37±1°C, 5±1% CO2 incubator.
Number of animals or in vitro replicates:
2 tissues were used for test compound and control.
Details on study design:
-Plate Reader Linearity Check:The linearity of the plate reader or spectrophotometer used for optical density (OD) determination will be verified prior to its use the same week the EIT assay is beingperformed.A dilution series of trypan blue or thiazolyl blue tetrazolium bromide (MTT) formazan will be prepared and 200 μl aliquots will be pipetted into a 96-well plate.The optical density of the plate wells will be measured at a wavelength of 570 nm (OD570), with no reference wavelength.A regression line and an R-squared value will be generated using Microsoft Excel®. Verification will be considered acceptable if the R-squared value is >0.999.Assessment of Direct MTTReduction:No assessment of the direct MTT (methyl thiazole tetrazolium) reduction potential of each test article will be made.-Assessment of Coloring or Staining Materials:No assessment of each test article’s ability to absorb light at the wavelength (570 nm) used for MTT determination will be made.- Pre-Incubation:EpiOcular™ tissues will be placed in six-well plates containing warmed assay media and will be equilibrated in a humidified 37±1°C, 5 ±1% CO2 incubator for at least one hour. The media will then be changed and the tissues incubated overnight (16-24 hours).Any tissues not being incubated the same day will be allowed to re-equilibrate at 37±1°C, 5±1% CO2 and will be stored at approximately 2-8°C..-Pre-Treatment:After the overnight incubation, the tissues will be moistened with 20 μl of phosphatebuffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.-Dosing:Whenever possible, solids should be ground to a fine powder before application. 50 mg of a solid test article will be applied topically to duplicate tissuesand incubated at 37±1°C, 5±1% CO2 for 6 hours ± 15 minutes.After dosing and incubation, the tissues will be thoroughly rinsed with PBS and soaked in 5 ml of room-temperature assay medium in a 12-well plate for the appropriate amount of time.Tissues will be soaked for 25±2 minutes.-MTT Extraction:Following the three-hour MTT incubation period, each tissue will be removed individually and gently rinsed with PBS to remove any residual MTT solution.The extraction plate will be covered and sealed to reduce evaporation of extractant.For solid, colored, or staining test articles, 2.0 ml of extractant solution will be used in a six-well plate, allowing extraction to occur through the bottom of the insert. Extraction Conditions:The extraction will be allowed to proceed overnight at room temperature in the dark.Alternatively, the extraction can proceed for at least two hours, with shaking, at room temperature.-Decant Extractant:Tissues immersed in extractant solution in a 24-well plate: After the extraction period is complete, the liquid within each tissue insert will be decanted back into the wellfrom which it was taken, i.e., the solution will be mixed with the extractant in the well.The tissue inserts will then be discarded.-Transferring to 96-Well Plate:Two 200 μl aliquots from each well of the extraction plate(s) will be pipetted into a 96- well microtiter plate.-Measuring Optical Density:The optical density of the extracted samples will be determined at a single wavelength of 570 nm and using eight 200 μl aliquots of the Extractant as blanks.Calculating Percent Viability:The percent viability of the test tissues will be determined using the following formula:% Viability = 100 x (ODsample / ODNegative Control)Quality Controls:Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.Tissue Variability: The difference in viability between identically treated tissues must be less than 20%. This applies to tissues treated with the same test article as well as living and killed controls.Ocular Irritation Potential:An irritant is predicted if the mean relative tissue viability of two individual tissues exposed to the test substance is less than or equal to 60% of the mean viability of thenegative control-treated tissue viability.In Vitro Result In Vivo Prediction (GHS3)Mean tissue viability ≤ 60% Category 1 / Hazard Code 318, or Category 2 / Hazard Codes 319 and 320Mean tissue viability > 60% No Category (Non-Irritating)Borderline Results:If the test article-treated tissue viability is 60±5%, a second EIT should be performed. If the results of the second test disagree with the first, then a third test should be performed. The conclusion will be based on the agreement of two of the three tests.Duration:The duration of the EpiOcularTM Eye Irritation Test is approximately five days.

Results and discussion

In vitro

Results
Irritation parameter:
other: mean % tissue viability
Run / experiment:
Run 1
Value:
109.5
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:
Negative Controls: The assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5.Positive Controls: The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability.Tissue Variability: The difference in viability between identically treated tissues must be less than 20%.

Any other information on results incl. tables

Test and Control Article Identity

 

Tissue Viability

Irritancy Classification

GHS Category

 

Mean

SD

84-54-8

109.5

5.95%

Non Irritant

No category

 

Test and control article identity

Tissue no.

Raw data

Blank corrected data

Mean of aliquots

% viability

OD

Viabilities (%)

MEAN

SD

Mean

SD

 

Aliq 1

Aliq 2

 

Aliq 1

Aliq 2

 

 

 

 

84-54 -8, solid

1

 

1.786

1.885

1.741

1.840

1.790

106.5

1.840

0.100

109.5

5.95

2

1.934

1.937

1.889

1.892

1.790

112.5

Applicant's summary and conclusion

Interpretation of results:
other: not irritating
Conclusions:
The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The mean % tissue viability of the test chemical was determined to be 109.5%. Thus,the test chemical was considered to be not irritating to MatTek EpiOcular Tisssue Model OCL-200.
Executive summary:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The MatTek EpiOcular™ model was used to assess the potential ocular 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 ocular irritation potential of test article. Tissues were exposed to 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 0.8 to 2.5 in run 1. Also, the positive control, methyl acetate, reduced tissue viability to be below 50% of negative control (for 6 hour exposures with solids) in run one.The mean % tissue viability of the test chemical was determined to be 109.5%.

Hence, under the experimental test conditions it was concluded that the test chemical was considered to be not irritating to the MatTek EpiOcular Tissue Model OCL-200 and being classified as “Not classified’’.