Registration Dossier

Administrative data

Description of key information

Skin Irritation:

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 103.7%.

Hence, under the experimental test conditions it was concluded that the test chemical was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

Eye Irritation:

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’’.

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:
December 04, 2017 to March 13, 2018
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 is to provide classification of dermal irritation potential of a chemical by using a three-dimensional human epidermis model, according to the OECD Test Guideline No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”.
GLP compliance:
yes
Test system:
human skin model
Remarks:
MatTek EpiDerm™ Tissue 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:
EpiDerm™ tissues, Lot No. 27646 Kits I and J, were received from MatTek on 12 Dec 2017, and Lot No. 27654 Kits O and P, were received from MatTek on 19 Dec 2017. All tissues were refrigerated at 2-8°C upon receipt. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium, for 18±3 hours. Equilibration medium was replaced with fresh medium before dosing.
Justification for test system used:
The EpiDerm™ Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology
Vehicle:
unchanged (no vehicle)
Details on test system:
EpiDerm™ Tissue SamplesEpiDerm™ tissues, Lot No. 27646 Kits I and J, were received from MatTek on 12 Dec 2017, and Lot No. 27654 Kits O and P, were received from MatTek on 19 Dec 2017 All tissues were refrigerated at 2-8°C upon receipt. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium, for 18±3 hours. Equilibration medium was replaced with fresh medium before dosing.Mesh CompatibilityFive of the test articles supplied were liquids. These test articles were assessed for compatibility with pre-cut nylon mesh supplied with the tissues. The mesh was placed on a slide and 30 μl of a liquid test articles or PBS (negative control) were applied. After 60 minutes of exposure, the mesh was checked microscopically. If no damage or other interaction was observed, indicating that the mesh was compatible with the test article, the mesh was used as a spreading aid.Tissue Viability (MTT Reduction)At the end of the incubation period, each EpiDerm™ tissue was rinsed with PBS and transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well for at least two hours, with shaking, at room temperature. Two aliquots of the extracted MTT formazan were measured at 540 nm using a plate reader (μQuant Plate Reader, Bio-Tek Instruments, Winooski, VT).For several tissues, the test article had stained the tissues. Therefore, the tissues were extracted with only 1.0 ml, allowing extraction to occur only through the bottom of the insert. After the extraction period, the tissue insert was removed and discarded and 1.0 ml of extraction solution were added to each well, bringing the volume to a total of 2.0 ml.Quality ControlsThe assay meets the acceptance criteria if the mean OD540 of the negative control tissues is between 1.0 and 2.5, inclusive, and the mean viability of positive control tissues, expressed as percentage of the negative control tissues, is at least 20%. In addition, the standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates must be less than 18%.Note: Chemicals that provide tissue viabilities in a range of 30% to 70% may provide high SD. If the high SD (above acceptance limits) is typical for the chemical and the classification of the chemical is consistent in all independent runs, MatTek recommends that this result be accepted, although it did not meet the Assay Acceptance Criterion.Analysis of DataSee Table 1 for Experimental Data. The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:% viability = 100 X (OD sample/OD negative control)Skin Irritation PredictionAccording to the EU1,2 and GHS3 classification (R38 / Category 2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is 50% or less of the mean viability of the negative controls.In vitro result In vivo ClassificationMean tissue viability ≤ 50% Category 2Mean tissue viability > 50% Non-irritant (NI)Assessment of direct MTT reduction and assessment of coloring or staining materials was not performed. Therefore, it cannot be fully assessed if the test articles interfered with MTT viability measurements.Retention of DataUpon signing the final report, all raw data, supporting documentation and reports are submitted to the Archivist by the Study Director. The raw data are filed at MB Research by project number. The final report is filed at MB Research by Sponsor name and MB project number.All data generated during the conduct of this study will be archived at MB Research for at least one year from the date of the final report and optionally longer at additional cost. The Sponsor will be contacted in writing to determine final disposition of the records.Any remaining test article will be discarded upon submission of the report.Amendment to the ProtocolThere were no amendments to the protocol. See Appendix C for the protocol in its entiretyEvaluation 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 article 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 solid test article, 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 each test article 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):Identity: Phosphate-Buffered Saline (PBS), Lot No. AC10239794Provided by:MatTekDate Received:12 Dec 2017 and 19 Dec 2017Expiration Date:18 Jul 2018Storage:Room temperature and humidityDescription:Clear colorless liquidSample Preparation:Used as receivedPositive Control (PC):Identity: 5% Sodium Dodecyl Sulfate (SDS), Lot No. 071817MABProvided by:MatTekDate Received:12 Dec 2017 and 19 Dec 2017Expiration Date:18 Jul 2018Storage:Room temperature and humidityDescription:Clear colorless liquidSample Preparation:Used as received- 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:
Tissues will be topically exposed to the test article and control articles for 60 minutes.
Duration of post-treatment incubation (if applicable):
After dosing, the tissues will be returned to the incubator for 35 ±1 minute, and then returned to the sterile hood for the remainder of the 60-minute exposure period.
Number of replicates:
All treatments with test articles and controls will be dosed in triplicate EpiDerm™ tissues.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Run 1
Value:
103.7
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:
All treatments with test articles and controls will be dosed in triplicate EpiDerm™ tissues.In vitro result In vivo ClassificationMean tissue viability ≤ 50% Category 2Mean tissue viability > 50% Non-irritant

Test and Control Article Identity

 

Tissue Viability

Irritancy Classification

GHS Category

 

Mean

SD

84-54-8

 

 

103.7%

5.85%

 

Non-Irritant

 

 

                No Category

 

Test and control article identity

Tissue no.

Raw data

Blank corrected data

Mean of aliquots

% viability

OD

Viabilities(%)

CV%

Classification

MEAN

SD

Mean

SD

 

 

Aliq 1

Aliq 2

 

Aliq 1

Aliq 2

 

 

 

84-54-8

1

2.048

1.862

2.003

1.817

1.910

110.0

1.801

0.102

103.7

5.85

5.64

 

Not irritating

2

1.713

1.795

1.668

1.750

1.709

98.4

 

3

2.153

1.505

2.108

1.460

1.784

102.7

Interpretation of results:
other: not irritating
Conclusions:
The dermal irritation potential of test article 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 chemical was determined to be 103.7%. Thus, the test chemical was considered to be not irritating to the human skin.
Executive summary:

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. 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 article 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 shows that 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.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 103.7%.

Hence, under the experimental test conditions it was concluded that the test chemical 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:
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.
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
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
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.
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%.

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

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’’.

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 reviewed to determine the level of dermal irritation caused by the test chemical in living organisms. These include in vitro and in vivo experimental data for the test chemicals. The results are summarized below:

The dermal irritation potential of test article was determined according to the OECD 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”. 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 article 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 shows that 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.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 103.7%.

Hence, under the experimental test conditions it was concluded that the test chemical was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

Skin irritation effects were also estimated by four different models i.e, Battery, Leadscope, SciQSAR and CASE Ultra used within Danish QSAR database for the test chemical. Based on estimation, no severe skin irritation effects were known when the test chemical was exposed to rabbit skin.

The estimated results are supported by Draize test performed in albino rabbits to evaluate the skin irritation potential of the test chemical. 6 albino New Zealand rabbits (3/sex) were used for the study. 0.5 ml (0.5 g) of the test material was applied to clipped areas of intact and abraded skin. The abrasions were longitudinal epidermal incisions sufficiently deep to penetrate the stratum corneum, but not so deep as to destroy the integrity of the derma. Applications were made under occlusive patches (1" x 1" gauze, covered by adhesive tape). Following application of the test chemical to the entire trunk of each animal was covered with an impermeable occlusive wrapping. The animals were then immobilized. The wrapping and test material were removed 24 hours following application. The sites were individually examined and scored separately for erythema and edema at 24 and 72 hours. The mean scores for 24 and 72 hours grading were averaged to determine final irritation indices.

The Primary Irritation Index after 24, 72 hours for the test chemical was 0.15.

Based on the score and scale of interpretation of PII scores, the test chemical can be considered not irritating to skin.

The above results are further supported by OECD 404 Guideline study performed to assess the skin irritation potential of the test chemical in rabbits.

0.5g Undiluted test chemical was applied to the intact skin of 3 male New Zealand albino rabbits under semi-occlusive conditions for 4 hours. The rabbits were observed for signs of irritation at 1, 24, 48 and 72 hours.

Orange staining due to the dye interfered with evaluation of erythema. No oedema was observed. The test chemical could potentially have provoked slight to moderate, but not severe irritation.

Hence, the test chemical can be considered not irritating to New Zealand albino rabbit skin.

The results of the in vitro and in vivo studies are in mutual agreement with each other, indicating a very strong possibility that the test chemical indeed lacks the potential to cause irritation to skin. Hence, the test chemical can be considered to be not irritating to skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Eye Irritation

Various studies have been reviewed to determine the extent of ocular damage caused by the test chemical in living organisms. These include in vitro and in vivo experimental data for the test chemicals. The results are summarized below:

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’’.

This is supported by the results of similar OECD 492 Guideline performed to determine the ocular irritation potential of test article. 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. Tissues were exposed to solid test articles and control for approx.6 hours, followed by a 25 minute post-soak and approximately 18 hours 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, passing the acceptance criteria.

The mean % tissue viability of test substance was determined to be 111.0%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human eyes and can thus be classified as "Not Classified" as per CLP Regulation

These results are supported an in vivo study performed to compare the immediate and delayed effects of the test chemical on the cornea—a tissue normally devoid of blood vessels with their effects on the vascular tissues of the sclera and eyelids.

White rabbits were used for the study. The test chemical(dry powder)was placed in the lower conjunctival sac of one eye and held there usually for one-half minute. This was repeated at intervals of one or several days. The other eye was treated with 0.7% solution of Sodium chloride and used as control. The eyes were examined for evidence of inflammation, and changes in the corneal structures were observed with the bio-microscope.

The test chemical produced only immediate sensory and inflammatory reaction (discomfort, blepharospasm, and conjunctival congestion) that disappeared very soon. The eyes appeared normal in a few hours.

Hence, it was concluded the irritation effects were due to mechanical action of the powder rather than toxic effects of the test chemical. Therefore, the test chemical can be considered not irritating to eyes.

The above results are supported by a Draize test performed to evaluate the ocular irritation potential of the test chemical in albino rabbits. 9 albino New Zealand rabbits (mixed sex), weighing approximately 1.8- 2.4 kg were used for the study. One tenth (0.1) of a millilitre of the test substance was instilled in the right eye; the left eye, remaining untreated served as a control. The treated eyes of six rabbits remained unwashed for 24 hours. The eyes of the remaining three (3) rabbits were irrigated 4 seconds following instillation of the test material (30 ml tap water at 37 degrees C). Readings facilitated by hand-held lenses were made 1, 2, 3, and up to 7 days after treatment.

The scores of washed and unwashed eyes of rabbits after 3 days of observation were 0, 0 respectively.

Based on the scores, the test chemical can be considered not irritating to eyes.

The results of the in vitro and in vivo studies are in mutual agreement with each other, indicating a very strong possibility that the test chemical indeed lacks the potential to cause irritation to eyes. Hence, the test chemical can be considered to be not irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.

Justification for classification or non-classification

The results of the in vitro and in vivo studies are in mutual agreement with each other, indicating a very strong possibility that the test chemical indeed lacks the potential to cause irritation to eyes and skin. Hence, the test chemical can be considered to be not irritating to eyes and skin. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Not Classified”.