Dihydrogen (ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium, aluminium salt

Endpoint summary

Description of key information

Skin Irritation

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

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 substance was determined to be 82.1%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

A skin irritation study was performed to evaluate the irritation potential of the test chemical on 207 human volunteers.

The test substance was applied in the concentration of 5% in aqueous solution for 12 days .No skin reaction were observed. Hence, the test chemical was considered to be not irritating to hu\man skin.

Eye Irritation

Ocular irritation potential of the test chemical was determined according to a modification of the Draize test (Draize, 1959).

All animals survived and were free of significant clinical signs of toxicity throughout the study

Ophthalmoscopic examinations revealed that all animals were free of abnormalities, all animals were free of significant signs of ocular irritation, staining and particle embedment. Thus it was considered that the test chemical was not irritating to eyes.

An in vitro OECD 492 Study was performed to determine the irritation potential of the test chemical.

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 69.7%. 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

Reference 1

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

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 Samples
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.

Mesh Compatibility
Five 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 Controls
The 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 Data
See 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 Prediction
According 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 Classification
Mean tissue viability ≤ 50% Category 2
Mean 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 Data
Upon 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 Protocol
There were no amendments to the protocol. See Appendix C for the protocol in its entirety
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 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) Controls
30 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 Article
For 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 treatment
After 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: 6/08/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Twice

MTT 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 data

NUMBER OF REPLICATE TISSUES: 3

CALCULATIONS and STATISTICAL METHODS
All data were background subtracted before analysis. MTT data are presented as % viable compared to negative control. Data were generated as follows:

MTT Assay
Blanks:
·        The optical density (OD) mean from all replicates for each plate (ODblank).

Negative Controls (NC):
Identity: Phosphate-Buffered Saline (PBS), Lot No. AC10239794
Provided by:MatTek
Date Received:12 Dec 2017 and 19 Dec 2017
Expiration Date:18 Jul 2018
Storage:Room temperature and humidity
Description:Clear colorless liquid
Sample Preparation:Used as received

Positive Control (PC):
Identity: 5% Sodium Dodecyl Sulfate (SDS), Lot No. 071817MAB
Provided by:MatTek
Date Received:12 Dec 2017 and 19 Dec 2017
Expiration Date:18 Jul 2018
Storage:Room temperature and humidity
Description:Clear colorless liquid
Sample 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: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): neat

POSITIVE 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:

82.1

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 Classification
Mean tissue viability ≤ 50% Category 2
Mean tissue viability > 50% Non-irritant

Test and control article identity	Tissue Viability		Irritancy Classification	GHS Category
	Mean	SD
68921-92-6	82.1%	8.78%	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
68921-42-6	1	1.278	1.319	1.233	1.274	1.254	72.2	1.425	0.153	82.1	8.78	10.70	Not irritating
	2	1.615	1.566	1.570	1.521	1.546	89.0
	3	1.542	1.500	1.497	1.455	1.476	85.0

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 82.1%. Thus, substance 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 substance was determined to be 82.1%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

Data is from safety assessment report

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Principles of method if other than guideline:

Equivalent or similar to OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

not specified

Species:

other: Human

Strain:

not specified

Details on test animals or test system and environmental conditions:

No dat available

Type of coverage:

not specified

Preparation of test site:

not specified

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

5% in aqueous solution

Duration of treatment / exposure:

12 days

Observation period:

Not specified

Number of animals:

207 human volunteers

Details on study design:

Not specified

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

other: 12 days

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No skin reaction observed

Interpretation of results:

other: Not irritating

Conclusions:

The test chemical was applied on the skin of 207 human volunteers in the concentration 5% in aqueous solution for 12 days . No skin reaction were observed . Hence, the test chemical was considered to be not irritating to human skin

Executive summary:

A skin irritation study was performed to evaluate the irritation potential of the test chemical on 207 human volunteers. The test chemical was applied in the concentration of 5% in aqueous solution for 12 days. No skin reaction were observed. Hence, the test chemical was considered to be not irritating to human skin.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: Draize test

Principles of method if other than guideline:

Ocular irritation was determined according to a modification of the Draize test (Draize, 1959). Test material (3%,wt./vol. in aqueous vehicle) was administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White rabbits.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 3.0-3.5 kg
- Housing: individually housed in stainless steel, wire mesh-floor cages.
- Diet (e.g. ad libitum): Certified Lab Rabbit Chow HF; Purina No. 5325 ;ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-21°C
- Humidity (%): 38%
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycle

Vehicle:

other: test substance was prepared daily as a 3% (w/v) suspension in aqueous vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose and 0.25% (w/v) laureth -10 acetate.

Controls:

yes, concurrent vehicle

Amount / concentration applied:

Test and vehicle control materials (30µl) were administered to the right eye of New Zealand White rabbits

Duration of treatment / exposure:

24 hrs.

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

6 of each sex per group (Total 12)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data
- Ophthalmic observations: 7 days and 24 hr prior to the initial dose, on days 3, 7 and 14 (prior to daily dosing), and at the end of the study.
SCORING SYSTEM: ocular irritation pretest
TOOL USED TO ASSESS SCORE: slit-lamp bimicroscopy (including examinations of fluorescein stain retention to evaluate integrity of the corneal epithelium)

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

other: 21 days

Reversibility:

not specified

Remarks on result:

no indication of irritation

Remarks:

All animals survived and were free of significant clinical signs of toxicity throughout the study. Ophthalmoscopic examinations revealed that all animals were free of abnormalities & free of signs of ocular irritation, staining and particle embedment.

Irritant / corrosive response data:

All animals were free of significant signs of ocular irritation, staining and particle embedment.

Other effects:

All animals survived and were free of significant clinical signs of toxicity throughout the study. Ophthalmoscopic examinations revealed that all animals were free of abnormalities considered to be of clinical importance.

Table 1: Experimental design in evaluation of cumulative eye irritation of the test chemical

Group	Test material	No. of animals	Concentration	Dose/application	Applications/day	No of days
I	Control	12	0%*	30 µl	1	21
II	test chemical	12	3% (w/v)	30 µl	1	21

* Aqueous vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose and 0.25% (w/v) laureth-10 acetate.

Table 2: Scoring scheme for ocular staining

Extent of reaction	Score
Intensity
No reaction	0
Barely perceptible (scattered or diffuse areas of staining)	+
Slight (easily discernible stained areas; details of iris clearly visible)	+ 1
Moderate (moderately stained conjunctivae, and/or no details of iris visible; size of pupil barely discernible)	+2
Marked (severely stained conjunctivae, and/or iris not discernible through staining)
Area of cornea involved (I or greater)
One-quarter (or less) but not zero	1
Greater than one-quarter, but less than one-half	2
Greater than one-half, but less than three-quarters	3
Greater than three-quarters, up to whole area	4

Table 3: Summary of ocular effects in evaluation of the (repeated application) eye irritation potential of the test chemical

Group	Eye	Observation
		Test day	1	7	14	21
I - Control	Test eye	Conjunctivae
		Redness --score of 1	-	-	-	-
		Discharge--score of 1	1	-	1	-
I - Control	Control eye	Conjunctivae
		Discharge – score of 1	1	-	1	-
		Apparent hemorrhage	-	-	-	1
test chemical	Test eye	Conjunctivae
		Redness --score of 1	-	-	-	-
		Discharge--score of 1	-	-	-	1
test chemical	Control eye	Conjunctivae
		Redness --score of 1	-	-	-	-
		Chemosis--score of 1	-	-	1	-

 

 

Interpretation of results:

other: Not irritating

Conclusions:

The test chemical when administered once daily, for 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits at a dose volume of 30 µl showed no significant signs of ocular irritation, staining and particle embedment. All animals survived and were free of significant clinical signs of toxicity throughout the study. Thus it was concluded that the test chemical was not irritating to eyes.

Executive summary:

Ocular irritation potential of the test chemical was determined according to a modification of the Draize test (Draize, 1959). The test chemical was prepared daily as a 3% (w/v) suspension in aqueous vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose and 0.25% (w/v) laureth -10 acetate. The test chemical (3% w/v in aqueous vehicle) was administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits (6 of each sex/ group) at a dose volume of 30µl. Control animals (6 of each sex) received 30/µl of the vehicle daily.Ocular irritation was determined according to a modification of the Draize test (Draize, 1959). Interpretation of observations and assignment of scores were consistent with those described by the Consumer Product Safety Commission (1972).All eyes were scored for ocular irritation pretest (8 days, 24 hr and immediately prior to the initial dose) and approximately 24 hr after each treatment, prior to the next instillation of test material; on days 1, 3, 7, 14 and 21, the eyes were also evaluated for irritation 1 hr after treatment. In addition, all readily observable ocular structures were evaluated for eye stain and particle embedment 24 hr after each treatment. All animals survived and were free of significant clinical signs of toxicity throughout the study Ophthalmoscopic examinations revealed that all animals were free of abnormalities, all animals were free of significant signs of ocular irritation, staining and particle embedment. Thus it was considered that the test chemical was not irritating to eyes.

Reference 2

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

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 / Hazard
Code 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-200

Storage: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 applicability
The 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

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

VEHICLE (no vehicle)
- Amount(s) applied (volume or weight with unit): none
- Concentration (if solution): none
- Lot/batch no. (if required): none
- Purity: none

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): neat

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 being
performed.
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 tissues
and 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 well
from 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 the
negative 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 320
Mean 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:

69.7

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:

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
CAS No. 68921-42-6	69.7	16.01%	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
CAS No. 68921-42-6	1	1.058	1.107	1.013	1.062	1.037	61.7	1.172.	0.269	69.7	16.01
	2	1.340	1.363	1.295	1.318	1.306	77.7

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 69.7%. 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 69.7%. 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 summarized to determine the degree of dermal irritation caused by the test chemical in living organisms. These include in vivo experimental studies on humans, rabbits as well as in vitro study for the test chemicals. They are summarized as follows:

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 substance was determined to be 82.1%. Hence, under the experimental test conditions it was concluded that test substance was considered to be not irritating to the human skin and being classified as “Not Classified'' as per CLP Regulation.

This is supported by a skin irritation study performed to evaluate the irritation potential of the test chemical on 207 human volunteers.

The test substance was applied in the concentration of 5% in aqueous solution for 12 days .No skin reaction were observed. Hence, the test chemical was considered to be not irritating to human skin.

A similar study was performed on 207 humans to assess the dermal irritation potential of the test chemical. 207 human volunteers were exposed to the test chemical in a 48 hours closed application. The volunteers were observed for signs of irritation. Moderate irritation observed in 14 of 207 volunteers after 48 hours exposure to the test chemical

Hence, the test chemical can be considered to be a skin irritant.

A skin irritation study was also conducted in rabbits to observe the irritation potential of the test chemical.

0. 5 ml of a saturated solution of the test chemical in saline was applied to rabbit skin (site not specified) and effects were observed (duration not specified).

The test chemical was observed to be not irritating to rabbit skin.

These results are supported by a skin irritation study performed to evaluate the skin irritation potential of the test chemical on 207 human volunteers.

The test substance was applied in the concentration of5% in aqueous solution for 12 days .No skin reactions were observed. Hence. the test chemical was considered to be not irritating to human skin.

 

Even though the test chemical caused signs of irritation to the skin of humans, but it failed to cause any irritation to skin of humans, rats and in the in vitro study. Taking all these factors into consideration, the test chemical can be considered to be not irritating to skin.  Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation.

Eye Irritation

In different studies, the test chemical has been investigated for potential for ocular irritation in living organisms to a greater or lesser extent. These include in vivo experimental studies performed on rabbits as well as in vitro experimental study for the test chemical. The results are summarized as follows:

Ocular irritation potential of the test chemical was determined according to a modification of the Draize test (Draize, 1959).

The test chemical was prepared daily as a 3% (w/v) suspension in aqueous vehicle containing 0.5% (w/v) hydroxypropyl methylcellulose and 0.25% (w/v) laureth -10 acetate.

The test chemical (3% w/v in aqueous vehicle) was administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits (6 of each sex/ group) at a dose volume of 30µl. Control animals (6 of each sex) received 30/µl of the vehicle daily.Ocular irritation was determined according to a modification of the Draize test (Draize, 1959). Interpretation of observations and assignment of scores were consistent with those described by the Consumer Product Safety Commission (1972).All eyes were scored for ocular irritation pretest (8 days, 24 hr and immediately prior to the initial dose) and approximately 24 hr after each treatment, prior to the next instillation of test material; on days 1, 3, 7, 14 and 21, the eyes were also evaluated for irritation 1 hr after treatment. In addition, all readily observable ocular structures were evaluated for eye stain and particle embedment 24 hr after each treatment.

All animals survived and were free of significant clinical signs of toxicity throughout the study

Ophthalmoscopic examinations revealed that all animals were free of abnormalities, all animals were free of significant signs of ocular irritation, staining and particle embedment. Thus it was considered that the test chemical was not irritating to eyes.

This is supported by an ocular irritation study performed to assess the potential of the test chemical. Groups of six or more albino rabbits received 0.2 ml of 10% aqueous solution of the test chemical in one eye twice daily, five times per week, for four weeks. Three days after the last application, two rabbits from each group were killed and their eyes and optic nerve trunks were examined grossly and microscopic examination of the upper eyelids was performed. One hour after each treatment, the eyes were examined for signs of irritation or staining. Draize method was used for scoring of ocular reactions.

The test chemical caused marked staining of the eyes. The test chemical failed to cause any eye irritation when examined prior to application on days 2, 5 10 and 20 of treatment and three days after the end of the treatment. Hence, the test chemical can be considered to be not irritating to eyes.

The above in vivo studies are further supported by an in vitro OECD 492 Study performed to determine the irritation potential of the test chemical. 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 69.7%. 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’’.

The in vivo and in vitro results are in mutual agreement with each other, indicating a very strong possibility that the test chemical is indeed 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

Even though the test chemical caused signs of irritation to the skin of humans, but it failed to cause any irritation to skin of humans, rabbits and in the in vitro study. Taking all these factors into consideration, the test chemical can be considered to be not irritating to skin.  Hence, the test chemical can be classified under the category “Not Classified” as per CLP Regulation

The in vivo and in vitro results are in mutual agreement with each other, indicating a very strong possibility that the test chemical is indeed 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”.