Phenylacetic acid

Endpoint summary

Description of key information

Skin Irritation

A preliminary irritation test was performed on guinea pigs to determine the maximum non-irritating concentration for the Open Epicutaneous test. 6-8 male/female guinea pigs were used for the study.

No signs of irritation were observed in the 24 hours observation period.

Hence, the test chemical was considered to be not irritating to guinea pig skin.

Eye Irritation

The potential of the test chemical to induce ocular irritation was evaluated in rabbits according to O.E.C.D. 405(24th February, 1987) and E.C. (92/69/E.E.C, B5- 31st July, 1992) Guidelines. 3 Male New Zealand White rabbits were used for the study.

Based on these observations, the test chemical was considered to be severely irritating to rabbit eyes.

An in vitro study was performed according to OECD 492 test guideline to evaluate the ocular irritation potential of test article.

he mean % tissue viability of test chemical was determined to be 1.5%. Thus, the test chemical was considered to be irritating to MatTek EpiOcular Tissue Model OCL-200.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin 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 journals

Qualifier:

according to guideline

Guideline:

other: Consumer Product Safety Commission of the USA (Code of Federal Regulations, Title 16, Sec. 1500.41)

Principles of method if other than guideline:

A preliminary irritation test was performed on guinea pigs to determine the maximum non-irritating concentration for the Open Epicutaneous tesrt

GLP compliance:

not specified

Species:

guinea pig

Strain:

not specified

Details on test animals or test system and environmental conditions:

- Weight at study initiation: 300-450g

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

other: ethanol, acetone, H20, petroleum, PEG and/or other suitable vehicles

Amount / concentration applied:

0.025 ml of each test concentration (e.g, 100, 30, 10 and 3%) in ethanol, acetone, H20, petroleum, PEG and/or other suitable vehicles

Duration of treatment / exposure:

24 hours

Observation period:

24 hours

Number of animals:

6

Details on study design:

Details on study design
TEST SITE
- Area of exposure: flanks
- % coverage: areas measuring 2 cm2 of the flank skin
- Type of wrap if used: no data available

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no data available
- Time after start of exposure: no data available

SCORING SYSTEM: no data available

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24 h

Score:

0

Reversibility:

not specified

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No signs of irritation were observed in the 24 hours observation period

Interpretation of results:

other: not irritating

Conclusions:

No signs of irritation were observed in the 24 hours observation period.
Hence, the test chemical was considered to be not irritating to guinea pig skin.

Executive summary:

A preliminary irritation test was performed on guinea pigs to determine the maximum non-irritating concentration for the Open Epicutaneous test. 6-8 male/female guinea pigs were used for the study.

0.025 ml of each test concentration (e.g, 100, 30, 10 and 3%) inethanol, acetone, H20, petroleum, PEG and/or other suitable vehicles was applied to theone of the areas measuring 2 cm2 of the flank skin previously clipped and marked with a circular stamp.Reactions are read 24 h after the application of the test material.The minimal irritating and the maximal nonirritating concentrations were determined by an all-or-none criterion. The minimal irritating concentration is defined as thelowest one causing skin irritation. The maximal nonirritating concentration is defined as the highest one not causing macroscopic skin reactions in any of the animals.

No signs of irritation were observed in the 24 hours observation period.

Hence, the test chemical was considered to be not irritating to guinea pig skin.

Reference 2

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:

33.3

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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
103-82-2	33.3%	6.48%	Irritant	Category 1/2

 

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
103-82-2	1	0.8	0.798	0.754	0.752	0.753	40.8	0.615	0.120	33.3	6.48	19.46	Irritant
	2	0.592	0.6	0.546	0.554	0.550	29.8
	3	0.585	0.590	0.539	0.544	0.542	29.3

 

 

Interpretation of results:

Category 2 (irritant) based on GHS criteria

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 33.3%. Based on these values, the test chemical can be considered to irritating. It can be further classified under the category "Category 1/2" as per CLP Regulation.

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 show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was <18% passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 33.3%. Based on these values, the test chemical can be considered to irritating. It can be further classified under the category "Category 1/2" as per CLP Regulation.

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:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Justification for type of information:

data is from NTRL Reports

Qualifier:

according to guideline

Guideline:

other: O.E.C.D. 405(24th February, 1987) and E.C. (92/69/E.E.C, B5- 31st July, 1992) Guidelines

Principles of method if other than guideline:

The potential of the test chemical to induce ocular irritation was evalualed in rabbits according to O.E.C.D. 405(24th February, 1987) and E.C. (92/69/E.E.C, B5- 31st July, 1992) Guidelines

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Sex:Male
- Source: Elevage Cunicole de Val de Selle. 80160 Prouzel. France
- Age at study initiation: no data
- Weight at study initiation: 2.7 ± 0.2 kg
- Housing: The animals were housed individually in polystyrene cages
- Diet (e.g. ad libitum): 112 C pelleted diet
- Water (e.g. ad libitum): Drinking water filtered by a EG. Millipore membrane (0.22 micron) was provided ad libitwn.
- Acclimation period: at least five days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour of filtered. non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h112 h

IN-LIFE DATES: From: To:

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100 mg

Duration of treatment / exposure:

The test substance was used in its original form. A single dose of 100 mg of the test substance was introduced into the conjunctival sac of the left eye after gently pulling the lower lid away from the eyeball. The lower and upper eyelids were held together for about one second to avoid any loss of test substance.

Observation period (in vivo):

Ocular reactions were observed approximately one hour, 24, 48 and 72 hours after the administration and then daily until the end of the study (day 22).

Number of animals or in vitro replicates:

3 animals

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): NO, The eyes were not rinsed after administration of the test substance.
- Time after start of exposure: NO

SCORING SYSTEM: Draize scale
Interpretation of the results:
Criteria for irritation
A substance or a preparation is considered irritant for the eyes if. when applied to the eye of the animal. significant severe ocular lesions are caused within 72 hours after exposure and which
persist for 24 hours or more after treatment with the test substance. All the scores at each reading time (24. 48 and 72 hours) and for an effect are used by calculating the respective mean values.

TOOL USED TO ASSESS SCORE: hand-slit lamp / biomicroscope / fluorescein

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24 h

Score:

2

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

48 h

Score:

4

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

72 h

Score:

2.3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48 h

Score:

1

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

3.3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

other: 48,72 hours

Score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #1

Time point:

24 h

Score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

48 h

Score:

2.7

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Time point:

72 h

Score:

3

Reversibility:

not specified

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

Slight to severe conjunctival reactions were observed in all animals: very slight to severe chemosis (grades 1 to 4). very slight to moderate redness of the conjunctivae (grade 1 to 3) and clear or whitish purulent discharge were noted. from day 1 up to day 17 at the latest.
A slight iritis (grade 1) was observed in two animals on day 2; it persisted up to day 12 or 14. Reading was masked by corneal opacity in the remaining animal.
A moderate or severe corneal opacity (grade 3 or 4) was noted in all animals on day 1: a slight to severe corneal opacity (grades 2 to 4) persisted up to the end of the observation period (day 22) in two animals. The remaining animal was killed on day 7 for ethical reasons, because of the severity of ocular reactions.
A whitening of the conjunctivae was noted in all animals on day 1; it persisted up to day 17 at the latest. Neovascularisation was noted in two animals from day 5 up to day 14 or from day 7 up to the end of the observation period (day 22) and alopecia around the eye was recorded in one animal, from day 11 up to day 20.

Individual ocular examinations and mean values of the scores recorded at each readings (24, 48 and 72 hours) for each animal

Rabbit number	Region of the eyes	Description of the ocular reactions	Scores				Mean irritation score	Interpretation (+)(-)
			1h D1	24h	48h	72h
1	Conjunctivae	chemosis	3	4	3	3	3.3	+
		Redness	2	3	3	3	3	+
		Discharge	2	S	S	S	(2)
	Iris		0	1	1	1	1	+
	Corneal opacity	intensity	3	2	2	2	2	+
		Area	2	4	4	3	3.7
	other		B	B	B	B
	Fluorescein		/	U	U	U
2	Conjunctivae	chemosis	3	3	3	3	3.0	+
		Redness	1	2	3	3	2.7	+
		Discharge	0	S	S	S	(2)
	Iris		OP	OP	OP	OP	(2)	(2)
	Corneal opacity	intensity	4	4	4	4	4	+
		Area	1	2	2	3	2.3
	other		B	B	B	B
	Fluorescein		/	U	U	U
3	Conjunctivae	chemosis	2	3	3	3	3	+
		Redness	2	3	3	3	3	+
		Discharge	0	S	S	S	(2)
	Iris		0	1	1	1	1	+
	Corneal opacity	intensity	4	3	2	2	2.3	+
		Area	2	2	2	2	(2)
	other		B	B	B	B
	Fluorescein		/	U	U	U

(1) = mean of scores on days 2, 3 and 4

h = hour

D = day

(+) = irritant according to EEC criteria

(-) = Non-irritant according to EEC criteria

(2) = not calculated.

U = Fluorescein batches Nos. 5691 and 7239

I = Fluorescein not used

S = Whitish purulent discharge

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Slight to severe conjunctival reactions were observed in all animals: very slight to severe chemosis (grades 1 to 4). very slight to moderate redness of the conjunctivae (grade 1 to 3) and clear or whitish purulent discharge were noted. from day 1 up to day 17 at the latest.
A slight iritis (grade 1) was observed in two animals on day 2; it persisted up to day 12 or 14. Reading was masked by corneal opacity in the remaining animal.
A moderate or severe corneal opacity (grade 3 or 4) was noted in all animals on day 1: a slight to severe corneal opacity (grades 2 to 4) persisted up to the end of the observation period (day 22) in two animals. The remaining animal was killed on day 7 for ethical reasons, because of the severity of ocular reactions.
Based on these observations, the test chemical was considered to be severely irritating to rabbit eyes.
A whitening of the conjunctivae was noted in all animals on day 1; it persisted up to day 17 at the latest. Neovascularisation was noted in two animals from day 5 up to day 14 or from day 7 up to the end of the observation period (day 22) and alopecia around the eye was recorded in one animal, from day 11 up to day 20.

Executive summary:

The potential of the test chemical to induce ocular irritation was evaluated in rabbits according to O.E.C.D. 405(24th February, 1987) and E.C. (92/69/E.E.C, B5- 31st July, 1992) Guidelines. 3 Male New Zealand White rabbits were used for the study. The test substance was used in its original form. A single dose of 100 mg of the test substance was introduced into the conjunctival sac of the left eye after gently pulling the lower lid away from the eyeball. The lower and upper eyelids were held together for about one second to avoid any loss of test substance. The right eye which remained untreated, served as a control. The eyes were not rinsed after administration of the test substance.

The eyes were examined approximately one hour, 24, 48 and 72 hours after administration of the test substance following the O.E.C.D. and E.C. guidelines:

· when there is no evidence of irritation after 1 hours. The study is ended.

· when there is persistent ocular irritation after 72 hours, the observation period is extended to a maximum of 21 days (until day 22) in order to determine the progress of the lesions and their reversibility.

· when severe irritant effects are observed, the animals are killed on humane ground.

Any change in the animals' behaviour was noted. Ocular reactions were observed approximately one hour, 24, 48 and 72 hours after the administration and then daily until the end of the study (day 22).

Slight to severe conjunctival reactions were observed in all animals: very slight to severe chemosis (grades 1 to 4). Very slight to moderate redness of the conjunctivae (grade 1 to 3) and clear or whitish purulent discharge were noted from day 1 up to day 17 at the latest.

A slight iritis (grade 1) was observed in two animals on day 2; it persisted up to day 12 or 14. Reading was masked by corneal opacity in the remaining animal.

A moderate or severe corneal opacity (grade 3 or 4) was noted in all animals on day 1: a slight to severe corneal opacity (grades 2 to 4) persisted up to the end of the observation period (day 22) in two animals. The remaining animal was killed on day 7 for ethical reasons, because of the severity of ocular reactions.

A whitening of the conjunctivae was noted in all animals on day 1; it persisted up to day 17 at the latest. Neovascularisation was noted in two animals from day 5 up to day 14 or from day 7 up to the end of the observation period (day 22) and alopecia around the eye was recorded in one animal, from day 11 up to day 20.

Based on these observations, the test chemical was considered to be severely irritating to rabbit eyes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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
103-82-2	1.5%	0.27%	Irritant	Category 1 or 2

 

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
103-82-2	1	0.068	0.069	0.023	0.024	0.023	1.4	0.025	0.004	1.5	0.27
	2	0.074	0.072	0.029	0.027	0.028	1.6

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The ocular irritation potential of test article was determined according to the OECD 492 test guideline. The mean % tissue viability of test chemical was determined to be 1.5%. Thus,the test chemical was considered to be irritating to MatTek EpiOcular Tissue 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 assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5. The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability. The difference in viability between identically treated tissues must be less than 20%.

The assay meets all the acceptance criteria mentioned.

The mean % tissue viability of test chemical was determined to be 1.5%. Thus,the test chemical was considered to be irritating to  MatTek EpiOcular Tissue Model OCL-200.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

In different studies, the test chemical has been investigated for potential to cause dermal irritation in living organisms to a greater or lesser extent. The studies are based on in vivo experiments in rabbits, guinea pigs, humans as well as estimated data and in vitro experiments for the test chemical. The results are summarized as follows:

A preliminary irritation test was performed on guinea pigs to determine the maximum non-irritating concentration for the Open Epicutaneous test. 6-8 male/female guinea pigs were used for the study.

0.025 ml of each test concentration (e.g, 100, 30, 10 and 3%) in ethanol, acetone, H20, petroleum, PEG and/or other suitable vehicles was applied to the one of the areas measuring 2 cm2 of the flank skin previously clipped and marked with a circular stamp. Reactions are read 24 h after the application of the test material. The minimal irritating and the maximal nonirritating concentrations were determined by an all-or-none criterion. The minimal irritating concentration is defined as the lowest one causing skin irritation. The maximal nonirritating concentration is defined as the highest one not causing macroscopic skin reactions in any of the animals.

No signs of irritation were observed in the 24 hours observation period.

Hence, the test chemical was considered to be not irritating to guinea pig skin.

This is supported by a study performed to determine the irritation potential of the test chemical in humans. 2% test chemical in petrolatum was applied to the skin of human volunteers in closed patch for 48 hours. The volunteers were observed for effect.

No signs of irritation were observed after 48 hours exposure to the test chemical. Hence, the test chemical can be considered to be not irritating to skin.

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. Hence, the test chemical can be considered not irritating to skin.

The dermal irritation potential of test article was further 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 show the assay quality controls were met, as the OD of the negative control tissues was between 1.195 and 1.430. Also, the positive control, 5% sodium dodecyl sulfate (SDS), reduced tissue viability to 4.5% of negative control and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was <18% passing the acceptance criteria.

The Mean % tissue viability compared to negative control (n=3) of the test chemical was determined to be 33.3%. Based on these values, the test chemical can be considered to irritating. It can be further classified under the category "Category 1/2" as per CLP Regulation.

The above in vitro result is further supported by a study performed according to OECD Test Guideline No. 431 “In Vitro Skin Irritation: Reconstructed Human Epidermis (RHE) Test Method” to determine the dermal corrosion potential of the test chemical. The EpiDermTM SCT allows discrimination between non-corrosive and corrosive chemicals according to the UN Globally Harmonized System of Classification and Labeling of Chemicals (GHS).

The MatTek EpiDermTM tissue cultures were obtained from MatTek In Vitro Life Science Laboratories. The MatTek EpiDermTM tissue cultures were placed in the refrigerator at 5°C. Prior to use, the tissues were incubated (37±1°C, 5±1% CO2) in 0.9 mL of fresh maintenance medium in 6 -well plates for ~one hour.

Before dosing, the tissues were moistened with 25microliter of sterile water to improve the contact of the tissue surface to the test article. Approximately 25 mg of solid test article was evenly applied to the apical surface of each tissue. Each treatment with test article or control was conducted in duplicate. The exposure period for the test articles and controls was 3 and 60 minutes. For the 60-minute exposure, the dosed tissues were placed in an incubator at 37±1°C, 5±1% CO2for the remainder of the 60-minute exposure period. Following the rinsing period, the MTT assay was performed by transferring the tissues to 24-well plates containing 300 microliter MTT medium (1.0 mg/mL). After 3 hours of incubation at 37±1°C, 5±1% CO2in a humified incubator, the blue formazan salt was extracted by submerging the tissues in 2 mL isopropanol (Lot #XXXX; provided by MatTek, Slovakia) in a 24 -well plate. The extraction time was approximately 2 hours and 05 minutes with gentle shaking. The optical density of the extracted formazan (200mL/well if a 96-well plate) was determined using a Thermo Scientific™ Multiskan™ FC Microplate Photometer at 570 nm. Relative cell viability was calculated for each tissue as % of the mean negative control tissue. A chemical was regarded to be corrosive if the relative tissue viability after 3 min treatment with a test chemical was decreased below 50%. In addition, materials were regarded to be non-corrosive after 3 min (viability>50%) were regarded to be corrosive if the relative tissue viability after 1-hour treatment with a test chemical was decreased below 15%. The assay passed all acceptance criteria as the ODs of the negative control exposed tissues were >0.8, the positive control exposed tissues were less than 15% viable and the standard deviation (SD) calculated from individual percent tissue viabilities of the test article exposed replicates was <30.

The mean tissue viability of the test chemical at 3minutes and 60 minutes exposure time were 90.7% and 9.1% respectively.

Based on these values, the test chemical can be considered to irritating. It can be further classified under the category "Category 1/2" as per CLP Regulation.

Even though the results of in vitro Guideline studies substantiate the claim that undiluted test chemical can indeed be irritating to skin, but the results from the remaining in vivo as well as estimation study present a stronger evidence of its not irritating properties.

Hence, taking all these factors into consideration, 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

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

The potential of the test chemical to induce ocular irritation was evaluated in rabbits according to O.E.C.D. 405(24th February, 1987) and E.C. (92/69/E.E.C, B5- 31st July, 1992) Guidelines. 3 Male New Zealand White rabbits were used for the study. The test substance was used in its original form. A single dose of 100 mg of the test substance was introduced into the conjunctival sac of the left eye after gently pulling the lower lid away from the eyeball. The lower and upper eyelids were held together for about one second to avoid any loss of test substance. The right eye which remained untreated, served as a control. The eyes were not rinsed after administration of the test substance.

The eyes were examined approximately one hour, 24, 48 and 72 hours after administration of the test substance following the O.E.C.D. and E.C. guidelines:

· when there is no evidence of irritation after 1 hours. The study is ended.

· when there is persistent ocular irritation after 72 hours, the observation period is extended to a maximum of 21 days (until day 22) in order to determine the progress of the lesions and their reversibility.

· when severe irritant effects are observed, the animals are killed on humane ground.

Any change in the animals' behaviour was noted. Ocular reactions were observed approximately one hour, 24, 48 and 72 hours after the administration and then daily until the end of the study (day 22).

Slight to severe conjunctival reactions were observed in all animals: very slight to severe chemosis (grades 1 to 4). Very slight to moderate redness of the conjunctivae (grade 1 to 3) and clear or whitish purulent discharge were noted from day 1 up to day 17 at the latest.

A slight iritis (grade 1) was observed in two animals on day 2; it persisted up to day 12 or 14. Reading was masked by corneal opacity in the remaining animal.

A moderate or severe corneal opacity (grade 3 or 4) was noted in all animals on day 1: a slight to severe corneal opacity (grades 2 to 4) persisted up to the end of the observation period (day 22) in two animals. The remaining animal was killed on day 7 for ethical reasons, because of the severity of ocular reactions.

A whitening of the conjunctivae was noted in all animals on day 1; it persisted up to day 17 at the latest. Neovascularisation was noted in two animals from day 5 up to day 14 or from day 7 up to the end of the observation period (day 22) and alopecia around the eye was recorded in one animal, from day 11 up to day 20.

Based on these observations, the test chemical was considered to be severely irritating to rabbit eyes.

The in vivo result is supported by an in vitro study performed according to OECD 492 test guideline to evaluate the ocular irritation potential of test article. 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 assay meets the acceptance criterion if the OD570 of the Negative Control is greater than 0.8 and less than 2.5. The assay meets the acceptance criterion if the mean relative viability of the positive control is below 50% of negative control viability. The difference in viability between identically treated tissues must be less than 20%.

The assay meets all the acceptance criteria mentioned.

The mean % tissue viability of test chemical was determined to be 1.5%. Thus, the test chemical was considered to be irritating to MatTek EpiOcular Tissue Model OCL-200.

The above in vitro result is further supported by another OECD 492 test guideline study 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 2.8%. Hence, under the experimental test conditions it was concluded that test substance was considered to be irritating to the human eyes and can thus be classified as ‘’Irritating to eyes in Category 2” as per CLP Regulation

The results from the Guideline studies indicate a very strong possibility that the test chemical causes moderate to severe irritation to eyes.The test chemical can be considered to be irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.

Justification for classification or non-classification

Even though the results of in vitro Guideline studies substantiate the claim that undiluted test chemical can indeed be irritating to skin, but the results from the remaining in vivo as well as estimation study present a stronger evidence of its not irritating properties.

Hence, taking all these factors into consideration, 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”.

The results from the Guideline studies indicate a very strong possibility that the test chemical causes moderate to severe irritation to eyes.The test chemical can be considered to be irritating to eyes. Comparing the above annotations with the criteria of CLP Regulation, the test chemical can be classified under the category “Category 2”.