Amides, adipic acid

Endpoint summary

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:

15 February 2019 to 22 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN model - reconstructed human epidermis

Details on animal used as source of test system:

reconstructed human epidermis

Vehicle:

physiological saline

Details on test system:

EpiDerm tissues, Lot no.: 29965 were received from MatTek and refrigerated at 2-8°C, Before use the tissues were incubated 37±1°C, 5±1% CO2 with assay medium for one hour equillibrium. The tissues were then moved to new wells with fresh medium for an additional overnight equillibrium, for approximately 18 h. Equillibrium medium was replaced with fresh medium before dosing.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Each test substance-treated tissue was moistened with 25 µL phosphate-buffered saline and 25 mg of test substance solid material were applied to each tisssue
Negative control: 30 µL of DPBS
positive control: 30 µL of 5% SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Vehicle:

physiological saline

Irritation / corrosion parameter:

% tissue viability

Value:

105

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Chemicals that provide tissue viabilities in a range of 30 – 70 % may provide high standard deviation (SD). If the high SD was typical for the chemical and the classification of the chemical was consistent in all independent runs, MatTek recommends that this result be accepted, although it did not meet the Assay Acceptance Criterion.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not irritating to skin.

Executive summary:

A study was conducted to determine the skin irritation potential of the test substance according to OECD Guideline 439 in compliance with GLP. The skin irritation potential was examined using the EpiDerm reconstructed human epidermis model following a treatment period of 60 min and a post-exposure incubation period of 42 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of the main test, tissues were treated with the test substance for an exposure period of 60 min. At the end of the exposure period, each tissue was rinsed before incubating for 42 h at 37 ±1°C, 5 ±1% CO2 in air. The relative mean viability of the test substance treated tissues was 105.0 %. Under the study conditions, the test substance was not irritating to skin (Troese, 2019).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 January 2018 to 11 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

EpiDerm™ tissues, Lot No. 27667 Kit F, were received from MatTek Corporation (Ashland, MA) and refrigerated at 2-8°C. Before use, tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test substance

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

dublicate

Details on study design:

Test Substance Reduction of MTT:
100 mg of the test substance were mixed with 1 mL of MTT solution (1 mg/mL Thiazolyl blue tetrazolium bromide diluted in Dulbecco's Modified Eagle's Medium (DMEM)). A negative control, 100 μL of tissue culture water was tested concurrently. The solutions were incubated at room temperature in the dark for 60 minutes. After incubation, the solutions were visually inspected for purple coloration, which indicates that the test substance reduced MTT. Since tissue viability is based on MTT reduction, direct reduction by a test substance can exaggerate viability, making a test substance seem less irritating than its actual irritation potential. The test substance did not reduce MTT and the assay continued as per the protocol.

Dosing:
The test substance was broken into small pieces using a mortar and pestle prior to application. 25 mg of the test substance, plus 25 μL of TCH2O, were applied to the top of each EpiDerm™ tissue. The test substance remained in contact with the EpiDerm™ tissue for 3 minutes at room temperature and 60 minutes at 37±1°C, 5±1% CO2. A negative control (50 μL of TCH2O) and a positive control (50 μL of KOH) were tested in the same manner. Nylon mesh was placed on top of each control to facilitate even distribution of the material. Each treatment with test substance or control was conducted in duplicate.

Tissue Viability (MTT Reduction):
At the end of the exposure period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well microplate 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 overnight at room temperature with 2.0 mL. of extractant solution (isopropanol) per well. The absorbency of an aliquot of the extracted MTT formazan was measured in triplicate at 540 nm using a microplate reader.

μQuant Plate Calibration:
To confirm optical performance of the microplate reader, a certified BioTek® Absorbance Test Plate is used to perform a Calibration Plate Analysis daily, before each use which confirms the alignment, optical density accuracy, linearity, repeatability, and wavelength accuracy of the μQuant. All calibrations passed during the course of the study.

Irritation / corrosion parameter:

% tissue viability

Value:

107

Negative controls validity:

valid

Remarks:

mean viability in 3 min and 60 min is 100%

Positive controls validity:

valid

Remarks:

Mean viability in 3 min and 60 min is 41.1% and 2.3% respectively

Remarks on result:

no indication of irritation

Quality Controls:

The mean OD of the negative control tissues was 1.597 at 3 minutes and 1.529 at 60 minutes, which met the acceptance criterion (OD = 0.8).

The mean relative tissue viability of the 60-minute positive control was 2.3%, which met the acceptance criterion <15%). Viability differences between the two identically treated tissues in all samples and controls at 3 minutes were 2.0% to 8.5%. Viability differences between the two identically treated tissues at 60 minutes were 0.7% to 6.5%. Inter-tissue viability differences at both time points met the acceptance criterion.

Test substance results:

The test article was tested using the MatTek EpiDermTM Skin Corrosivity Test . Optical Density values include the background subtraction of an isopropanol (blank) value.

 

Test and control substance	Mean viability (3 min) (%)	Mean viability (60 min) (%)	Predicted corrosivity
Test substance	106.8	107.1	Non-corrosive
Negative Control)	100.0	100.0	Non-corrosive
Positive Control	41.1	2.3	corros

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not corrosive to skin.

Executive summary:

A study was conducted to determine the skin corrosivity potential of the test substance according to OECD Guideline 431, in compliance with GLP. The skin corrosive potential was examined using the EpiDerm reconstructed human epidermis model following a treatment period of 3 min and 60 min .The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of the main test, duplicate tissues were treated with the test substance for an exposure period of 3 min a room temperature and 60 min 37±1°C, 5±1% CO2. A negative control (50 μL of TCH2O) and a positive control (50 μL of KOH) were tested in the same manner. Nylon mesh was placed on top of each control to facilitate even distribution of the material. At the end of the exposure period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well microplate containing 300 μL of MTT solution. The tissues were then returned to the incubator for a 3 h MTT incubation period. The percent mean viability of test substace for 3 min and 60 min was found to be 106.8% and 107.1% respectively. Under the study conditions, the test substance was not corrosive to skin ( Troese, 2019).

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 February, 2020 to 28 February, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

Albino

Details on test animals or test system and environmental conditions:

Number of Animals: 3
Sex: Female, nulliparous and non-pregnant.
Species/Strain: Rabbit/New Zealand albino.
Age/Body Weight: Young adult (14 weeks)/2172-2223 grams at experimental start.
Source: Received from Robinson Services, Inc. on February 19, 2020.

Husbandry
Housing: The animals were singly housed in suspended stainless steel caging, which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.
Animal Room Temperature and Relative Humidity Ranges: 18-25°C and 48-61%, respectively.
Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs.
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 6 days
Food: PMI 5326 High Fiber Rabbit Diet. A designated amount of the diet (approximately 150 grams/day) and alfalfa hay cubes (Urainland Select Alfalfa Cubes) were available to each rabbit.
Water: Filtered tap water was supplied ad libitum.
Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs.

Identification
Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal. This number, together with a sequential animal number assigned to study 52413, constituted unique identification. Only the sequential animal number is presented in this report.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

no

Amount / concentration applied:

The test substance, as received, was a solid. Prior to use, the test substance was ground in a coffee mill. In order to ensure adequate contact with the skin, the test substance was applied as a dry paste (90% w/w mixture in distilled water). Preliminary sample preparation assessments conducted by PSL indicated that mixtures in excess of 90% (i.e., 95%) was too dry to assure adequate skin contact.

Duration of treatment / exposure:

4 h

Observation period:

Individual dose sites were scored according to the Draize scoring system at approximately 30-60 minules, 24, 48, and 72 hours after patch removal.

Number of animals:

3

Details on study design:

Preparation and Selection of Animals
On the day before application, a group of animals was prepared by clipping the dorsal area of the trunk. On the day of dosing, but prior to application, the animals were examined for health and the skin checked for any abnormalities. Three healthy, naive animals (not previously tested) without pre-existing skin irritation were selected for test.

Application of Test Substance
Five-tenths of a gram of the test substance (0.56 g of the prepared test mixture) was applied to one 6-cm2 intact dose site on each animal and covered with a 1-inch x 1-inch, 4-ply gauze pad. The pad and entire trunk of each animal were then wrapped with semi-occlusive 3-inch Micropore tape to avoid dislocation of the pad. Elizabethan collars were placed on each rabbit and they were returned to their designated cages. After 4 hours of exposure to the test substance, the pads and collars were removed and the dose sites were gently cleansed with a 3% soap solution to remove test substance followed by tap water and a clean paper towel to remove any residual test substance.

Evaluation of Dose Sites
Individual dose sites were scored according to the Draize scoring system at approximately 30-60 minules, 24, 48, and 72 hours after patch removal. The classification of irritancy was obtained by adding the average erythema and edema scores for the 30-60 minute, 24, 48, and 72-hour scoring intervals and dividing by the number of evaluation intervals. The resulting Primary Dermal Irritation Index (PDII) was classified as follows:

PDII Classification
0 Non-irritating
>0 - 2 Slighlty irritating
2.1 - 5 Moderately irritating
>5 Severly Irritating

In-life observation
The animals were observed for signs of gross toxicity and behavioral changes at least once daily during the test period.

Body Weights
Individual weights of animals were recorded shortly before application of the test substance (initial) and at the completion of testing (terminal).

Study Termination
Once testing was complete, the animals were released for euthanasia and humanely euthanized.

Statistical analysis
Statistical analysis was limited to the calculation of the mean irritation scores.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

Within 30-60 minutes of patch removal, three treated sites exhibited very slight erythema and/or very slight edema. At the 24-hour scoring interval one rabbit had very slight erythema and all animals were free of dermal irritation by 48 hours. The Primary Dermal Irritation Index for test substabce was 0.5.

Other effects:

All animals appeared active and healthy and gained body weight during the study. Apart from the dermal irritation noted, there were no other signs of gross toxicity, adverse clinical effects, or abnormal behavior.

Results

Table 1: Individual body weight

Animal No.	Sex	Body weight (g)
		Initial	Terminal
3501	F	2199	2366
3502	F	2172	2239
3503	F	2223	2639

Table 2: In-life observation

Animal Number	Animal Sex	Observation	Day of Observation (x=observation is present)
			0	1	2	3
3501	F	Active and healthy	X	X	X	X
3502	F	Active and healthy	X	X	X	X
3503	F	Active and healthy	X	X	X	X

Table 3: Erythem/Edema scores

Animal No.	Sex	Time After Patch Removal
		30-60 min	24 hrs	48 hrs	72 hrs
3501	F	1/0	1/0	0/0	0/0
3502	F	1/1	0/0	0/0	0/0
3503	F	1/1	0/0	0/0	0/0
Total		3/2	1/0	0/0	0/0
Mean		1.0/0.7	0.3/.0	0/0	0/0

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not irritating to rabbit skin.

Executive summary:

A study was conducted to determine the dermal irritation potential of  the test substance in rabbits, according to the OECD Guideline 404, in compliance with GLP. Five-tenths of a gram of the test substance was moistened with distilled water and applied to the clipped skin of three healthy female rabbits for 4 h. Following exposure, dermal irritation was evaluated by the Draize method of scoring after patch removal at 30-60 mins, 24, 48 and 72 h. Within 30-60 minutes of patch removal, three treated sites exhibited very slight erythema and/or very slight edema. At the 24-hour scoring interval one rabbit had very slight erythema and all animals were free of dermal irritation by 48 hours. All animals appeared active and healthy and gained body weight during the study. Apart from  the dermal irritation noted, there were no other signs of gross toxicity, adverse clinical effects, or abnormal behavior. The Primary Dermal Irritation Index (PDII) calculated for this test substance was 0.5. Under the study conditions, the test substance was not irritating to rabbit skin (Durando, 2020).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2019 to 22 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

GLP compliance:

yes

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ tissues, Lot No. 27466 Kit B, were received from MatTek and refrigerated at 2-8°C. Before use, the tissues were incubated (37±1°C, 5 ±1 % CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium for an additional overnight equilibration of 16 to 24 .After the overnight incubation, the tissues were moistened with 20 μl of phosphate-buffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 min.

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test substance was ground to a fine powder and an aliquot of 50 mg of the test substance were mixed with 1 mL of MTT solution (1 mg/mL methyl thiazole tetrazolium diluted in Dulbecco's Modified Eagle's Medium.

Duration of treatment / exposure:

6 h ± 15 min

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

duplicate

Irritation parameter:

other: tissue viability

Remarks:

The tissue viability of tissue substace was found to be 98.1%

Negative controls validity:

valid

Remarks:

The mean OD570 of the negative control tissues was 1.972, which met the acceptance criteria of greater than 0.8 and less than 2.5

Positive controls validity:

valid

Remarks:

The mean relative viability of the positive control tissues was 30.2%, which met the acceptance criterion of less than 50%.

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The differences in viability between identically treated tissues were 1.24% to 11.76%, which met the acceptance criterion of less than 20%.

The R-squared value calculated for the plate reader linearity check was 0.9997,which met the acceptance criterion of greater than 0.999. All other quality controls passed the acceptance criteria.

The experimental data and summarized results and irritation classifications are as follows

Test and Control Substance Identity	Tissue No.	Mean of Aliquots	% Viability	OD		Viabilities (%)		Irritancy classification
				Mean	S.D.	Mean	S.D.
TR063A L#252-PE	1	1.818	92.2	1.934	0.232	98.1	11.76	Non-irritant
	2	2.050	104.0
TCH2O (Negative Control)	1	1.859	94.3	1.972	0.226	100.0	11.46	Non-irritant
	2	2.085	105.7
Methyl Acetate (Positive Control)	1	0.583	29.6	0.596	0.025	30.2	1.24	Irritant
	2	0.608	30.8

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not irritating to eye.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 492 in compliance with GLP. Eye irritation potential was examined using reconstructed Human Cornea-like Epithelium (RhCE) following a treatment period of 6 h and a post-exposure incubation period of 18 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed Human Cornea-like Epithelium cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of dosing, duplicate tissues were treated with the test substance for an exposure period of 6 h at 37 ±1°C, 5% ±1 CO2 in air. At the end of the exposure period, each tissue was rinsed and soaked tissue were then incubated in fresh assay medium at incubated at 37 ±1°C, 5 ±1°% CO2 for 18 h. At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μL of MTT solution. The tissues were then returned to the incubator for a 3 h MTT incubation period. The relative mean viability of the test substance treated tissues was 98.1 %. Under the study conditions, the test substance was not irritating to eye (Troese, 2019).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 January 2018 to 11 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes

Species:

cattle

Strain:

other: in vitro bovine cornea study

Details on test animals or tissues and environmental conditions:

Bovine eyes (greater than 35 weeks of age) were obtained from an abattoir and transported to the MB Research in a refrigerated container containing Hanks’ Balanced Salt Solution (HBSS) with penicillin-streptomycin. The bovine eyes were transported to MB Research on 11 Jan 2018, within 24 hours of harvest.

Vehicle:

other: 0.9% Sodium Chloride Irrigation, USP (saline), Lot No. J6S983

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Three bovine corneas per group were dosed with 0.75 g of TR063A L#239, 0.75 mL of Minimal Essential Media (MEM, negative control), or a 20% formulation of imidazole in 0.9% saline (positive control).

Duration of treatment / exposure:

4 h

Number of animals or in vitro replicates:

Three corneas were allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substance.

Details on study design:

Test System:
Bovine eyes (greater than 35 weeks of age) were obtained from an abattoir and transported to the MB Research in a refrigerated container containing Hanks’Balanced Salt Solution (HBSS) with penicillin-streptomycin. The bovine eyes were transported to MB Research on 11 Jan 2018, within 24 h of harvest.

Pretest Procedures:
Fresh assay solutions were prepared prior to use. MEM solution was prepared by stirring together one jar (9.3 g) of MEM powder (sufficient to make one liter of solution), 2.2 g of sodium bicarbonate, 0.292 g Lglutamine, 10 mL of fetal bovine serum (FBS) and brought to a final volume of 1000 mL with distilled water.
The MEM solution was kept in a 32 (±1)°C incubator for the duration of testing. HBSS was prepared by stirring together HBSS powder (sufficient to make one liter) and 0.35 g of sodium bicarbonate and brought to a final volume of 1000 mL with distilled water. HBSS was maintained at room temperature.
In addition, MEM solution with phenol red was prepared by stirring together 9.3 g of MEM with phenol red (sufficient to make 1 L), 2.2 g of sodium bicarbonate, 0.292 g L-glutamine, 10 mL of fetal bovine serum (FBS) and brought to a final volume of 1000 mL with distilled water. The MEM solution with phenol red was kept in a 32 (±1)°C incubator for the duration of testing. The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded.
Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders (MC2, formerly Electro-Design – the manufacturer of the Op-KIT opacitometer) that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium.The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects. Prior to collection of the pretest opacity scores, the OP-KIT opacitometer (Electro-Design Corporation,
RIOM, France) was calibrated. The calibrator was placed into the calibration chamber of the opacitometer. The opacitometer was first calibrated to zero opacity units by measuring the opacity without a calibrator (i.e., a polyester filter insert provided with the OP-KIT). Then the opacitometer is calibrated to 75 opacity units using calibrator filter 1. During the test, if the opacity scores exceed 75, the opacitometer is recalibrated in the same manner, using the other calibrators provided with the OP-KIT equipment (i.e., calibrator filter 2 or 3 that represent opacity of 150 and 225, respectively). A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied by the opacitometer. Fresh MEM was added before taking these baseline opacity readings. Any cornea with a value greater than 7 units was discarded.
The entire holder was incubated at 32 (±1)°C and allowed to equilibrate for at least one hour, but not longer than 2 h.

Study Procedure
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 g of the test article, 0.75 mL of 20% (w/v) imidazole formulation in saline, MEM solution or 0.9% saline was applied to the epithelium of each of the three test substance corneas, three positive control corneas and three negative control corneas in a manner which ensured that the entire cornea was covered. The test substance corneas were dosed via the open-chamber method. The negative, positive, and vehicle controls were dosed via the closed-chamber method.
All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C water jacketed incubator (VWR, model: 1815 TC) incubator. After 4 h (±10 min), the 20% test substance formulation, 20% imidazole formulation, MEM or 0.9% saline was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations.
Immediately following the four-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 mL of 0.5% sodium fluorescein solution in Dulbecco's phosphate buffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea.
The Spectronic 20-D Colorimeter Spectrophotometer (Milton/Roy, model: 333175) was calibrated prior to use on the same day of dosing. Calibration entailed first adjusting the wavelength to 490 nm and the transmittance to 0.0%. The transmittance for a sample of fresh distilled water (in a cuvette, inserted into the sample compartment) was adjusted to 100.0% on the spectrophotometer. The mode was then switched to absborance before collecting optical density study data. After 90 (±5) minutes, the fluid from the posterior chamber of each corneal holder was removed and the amount of dye that passed through the cornea (permeability) was measured as the optical density at 490nm (i.e., the OD490 nm) by a spectrophotometry.

Irritation parameter:

in vitro irritation score

Run / experiment:

The two endpoints opacity and permeability were combined in an empirically derived formula to generate an in vitro irritancy score (IVIS

Value:

4.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

In vitro irritancy score is 1.78

Positive controls validity:

valid

Remarks:

In vitro irritancy score is 74.78

Remarks on result:

other: The result of this study has identified the test substance has not causing serious eye damage , but they do not permit conclusion that the test substance does not require classification for eye irritation.

Calculated in vitro irritancy scores

Scores	Test substance	Negative control	Positive control
4-Hour Corrected Mean Opacity Score	3.67 + (15 x 0.027)	1.33 + (15 x 0.030)	61.67 + (15 x 0.874)
	3.67 + 0.405	1.33 + 0.45	61.67 + 13.11
IVIS	4.08	IVIS = 1.78	IVIS = 74.78

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, no conclusion on eye irritation potential could be made.

Executive summary:

A study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were greater than 35 weeks old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. 0.75 g of the test substancewas applied to the epithelium of each of the three test substance corneas.The test substance corneas were dosed via the open-chamber method.All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C water jacketed incubator. After 4 h (±10 minutes), the test substance formulation, imidazole formulation, MEM or 0.9% saline was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations. Immediately following the 4 h opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco's phosphate buffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior side up)ensuring contact of the fluorescein with the cornea.

An in vitro irritancy score (IVIS) was calculated using corneal opacity and permeability test. The in vitro irritancy scores are summarized as follows: for the test substance: 4.08, negative control: 1.78, positive control: 74.78. Under the study conditions, the test substance cause mild irritation to the bovine corneas,. However, based on the results, no prediction for eye irritation potential could be made (Yasso, 2018).

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Number of Animals: 3
Sex: Female, nulliparous and non-pregnant.
Species/Strain: Rabbit/New Zealand albino.
Age/Body Weight: Young adult (15 weeks)/2466-2766 grams at experimental start.
Source: Received from Robinson Services, Inc. on February 19, 2020.

Husbandry
Housing: The animals were singly housed in suspended stainless steel caging, which conforms to the size recommendations in the most recent Guide for the Care and Use of Laborat01y Animals (Natl. Res. Council, 2011). Enrichment (e.g., toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.
Animal Room Temperature and Relative Humidity Ranges: 19-23°C and 45-55%, respectively.
Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs.
Photoperiod: 12-hour light/dark cycle
Acclimation Period: 12 days
Food: PMI 5326 High Fiber Rabbit Diet. A designated amount of the diet (approximately 150 grams/day) and alfalfa hay cubes (Urainland Select Alfalfa Cubes) were available to each rabbit.
Water: Filtered tap water was supplied ad libitum.
Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs.

Identification
Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
Animal: A number was allocated to each rabbit on receipt and a stainless steel ear tag bearing this number was attached to the animal. This number, together with a sequential animal number assigned to study 52412, constituted unique identification. Only the sequential animal number is presented in this report.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.071 gm

Duration of treatment / exposure:

72 h

Observation period (in vivo):

72 h

Number of animals or in vitro replicates:

3

Details on study design:

Preparation and Selection of Animals
Prior to test initiation, both eyes of a group of animals were examined using a white light source and a fluorescein dye procedure. One drop of ophthalmic fluorescein sodium dye was instilled into both eyes of each rabbit. The eyes were rinsed with physiological saline (0.9% NaCl) after instillation of the fluorescein and then evaluated for corneal damage using an ultraviolet light source. Prior to test substance instillation, the eyes were re-examined and scored for abnormalities according to the "Scale for Scoring Ocular Lesions" (Draize et al., 1944). Three healthy, naive animals (not previously tested) without pre-existing ocular irritation were selected for test.

A systemic analgesic (Buprenorphine SR®) was administered to relieve potential discomfort associated with eye irritation which provides therapeutic relief for periods of up to 76 hours. Prior to test substance instillation, 0.1 mg/kg of body weight of the analgesic was administered to the animals and at appropriate intervals to maintain therapeutic blood levels.

Preparation of Test Substance
The test substance, as received, was a solid. Prior to use, the test substance was ground in a coffee mill. The pH was determined for the test substance prior to the instillation and was within a pH range of 2 and 11.5, therefore testing proceeded. The procedure used and the results are retained in the raw data.

Instillation
Prior to instillation, 1-2 drops of ocular anesthetic (Tetracaine Hydrochloride Ophthalmic Solution USP, 0.5%) were placed into both the treated and control eye of each animal. One-tenth of a milliliter (0.071 grams) of the test substance was then instilled into the conjunctiva! sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently held together for about one second before releasing to minimize loss of the test substance. The other eye of each rabbit remained untreated and served as a control. The rabbits were then returned to their designated cages.

Ocular Scoring
Ocular irritation was evaluated using a white light source in accordance with the Draize method of scoring at 1, 24, 48, and 72 hours post-instillation. The fluorescein dye evaluation procedure was used in the treated eye at 24 hours to verify the absence of corneal damage. Individual scores were recorded for each animal. In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted. The average score for all rabbits at each scoring period was calculated to aid in data interpretation.

Classification of Eye Scores
The time interval with the highest mean score (Maximum Mean Total Score - MMTS) for all rabbits was used to classify the test substance by the system of Kay and Calandra (Kay & Calandra, 1962).

In-life Observations
The animals were observed for signs of gross toxicity and behavioral changes at least once daily during the test period.

Body Weights
Individual weights of animals were recorded shortly before instillation of the test substance (initial) and at the completion of testing (terminal).

Study Tennination
Once testing was complete, the animals were released for euthanasia and humanely euthanized.

Statistical analysis
Statistical analysis was limited to the calculation of the mean irritation scores.

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

24/48/72 h

Score:

ca. 6.7

Max. score:

110

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible

Irritant / corrosive response data:

One hour after test substance instillation, one treated eye exhibited 'positive' conjunctival chemosis and the remaining two treated eyes exhibited minimal conjunctival redness and disch arge. There was no corneal opacity or iritis observed in any treated eye during this study. The overall incidence and severity of irritation (conjunctival redness, chemosis and discharge) decreased with time. Positive irritation (conjunctiva! chemosis) cleared from the treated eye by 24 hours. All animals were free of ocular irritation by 48 h. The Maximum Mean Total Score of test substance was 6.7. Under the study conditions, the test substance is minimally irritating to the eye. Based on the results of this study, the test substance is not classified according to the criteria of CLP.

Other effects:

All animals appeared active and healthy. Although one animal lost body weight, it was not considered to be significant. The two remaining animals gained body weight during the study.

Results

Table 1: Body weights

Animal No.	Sex	Body weight (g)
		Initial	Terminal
3401	F	2466	2763
3402	F	2766	2764
3403	F	2685	2767

Table 2: In-life observations

Animal Number	Animal Sex	Observation	Day of Observation (x=observation is present)
			0	1	2	3
3401	F	Active and healthy	X	X	X	X
3402	F	Active and healthy	X	X	X	X
3403	F	Active and healthy	X	X	X	X

Table 3: Scores for eye irritation

	Rabbit No.: 3401 (Female)				Rabbit No.: 3402 (Female)				Rabbit No.: 3403 (Female)
	Hour				Hour				Hour
	1	24	48	72	1	24	48	72	1	24	48	72
I.Cornea
A.Opacity	0	0*	0	0	0	0*	0	0	0	0*	0	0
B. Area	4	4	4	4	4	4	4	4	4	4	4	4
(AxB)xS	0	0	0	0	0	0	0	0	0	0	0	0
II. Iris
A.Values	0	0	0	0	0	0	0	0	0	0	0	0
Ax5	0	0	0	0	0	0	0	0	0	0	0	0
III.Conjunctivae
A.Redness	1	1	0	0	1	1	0	0	1	1	0	0
B. Chemosis	1	0	0	0	2	0	0	0	1	0	0	0
C. Discharge	1	0	0	0	1	0	0	0	1	0	0	0
(A+B+C)x2	6	2	0	0	8	2	0	0	6	2	0	0
Total	6	2	0	0	8	2	0	0	6	2	0	0

* Ophthalmic fluorescein sodium dye was used to verify the absence of corneal opacity

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was not irritating to rabbit eye.

Executive summary:

A study was conducted to determine the eye irritation potential of the test substance in rabbits according to the OECD Guideline 405, in compliance with GLP. Three healthy, naive animals without pre-existing ocular irritation were selected for the study. Prior to instillation, 1-2 drops of ocular anesthetic (Tetracaine hydrochloride ophthalmic solution USP, 0.5%) were placed into both the treated and control eye of each animal. One-tenth of a milliliter (0.071 g) of the test substance was then instilled into the conjunctival sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently held together for about one second before releasing to minimize loss of the test substance. The other eye of each rabbit remained untreated and served as a control. Ocular irritation was evaluated using a white light source in accordance with the Draize method of scoring at 1, 24, 48 and 72 h post-instillation. The fluorescein dye evaluation procedure was used in the treated eye at 24 h to verify the absence of corneal damage. Individual scores were recorded for each animal. In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted. The average score for all rabbits at each scoring period was calculated to aid in data interpretation. The time interval with the highest mean score (Maximum Mean Total Score - MMTS) for all rabbits was used to classify the test substance by the system of Kay and Calandra. The animals were observed for signs of gross toxicity and behavioral changes at least once daily during the test period. Individual weights of animals were recorded shortly before instillation of the test substance (initial) and at the completion of testing (terminal). All animals were free of ocular irritation by 48 h. The MMTS of the test substance was calculated to be 6.7. All animals appeared active and healthy. One hour after test substance instillation, one treated eye exhibited positive conjunctival chemosis and the remaining two treated eyes exhibited minimal conjunctival redness and discharge. There was no corneal opacity or iritis observed in any treated eye during this study. The overall incidence and severity of irritation (conjunctival redness, chemosis and discharge) decreased with time. Positive irritation (conjunctival chemosis) cleared from the treated eye by 24 h. Under the study conditions, the test substance was not irritating to rabbit eye (Ellis, 2020).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation, in vitro, human skin model (EpiDerm reconstructed human epidermis)  

 

A study was conducted to determine the skin irritation potential of the test substance according to OECD Guideline 439, in compliance with GLP. The skin irritation potential was examined using the EpiDerm reconstructed human epidermis model following a treatment period of 60 min and a post-exposure incubation period of 42 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of the main test, tissues were treated with the test substance for an exposure period of 60 min. At the end of the exposure period, each tissue was rinsed before incubating for 42 h at 37 ± 1°C, 5 ± 1% CO2 in air. The relative mean viability of the test substance treated tissues was 105.0 %. Under the study conditions, the test substance was not irritating to skin (Troese, 2019). 

 

Skin Corrosivity Test, in vitro, human skin model  

 

A study was conducted to determine the skin corrosivity potential of the test substance according to OECD Guideline 431, in compliance with GLP. The skin corrosive potential was examined using the EpiDerm reconstructed human epidermis model following a treatment period of 3 min and 60 min .The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of the main test, duplicate tissues were treated with the test substance for an exposure period of 3 min a room temperature and 60 min 37± 1°C, 5± 1% CO2. A negative control (50μL of TCH2O) and a positive control (50μL of KOH) were tested in the same manner. Nylon mesh was placed on top of each control to facilitate even distribution of the material. At the end of the exposure period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well microplate containing 300μL of MTT solution. The tissues were then returned to the incubator for a 3 h MTT incubation period. The percent mean viability of test substance for 3 min and 60 min was found to be 106.8% and 107.1% respectively. Under the study conditions, the test substance was not corrosive to skin (Troese, 2019). 

 

Skin irritation, in vivo, rabbit study

 

A study was conducted to determine the dermal irritation potential of the test substance in rabbits according to OECD Guideline 404, in compliance with GLP. Five-tenths of a gram of the test substance was moistened with distilled water and applied to the clipped skin of three healthy female rabbits for 4 h. Following exposure, dermal irritation was evaluated by the Draize method of scoring after patch removal at 30-60 minutes, 24, 48 and 72 h. Within 30-60 minutes of patch removal, three treated sites exhibited very slight erythema and/or very slight edema. At the 24 h scoring interval, one rabbit had very slight erythema and all animals were free of dermal irritation by 48 h. All animals appeared active and healthy and gained body weight during the study. Apart from the dermal irritation noted, there were no other signs of gross toxicity, adverse clinical effects or abnormal behaviour. The Primary Dermal Irritation Index (PDII) was 0.5. Under the study conditions, the test substance was not irritating to rabbit skin (Durando, 2020). 

 

Eye irritation, in vitro, human eye model (reconstructed human cornea-like epithelium)  

 

A study was conducted to determine the eye irritation potential of the test substance according to OECD Guideline 492, in compliance with GLP. Eye irritation potential was examined using reconstructed Human Cornea-like Epithelium (RhCE) following a treatment period of 6 h and a post-exposure incubation period of 18 h. The principle of the assay is based on the measurement of cytotoxicity in reconstructed Human Cornea-like Epithelium cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue formazan salt in the substance treated tissues relative to the negative controls. To identify possible interference of the test substance with the MTT endpoint, the test substance was checked for the ability to directly reduce MTT prior to the main assay. On the day of dosing, duplicate tissues were treated with the test substance for an exposure period of 6 hat 37±1°C, 5%±1CO2 in air. At the end of the exposure period, each tissue was rinsed and soaked tissue were then incubated in fresh assay medium at incubated at 37±1°C, 5±1°% CO2 for 18 h. At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300μL of MTT solution. The tissues were then returned to the incubator for a 3 h MTT incubation period. The relative mean viability of the test substance treated tissues was 98.1 %. Under the study conditions, the test substance was not irritating to eye (Troese, 2019).  

 

Eye irritation, in vitro bovine corneal opacity and permeability test (BCOP)  

 

A study was conducted to determine the corneal damage potential of the test substance according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage is assessed by quantitative measurements of changes in corneal opacity and permeability. Bovine corneas were collected from slaughtered cattle which were greater than 35 weeks old. One valid experiment was performed with three replicates for negative control, positive control and the test substance. 0.75 g of the test substance was applied to the epithelium of each of the three test substance corneas. The test substance corneas were dosed via the open-chamber method. All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C water jacketed incubator. After 4 h (±10 minutes), the test substance formulation, imidazole formulation, MEM or 0.9% saline was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations. Immediately following the 4 h opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco’s phosphate buffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior side up)ensuring contact of the fluorescein with the cornea. An in vitro irritancy score (IVIS) was calculated using corneal opacity and permeability test. The in vitro irritancy scores are summarized as follows: for the test substance: 4.08, negative control: 1.78, positive control: 74.78. Under the study conditions, the test substance cause mild irritation to the bovine corneas. However, based on the results, no prediction for eye irritation potential could be made (Yasso, 2018). 

 

In vivo eye irritation test, rabbit study 

 

A study was conducted to determine the eye irritation potential of the test substance in rabbits according to the OECD Guideline 405, in compliance with GLP. Three healthy, naive animals without pre-existing ocular irritation were selected for the study. Prior to instillation, 1-2 drops of ocular anaesthetic (Tetracaine hydrochloride ophthalmic solution USP, 0.5%) were placed into both the treated and control eye of each animal. One-tenth of a milliliter (0.071 g) of the test substance was then instilled into the conjunctival sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently held together for about one second before releasing to minimize loss of the test substance. The other eye of each rabbit remained untreated and served as a control. Ocular irritation was evaluated using a white light source in accordance with the Draize method of scoring at 1, 24, 48 and 72 h post-instillation. The fluorescein dye evaluation procedure was used in the treated eye at 24 h to verify the absence of corneal damage. Individual scores were recorded for each animal. In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted. The average score for all rabbits at each scoring period was calculated to aid in data interpretation. The time interval with the highest mean score (Maximum Mean Total Score - MMTS) for all rabbits was used to classify the test substance by the system of Kay and Calandra. The animals were observed for signs of gross toxicity and behavioural changes at least once daily during the test period. Individual weights of animals were recorded shortly before instillation of the test substance (initial) and at the completion of testing (terminal). All animals were free of ocular irritation by 48 h. The MMTS of the test substance was calculated to be 6.7. All animals appeared active and healthy. One hour after test substance instillation, one treated eye exhibited positive conjunctival chemosis and the remaining two treated eyes exhibited minimal conjunctival redness and discharge. There was no corneal opacity or iritis observed in any treated eye during this study. The overall incidence and severity of irritation (conjunctival redness, chemosis and discharge) decreased with time. Positive irritation (conjunctival chemosis) cleared from the treated eye by 24 h. Under the study conditions, the test substance was not irritating to rabbit eye (Ellis, 2020). 

Justification for classification or non-classification

Based on the results of in vitro as well as in vivo skin irritation/corrosion studies, no classification for skin and eye irritation is warranted according to EU CLP (Regulation EC/1272/2008) criteria.