N-glycylglycine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 18 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

18 June 2019.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) SOP; For the prediction of acute ocular irritation of chemicals. For use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model

Version / remarks:

10 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-glycylglycine

EC Number:

209-127-8

EC Name:

N-glycylglycine

Cas Number:

556-50-3

Molecular formula:

C4H8N2O3

IUPAC Name:

N-glycylglycine

Test material form:

solid: crystalline

Details on test material:

Batch Number: S119 0711
Storage conditions: room temperature

Specific details on test material used for the study:

Batch-No.: S119 0711
Storage: room temperature, protected from light

Test animals / tissue source

Species:

other: EpiOcular™ human cell construct (MatTek In Vitro Life Science Laboratories)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

POSITIVE CONTROL
- Amount applied: 50µL

NEGATIVE CONTROL
- Amount applied: 50µL

Duration of treatment / exposure:

6 hours (± 15 min)

Duration of post- treatment incubation (in vitro):

25±2 minutes immersion incubation (Post-Soak)
18 hours ± 15 minutes (Post-treatment Incubation)

Number of animals or in vitro replicates:

two replicates per test item and controls, respectively

Details on study design:

DETAILS ON TEST SYSTEM:
- RhCE tissue construct used: EpiOcular™ (OCL-200-EIT)
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovakia
- Lot No.: 30663
- Expiry date: 18 June 2020
- Storage: The preparation of tissues for treatment was started after arrival in Toxi-Coop ZRT.’s Laboratory. Therefore, the storage of EpiOcular™ (OCL-200-EIT) units was not necessary. The assay medium supplied with the kits was stored at refrigerator (2-8 °C).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
The EpiOcular™ Eye Irritation Test (EIT) was validated by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and Cosmetics Europe between 2008 and 2013. From this validation study and its independent peer review it was concluded that the EpiOcular™ EIT is able to correctly identify chemicals (both substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage according to UN GHS, and the test method was recommended as scientifically valid for that purpose. The EpiOcular™ EIT is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS No Category) without further testing, however, a drawback of this test is the inability to distinguish between Category 1 (corrosive to eye) and Category 2 (eye irritant). Thus, in case of a positive result further testing is required.

DETAILS ON THE TEST PROCEDURE USED:
- Preparation of EpiOcular™ Tissues for Treatment
After the test kit arrival, the tissues were equilibrated to room temperature for 15 minutes. The Assay Medium was pre-warmed to 37±1 °C. The appropriate number of an assay plate wells (6-well plates) was filled with the pre-warmed medium (1 mL per well). The insert was transferred aseptically into the 6-well plates and pre-incubated at 37±2 °C in an incubator with 5±1 % CO2, > or = 95 % humidified atmosphere for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 - 24 hours).

- Application
Two replicates were used for the test item and control(s) respectively.
*Pre-Treatment: After the overnight incubation, the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS. If the Ca++Mg++Free-DPBS did not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at standard culture conditions for 30±2 minutes.
*Test Item: 50 mg test item was applied (each test item treated insert) topically onto the EpiOcular™ tissues. To avoid that test item is spilled into the medium under the tissue inserts, the tissue insert was removed from the medium, placed onto a sterile surface and dosed by pouring the solid test item onto the tissue surface so that the surface of the tissue was completely covered by the test item. The insert was gently shaken from side to side to ensure that tissue was completely covered by the test item. After this procedure the tissue was then returned to its medium.
*Positive and Negative Control: A volume of 50 µL positive control (methyl acetate) or negative control (sterile deionized water) was applied on the tissues surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the EpiOcular™ tissues surface.

- Exposure
The plates with the treated tissue units were incubated for the exposure time of 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, > or = 95 % humidified atmosphere).

- Rinsing
After the incubation time the EpiOcular™ units were removed and rinsed thoroughly with Ca++Mg++ Free-DPBS as follows: Three clean beakers (glass with minimal 150 mL capacity), containing a minimum of 100 mL each of Ca++Mg++ Free-DPBS was used per test item and controls. Each test item and controls utilized a different set of three beakers. The inserts containing the tissue was lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps (taking care not to damage the tissues by the forceps). The test or control items were decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the tissues were dipped into the first beaker of DPBS and were swirled in a circular motion in the liquid for approximately 2 seconds and thereafter were lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The tissue was rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle and touching the upper lip to the absorbent material (to break the surface tension).

-Post-Soak and Post-incubation
After rinsing, the tissues were transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the Post-Soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the insert was blotted on absorbent material, and afterwards transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of warm (37 °C) Assay Medium. The tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-exposure Incubation).

- MTT Test After Post-incubation
After the post-incubation the EpiOcular™ units were transferred into the 24-well plate filled with MTT ready to use solution (300 µL of 1 mg/mL MTT per well) and then incubated for 3 hours (± 15 min) at 37±2 °C in an incubator with 5±1 % CO2 protected from light, > or = 95 % humidified atmosphere.

- Formazan Extraction
Inserts were removed from the 24-well plate after 3 hours ± 15 minutes; the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol was flowing into the insert. The plate was sealed with parafilm and extracted.
To extract the MTT, the plates were placed on an orbital plate shaker and shaken (~120 rpm) for ap proximately 2 hours at room temperature. At the end of the extraction period, the tissue was not pierced. The corresponding negative and positive controls were treated identically.

- Cell viability measurements
Following the formazan extraction, 200 µL sample(s) from each tube (2×200 µL) was placed into the wells of a 96-well plate and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer at the wavelength of 570 nm using isopropanol solutions as the blank (8×200 µL).

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Value:

90

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Possible direct MTT reduction with test item:
No colour change was observed after three hours of incubation during the check for possible direct MTT reduction by the test item. The test item did not interact with MTT, therefore additional controls and data corrections were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test item:
The test item showed no ability to become coloured in contact with water and isopropanol. The intrinsic colour of test item is white and therefore considered not to be able to significantly stain the tissues and lead false estimate of viability. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: mean OD value 1.094
- Acceptance criteria met for positive control: 9 % viability at 6 hours exposure
- Difference of viability between the two tissue replicates: 0.1 to 1.7 %

Any other information on results incl. tables

Cell Viability

 

The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:

 

OD values and viability percentages of the controls:

 

Controls		Optical Density (OD)	Viability (%)	¿ %
Negative Control: Sterile deionized water	1 2 mean	1.094 1.095 1.094	100 100 100	0.1
Positive Control: Methyl acetat	1 2 mean	0.089 0.108 0.098	8 10 9	1.7

 

OD values and viability percentages of the test item:

Test item		Optical Density (OD)	Viability (%)	¿ %
Glycyl-glycine	1 2 mean	0.983 0.976 0.980	90 89 90	0.7

 

Remark:

- ¿%: The difference of viability between the two relating tissues

- Mean of the blank OD values was 0.039

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro eye irritation test, using the EpiOcular™ Model, with the test item Glycyl-glycine (CAS 556-50-3) indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, Glycyl-glycine is, thus, considered as non-irritant to eye (UN GHS / CLP No Category).

Executive summary:

The purpose of this study was to determine the acute ocular irritation potential of the test item Glycyl-glycine (CAS 556-50-3) on three-dimensional RhCE tissue in the EpiOcular™ model in vitro.

Before the treatment the tissues were pre-wetted with approximately 20 µL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30±2 minutes. Disks of EpiOcular™ (two units) were treated with the test item and incubated for 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator with 5±1 % CO2, = 95 % humidified atmosphere).

Exposure of the test item was terminated by rinsing with Ca++Mg++ Free-DPBS solution. After rinsing, the tissues were incubated for a 25±2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, the test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). Fresh Assay Medium was used during the Post-Soak and Post-incubation. The viability of each disk was assessed by incubating the tissues for 3 hours (± 15 min) with MTT solution at 37±2 °C in an incubator with 5±1 % CO2 protected from light, > or = 95 % humidified atmosphere. The precipitated formazan was then extracted using isopropanol and quantified spectrophotometrically.

Sterile deionized water and methyl acetate treated tissues were used as negative and positive controls, respectively. The Disks of EpiOcular™ (two units / control) were treated with positive and negative control and incubated for 6 hours ± 15 minutes.

The test item Glycyl-glycine did not show significantly reduced cell viability in comparison to the negative control (Mean tissue viability: 90 %). All obtained test item viability results were above 60 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to eye.

Positive control viability results (mean tissue viability: 9 %) were below 50 % when compared to the viability values obtained from the negative control. Furthermore, the mean OD value of the two negative control tissues (mean OD value: 1.094) were between 0.8 and 2.8. So, the positive and negative controls showed the expected values within acceptable limits. The experiment was considered to be valid.

The results obtained from this in vitro eye irritation test, using the EpiOcular™ Model, with the test item Glycyl-glycine (CAS 556-50-3) indicated that the test item reveals no eye irritation potential under the applied testing conditions. According to the current OECD Guideline No. 492, Glycyl-glycine is, thus, considered as non-irritant to eye (UN GHS / CLP No Category).