N-glycylglycine

Endpoint summary

Description of key information

Skin irritation/corrosion:

- EpiDerm^TM test for skin irritation (OECD 439): 109% cell viability: non-irritant

 

Eye irritation/ damage:

- Epiocular^TM (OECD 492): 90 % cell viability, non-irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 19 June 2020

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)

Version / remarks:

18 June 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

Commission Regulation (EU) No 2019/1390 of 31 July 2019 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), B.46. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

Deviations:

no

Guideline:

other: In Vitro EpiDermTM Skin Irritation Test (EPI-200-SIT), For use with MatTek Corporation’s Reconstructed Human Epidermal Model EpiDermTM (EPI-200-SIT)

Version / remarks:

10 February 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Test system:

human skin model

Remarks:

EPI-200, MatTek, Ashland, MA, USA and Bratislava, Slovakia)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm™ model has been validated for irritation testing in an international trial. The validation trial was in accordance with the principles and criteria documented in OECD Guidance Document No. 34 on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment and ECVAM (2007) Performance Standards for applying human skin models to in vitro skin irritation. In 2008, ESAC concluded that the Modified EpiDerm™ SIT has sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non-skin irritating) test substances. The Modified EpiDerm™ SIT is an in vitro procedure that, depending on information requirements, allows determining the skin irritancy of chemicals as a stand alone replacement test, as a screen, or within a testing strategy in combination with, if appropriate, a weight of evidence approach.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDermTM reconstructed human epidermal model
- Supplier: MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, Bratislava, Slovakia
- Tissue batch number(s): 30874
- Expiry date: June 19, 2020
- Date of initiation of testing: June 17, 2020

PRE-INCUBATION
After the quality check of the inserts by unaided eye assessment, the inserts were transferred into the 6-well plates (pre-filled with 0.9 mL medium, upper row) and pre-incubated (60±5 minutes) at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere.
After incubation of one hour, every tissue was transferred into the wells of the lower rows of the 6-well plates (pre-filled with 0.9 mL medium). Then, all 6-well plates were incubated at standard culture conditions (37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere) overnight (18±3 hours).

EXPOSURE
- Test Item: First, 25 µL of sterile DPBS was applied to the epidermal surface in order to improve further contact between powder and epidermis. Subsequently, 25 mg of the test item was applied evenly to the epidermal surface. The insert was gently shaken from side to side to ensure that the tissue was completely covered by the test item.
- Positive and negative control: A volume of 30 µL positive control (SDS 5 % aq.) or negative control (1x DPBS) were applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface. Application of the nylon mesh was not necessary.
- Exposure times: The total exposure time was 60 minutes, which included two different incubation conditions. Each plate with the treated epidermis units was incubated for 35 minutes (± 1 min) at standard culture conditions (37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere). Furthermore, each plate was incubated for 25 minutes (± 1 min) at room temperature.

NUMBER OF REPLICATE TISSUES FOR TEST ITEMS AND CONTROLS:
Three replicates were used for the test item and positive and negative controls, respectively.

REMOVAL OF TEST MATERIAL AND CONTROLS
After the exposure time (60±1 min) the EpiDermTM units were rinsed thoroughly with 1x DPBS sterile solution to remove all of the test material from the epidermal surface. The constant stream of 1x DPBS was applied from approximately 1.5 cm distance and filling and emptying the tissue insert was completed 15 times. After the 15th rinse from the washing bottle, the insert was completely submerged three times in clean beakers containing a minimum of 150 mL 1x DPBS solution. Finally, the insert was rinsed once from inside and once from outside with 1x DPBS solution. The rest of the 1x DPBS was decanted onto the absorbent material. Remaining 1x DPBS and/or test item was removed from the tissues by gently sweeping the tissues surface with a sterile cotton tipped swab (care was taken to avoid the damage of tissues).
- Observable damage in the tissue due to washing: none

POST-INCUBATION
After rinsing the inserts were placed into the 6-well plates (pre-filled with 0.9 mL assay medium per well) in the upper row and then incubated for 24 hours (± 2 h) at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere. After incubation of 24±2 hours, the lower row of the 6-well plates was pre-filled with fresh assay medium (0.9 mL per well) and then the inserts were transferred from the upper row to the lower row. Finally, the plates were placed back into the incubator and were incubated for 18±2 hours at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere.

MTT TEST
After the post-incubation period the EpiDerm™ 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 (± 5 min) at 37±1 °C in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere.

FORMAZAN EXTRACTION
After the MTT incubation, the MTT medium was removed (gently aspirated) from all the wells with a Pasteur pipette (or suitable pipette tip), linked to a vacuum source. After removing all the possible amount of MTT medium the wells were refilled with 1x DPBS solution and were aspirated again. This rinsing process with 1x DPBS was repeated twice and after the last aspiration the dryness of the tissue surface was checked and the inserts were transferred into the new 24-well plate. After the transportation, 2 mL extractant solution MTT-100-EXT was pipetted into each well (extractant solution was flowing into the insert on the tissue surface). The plates were sealed with parafilm and extracted.
To extract the formazan the plate was placed on an orbital plate shaker and shaken (~120 rpm) for 2 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.

CELL VIABILITY MEASUREMENTS:
Following the formazan extraction, the extracts were homogenized by pipetting them up and down 3 times, then 2x200 µL samples from each well from the 24-well plate was placed into the wells of a 96-well plate. The Absorbance / Optical Density (OD) of the samples was read in the spectrophotometer at the wavelength of 570 nm using MTT-100-EXT (isopropanol) solution as the blank (6×200 µL).

PREDICTION MODEL / DECISION CRITERIA:
The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative tissue viability after 60 minutes exposure and post incubation is less or equal (=) to 50 % of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25mg

NEGATIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: sterile 1x DPBS (Phosphate Buffered Saline)

POSITIVE CONTROL
- Amount(s) applied: 30 µL
- Concentration: Sodium Dodecyl Sulphate (SDS) 5% aq. solution

Duration of treatment / exposure:

60±1 min

Duration of post-treatment incubation (if applicable):

42±2 h

Number of replicates:

Three replicates were used for the test item and controls, respectively.

Irritation / corrosion parameter:

% tissue viability

Value:

109

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: mean viability value of three tissues

Other effects / acceptance of results:

OTHER EFFECTS
- Possible direct MTT reduction with test item:
No colour change was observed after one hour of incubation. The test item did not interact with the MTT, therefore additional controls and data calculations 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. The intrinsic colour of test item is white and therefore considered not to be able to significantly stain the tissues and lead to a 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 2.172 and standard deviation value (SD) 6.54 for the100 % viability (The mean OD value of the three negative control tissues should be between 0.8 and 2.8 and the standard deviation value (SD) of the % viability should be < or = 18.)
- Acceptance criteria met for positive control: Mean OD value 0.049 and standard deviation value (SD) 0.23 for the 2 % viability (The acceptable percentage viability for positive control (mean of three tissues) is < 20 % and the standard deviation value (SD) of the % viability should be < or = 18)
- For the test item, the standard deviation value (SD) of the % viability was 0.43 (should be < or = 18).

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:

Substance		Optical Density (OD)	Viability (%)
Negative Control 1x DPBS	1 2 3 mean standard deviation (SD)	2.102 2.336 2.080 2.172	97 708 96 100 6.54

Positive Control SDS (5% aq.)

1 2 3 mean standard deviation (SD)

0.046 0.048 0.055 0.049

2 2 3 2 0.23

OD values and viability percentages of the test item:

Test Item: Glycyl-glycine

1 2 3 mean standard deviation (SD)

2.387 2.372 2.369 2.376

110 109 109 109 0.43

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EpiDermTM model, with the test item Glycyl-glycine (CAS 556-50-3) , indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Glycylglycine (CAS 556-50-3) is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

Executive summary:

EpiDermTM Model test of Glycyl-glycine (CAS 556-50-3) has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 18 June 2019.

Disks of EpiDermTM (three units) were treated with the test item and incubated for 25 minutes at room temperature and 35 minutes at standard culture conditions (37±1 °C, 5±1 % CO2, 90±10 % humidified atmosphere). The 60 minutes exposure of the test item was terminated by rinsing with 1x DPBS solution. Epidermis units were then incubated at 37±1 °C for approximately 24 hours in an incubator with 5±1 % CO2, 90±10 % humidified atmosphere.

After incubation of approximately 24 hours, the tissues were transferred into fresh medium and the incubation process was continued for approximately 18 hours at standard culture conditions. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37±1 °C in 5±1 % CO2, 90±10 % humidified atmosphere. The precipitated formazan was then extracted using extractant solution MTT-100-EXT (isopropanol) and

quantified spectrophotometrically.

SDS (5 % aq.) and 1× DPBS treated (three units / positive and negative control) epidermis were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

A test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 60 minutes exposure and post incubation is less or equal (=) to 50 % of the negative control.

In this in vitro skin irritation test using the EpiDermTM model, the test item Glycyl-glycine did not show significantly reduced cell viability in comparison to the negative control (mean value: 109 %). All obtained test item viability results were far above 50 %, when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.

Positive and negative controls showed the expected optical density (OD) and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EpiDermTM model, with the test item Glycyl-glycine (CAS 556-50-3) , indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Glycylglycine (CAS 556 -50 -3) is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 - 18 June 2020

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)

Version / remarks:

18 June 2019.

Deviations:

no

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)

Specific details on test material used for the study:

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

Species:

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

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).

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 %

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Additional information

Skin irritation:

A valid EpiDerm^TM test of Glycyl-glycine was performed under GLP to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439, 18 June 2019. EpiDerm discs were exposed in triplicate to Glycyl-glycine for 60 min (25 min at 37±1 °C and 25 min at room temperature). The EpiDerm discs treated with the test item did not show significantly reduced cell viability (109% viability compared to the negative controls). Therefore, Glycyl-glycine is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

 

Eye irritation:

A valid study was performed under GLP to determine the acute ocular irritation potential of the test item Glycyl-glycine on three-dimensional RhCE tissue  (EpiOcular™ model) in vitro, according to OECD guideline No. 492, 18 June 2019. EpiOcular™ (two units) were treated with 50 mg/units test item for 6 hours (± 15 min) at standard culture conditions (37±2 °C in an incubator) with 5±1 % CO2, ≥ 95 %  humidified atmosphere). The test item Glycyl-glycine showed no significantly reduced cell viability (≤ 60%), in comparison to the negative control (mean relative viability: 90 %). 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 and therefore not classified (UN GHS / CLP No Category).

 

Justification for classification or non-classification

For Skin Irritation:

According to the results obtained with the EpiDerm^TM SM test (OECD 439) Glycyl-glycine is considered to be non-irritant to skin and is therefore not classified (UN GHS / CLP No Category).

For Eye Irritation:

The Epiocular test (OECD 492) 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 and is therefore not classified (UN GHS / CLP No Category).