N-glycylglycine

Skin irritation / corrosion

Administrative data

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

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

Version / remarks:

18 June 2019

Deviations:

no

Test guideline 2

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

Test guideline 3

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)

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

In vitro test system

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.

Results and discussion

In vitro

Results

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).

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:

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

Applicant's summary and conclusion

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).