5-potassium hydrogen L-glutamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, No.431 (18 June 2019) “In Vitro Skin Corrosion:
Reconstructed Human Epidermis (RhE) Test Method”

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

5-potassium hydrogen L-glutamate

EC Number:

243-094-0

EC Name:

5-potassium hydrogen L-glutamate

Cas Number:

19473-49-5

Molecular formula:

C5H9NO4.K

IUPAC Name:

potassium 5-oxido-5-oxo-L-norvaline

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

other: EpiDerm (Source: MatTek Corporation, USA) is a three-dimensional human epidermis model.

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

3.3.1. Human skin
EpiDerm (Source: MatTek Corporation, USA) is a three-dimensional human epidermis model.
It consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured
to form a multilayered, highly differentiated model of the human epidermis. The NHEK, which
are cultured on specially prepared cell culture inserts using serum free medium, attain levels of
differentiation. Ultrastructurally, the EpiDerm Skin Model closely parallels human skin, thus
providing a useful in vitro means to assess dermal irritancy and toxicology [5].
The EpiDerm Skin Model exhibits in vivo-like morphological and growth characteristics which
are uniform and highly reproducible. EpiDerm consists of organized basal, spinous, granular and
cornified layers analogous to those found in vivo. EpiDerm is mitotically and metabolically
active. Markers of mature epidermis specific differentiation such as profilaggrin, the K1/K10
cytokeratin pair, involucrin, and type I epidermal transglutaminase have been localized in the
model. Ultrastructural analysis has revealed the presence of keratohyalin granules, tonofilament
bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar
lipid layers arranged in patterns characteristic of in vivo epidermis [5].
Its use for skin corrosivity testing involves topical application of test materials to the surface of
the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

3.3.2. Justification for selection of the test system
The EpiDerm model is one of the five RhE test methods recommended by the OECD 431
guideline. The test has been validated for in vitro corrosivity testing in an international trial
[6, 7]; it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

3.3.3. Number of replicate wells
In this assay, two replicates per time point were used. Two negative controls and two positive
controls were run additionally. Each test item was tested for a 3-minute and a 60-minute
exposure.
3.3.4. Quality Control
EpiDerm kits are manufactured according to defined quality assurance procedures (certified ISO
9001). All biological components of the epidermis and the kit culture medium have been tested
for the presence of viruses, bacteria and mycoplasma. The quality of the final product was
assessed by undertaking a MTT cell viability test, and a cytotoxicity test with 1% Triton X-100
Solution.
These quality control experiments were conducted at MatTek (supplier of the EpiDerm test kits
used in the present study) and are documented in Appendix 3.
3.3.5. Kit Contents
One EPI-200-SCT Kit and one MTT-100 Assay Kit was used for the test item.
Name: EpiDerm™ tissues (EPI-200 ver. 2.0)
Supplier: MatTek Corporation
Lot number: 34164
Name: Assay Medium (EPI-100-ASY) *
Batch number: 060321LHB
Expiry date: 17 June 2021
* Also used as Maintenance Medium in the test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test item was applied evenly to the epidermal surface with a spoon, and 25 µL of distilled water was pipetted onto it to wet the test item in order to ensure a good contact with the skin surface.

Duration of treatment / exposure:

3-minute exposure: After the 1-hour pre-incubation, each tissue was treated with an exposure
time of 3 minutes at room temperature.
60-minute exposure: After the 1-hour pre-incubation, the “Maintenance Medium” was replaced
with fresh medium (0.9 mL per well). Each tissue was treated with an exposure time of
60 minutes. The tissues were placed at 37 °C in an incubator with 5% CO2, in a ≥95%
humidified atmosphere for the exposure time.

Duration of post-treatment incubation (if applicable):

The viability of each
tissue was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated
formazan crystals were then extracted with isopropanol and then quantified with a
spectrophotometer at 570±10 nm.

Number of replicates:

Two tissues per exposure time were treated with the test item for 3 minutes or 60 minutes.
Exposure to test material was terminated by rinsing with DPBS solution.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

>= 73.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

>= 74.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

As no colour change was observed after one hour of incubation of the test item in MTT solution,
it is considered that the test material did not interact with MTT. Therefore, additional controls
and data calculations were not necessary to exclude the false estimation of viability.
In the lack of intrinsic colour or colouring potential, no additional controls and data calculations
were necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated
relative viability % values are presented below. The mean OD value for the test item treated skin
tissues showed 73.5% and 74.1% relative viability compared to the negative control, after the 3
minutes and the 60 minutes exposure, respectively. Positive control OD values were within the
historical control range therefore all validity criteria met according to OECD 431.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EpiDerm™ model test with L-Glutamic acid potassium salt monohydrate, the results indicate that the test item is not corrosive to the skin.

Executive summary:

An in vitro skin corrosivity test with the test item was performed in a reconstructed human

epidermis model. EpiDerm™ is designed to predict and classify the corrosive potential of

chemicals by measuring its cytotoxic effect as reflected in the MTT [3-(4,5-Dimethylthiazol-

2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] assay. The

corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Two tissues per exposure time were treated with the test item for 3 minutes or 60 minutes.

Exposure to test material was terminated by rinsing with DPBS solution. The viability of each

tissue was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated

formazan crystals were then extracted with isopropanol and then quantified with a

spectrophotometer at 570±10 nm.

Distilled water and 8N Potassium hydroxide solution treated tissues were used as negative and

positive controls, respectively (two tissues per exposure time). For each treated tissue, cell

viability was expressed as a % relative to the concurrent negative control.

Following exposure with the test item, the mean cell viability was 73.5% after the

3-minute exposure, and 74.1% after the 60-minute exposure, compared to the concurrent

negative control. This is above the thresholds of 50% after 3-min exposure, and higher than 15%

after 60-min exposure, therefore the test item was considered as being Non-corrosive according

to the UN GHS/EU CLP Classification. The experiment met all the validity criteria, therefore the

study was considered to be valid.

In conclusion, in this in vitro EpiDerm™ model test with L-Glutamic acid potassium salt

monohydrate, the results indicate that the test item is not corrosive to the skin.