5-potassium hydrogen L-glutamate

Endpoint summary

Key value for chemical safety assessment

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

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 (incl. QA statement)

Specific details on test material used for the study:

8.1. TEST ITEM
Name: L-Glutamic acid potassium salt monohydrate2
Lot number3: VG29748063
CAS number: 6382-01-0
Description: White powder
Expiry date: 01 December 2022
Purity*: 100 %
Storage conditions: Room temperature (15-25 ºC, below 70% relative humidity)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face
mask) for unknown materials were applied to assure personnel
health and safety.
In agreement with the Sponsor, no correction for purity of the test item was applied.
The certificate of analysis is included in Appendix 2.

Species:

other: EpiOcularTM human cell

Details on test animals or tissues and environmental conditions:

8.3.1. EpiOcularTM
The EpiOcularTM human cell construct (MatTek Corporation, MatTek IVSL) was used in the
assay. MatTek’s EpiOcular corneal model consists of normal, human-derived epidermal
keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that
found in the cornea. EpiOcular consist of highly organized basal cells which progressively flatten
out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal
epithelium. EpiOcular is mitotically and metabolically active and releases pro-inflammatory
agents (cytokines) known to be important in ocular irritation and inflammation.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

After the 30±2 minute period of pre-treatment, the tissues were treated
topically with 50 µL of both the negative and positive control. The test item was tested by
topically applying one levelled spoonful of the test item (≈50 mg).

Duration of treatment / exposure:

6 hr ± 15 minutes
At the end of the 6 hr ± 15 minutes treatment time, the test item was
removed by extensively rinsing the tissues with Ca2+Mg2+ free DPBS (brought to room
temperature) in three phases using three clean beakers per tissue, each of them containing
at least 100 mL of the rinsing solution.

Duration of post- treatment incubation (in vitro):

18 hours±15 minutes at SCC.

Number of animals or in vitro replicates:

In this assay, two replicates per compound (test item, positive and negative control items in the
same run) were used.

Details on study design:

8.4. EXPERIMENTAL PROCEDURE
8.4.1. Preparation of EpiOcular tissues for treatment on the day of the receipt (Day 0)
 The tissues were equilibrated (in its 24-well shipping container) at room temperature for
about 15 minutes.
 An appropriate volume of EpiOcularTM Assay Medium was equilibrated to room
temperature (20-25°C). One (1) mL of Assay Medium was aliquoted into the appropriate
wells of pre-labelled 6-well plates. The 6-well plates were labelled with the code of the
controls, test items and exposure times at least.
 The shipping containers of the tissues were opened under sterile conditions, and tissues
were inspected for air bubbles between the agarose gel and the inserts. The tissues were
transferred from the transport container into 6-well plates prefilled with Assay Medium,
and were incubated at standard culture conditions (SCC hereafter)2.
 After one hour, the Assay Medium was replaced with 1 mL fresh Assay Medium, and
tissues were incubated at SCC overnight.
8.4.2. Exposure to the test Item (Day 1)
The test item and control items were tested on two tissues.
 Pre – treatment: After the overnight incubation, the tissues were pre-wetted with 20 µL
of Ca2+Mg2+ free DPBS. To ensure the good covering of the whole surface of the tissues
with DPBS, the tissues were tapped. The tissues were incubated under SCC for 30±2
minutes.
Treatment: After the 30±2 minute period of pre-treatment, the tissues were treated
topically with 50 µL of both the negative and positive control. The test item was tested by
topically applying one levelled spoonful of the test item (≈50 mg). To avoid that the test
item is spilled into the medium under the tissue inserts, the inserts were removed from the
medium and placed onto a sterile surface, and dosed by pouring the test item onto the
surface of the tissue so that the surface of the tissue is completely covered by the test
item. The tissues were placed back onto the culture medium, and incubated under SCC
for 6 hr ± 15 minutes. Care was taken to clean the outer part of the tissue inserts if any
test item particle adheres onto it. The test item was ground with a mortar and pestle to
guarantee better contact to the tissue.
 Rinsing: At the end of the 6 hr ± 15 minutes treatment time, the test item was
removed by extensively rinsing the tissues with Ca2+Mg2+ free DPBS (brought to room
temperature) in three phases using three clean beakers per tissue, each of them containing
at least 100 mL of the rinsing solution.
 Post-soak: After rinsing, the tissues were transferred immediately, and immersed in
5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12 well
plate for a 25±2 minutes immersion incubation at room temperature.
 Post incubation: 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 inserts were blotted
on absorbent material, and transferred to the appropriate well of a pre-labelled 6-well
plate, containing 1 mL of warm Assay Medium. The tissues were incubated for 18
hours±15 minutes at SCC.
8.4.3. MTT assay (Day 2)
 A 300 µL of 1 mg/mL MTT solution1 was added into each designated well a of a prelabelled 24-well plate.
 At the end of the post-treatment incubation, each insert was removed from the 6 wellplate, gently blotted on an adsorbent material. The tissues were placed into a 24-well
plate containing 0.3 mL of MTT solution per well. Following the transfer, the tissues
were placed into the 24-well plate, for an incubation for 175 minutes at SCC.
 Extracting only from beneath was performed, in order to exclude the incidental
contamination of the isopropanol extraction solution with the test item. Inserts were
removed from the 24-well plate following the 180±10 minutes MTT incubation. The
bottom of the inserts was blotted on an adsorbent material, and then transferred to a prelabelled 6-well plate containing 1 mL of isopropanol in each well so that no isopropanol
is flowing into the inserts. The plates were sealed with parafilm and were immediately
extracted. For the extraction, plates were placed onto an orbital plate shaker, and shaken
for approximately 2 hours at room temperature. At the end of this non-submerged
extraction, the inserts and tissues were discarded without piercing, and 1 mL isopropanol
was added into each well. The extract solution was mixed and two 200 µL aliquots were
transferred into the appropriate wells of a 96-well plate.
 The absorbance of the samples was measured by a plate reader at 570 nm. No reference
wavelength measurement was used.

Irritation parameter:

percent tissue viability 

Run / experiment:

1

Value:

ca. 11.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Following exposure with the test item, the mean cell viability was 11.2% after the
6-hours exposure, compared to the concurrent negative control. This is below the threshold of
60%, therefore the test item was considered as being irritant according to OCL-200-EIT
classification, and no prediction can be made 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 EpiOcular™ Eye Irritation Test with L-Glutamic acid
potassium salt monohydrate, the results indicate that the test item is irritative to the eye.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

H319

Conclusions:

In conclusion, in this in vitro EpiOcular™ Eye Irritation Test with L-Glutamic acid
potassium salt monohydrate, the results indicate that the test item is irritative to the eye.

Executive summary:

An in vitro eye irritation test with L-Glutamic acid potassium salt monohydrate (test item) was
performed in a reconstructed human epidermis model. EpiOcularTM Eye Irritation Test
(OCL-200-EIT) is designed to predict and classify the irritancy potential of chemicals by
measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay. The irritating potential of the test item was evaluated
according to the OECD No. 492 guideline.
Two tissues per chemicals (the test item, positive and negative controls) were treated for 6 hours.
Exposure of 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 using an extractant (isopropanol) and then quantified
spectrophotometrically at 570±10 nm.
Sterile deionized water and Methyl acetate treated tissues were used as negative and positive
controls, respectively (two tissues per compound). 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 11.2% after the
6-hours exposure, compared to the concurrent negative control. This is below the threshold of
60%, therefore the test item was considered as being irritant according to OCL-200-EIT
classification, and no prediction can be made 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 EpiOcular™ Eye Irritation Test with L-Glutamic acid
potassium salt monohydrate, the results indicate that the test item is irritative to the eye.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)