5-potassium hydrogen L-glutamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

14 June 2021

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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

Specific details on test material used for the study:

Name: L-Glutamic acid potassium salt monohydrate
Batch number: VG29748063 / Vendor Lot: SLCJ 1327
CAS number: 6382-01-0
Expiry date: 01 December 2022
Molecular weight: 203.23 g/mol
Purity: 100 %
Appearance: white powder
Storage: room temperature
Safety precautions: According to the SDS

In chemico test system

Details on the study design:

HPLC System Conditions

HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Serial number: USRY003976

Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7µL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradient twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow

Mobile phases for HPLC:

Mobile Phase A – 0.100 % (v/v) trifluoroacetic acid in ultrapure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

1:10 ratio cysteine peptide0.5 mM peptide, 5 mM test item:
750 µL cysteine peptide stock solution
(or phosphate buffer for the co-elution control)
200 µL acetonitrile
50 µL 100mM test item solution
(or solvent for the reference controls A,B,C or
100 mM positive control solution for the positive control)

1:50 ratio lysine peptide 0.5 mM peptide, 25 mM test item:
750 µL lysine peptide stock solution (or ammonium acetate buffer for the co-elution control)
250 µL 100mM test item solution
(or solvent for the reference controls A,B,C or
100 mM positive control solution for the positive control)




The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5°C - 30°C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches were consisted of 2 parts: one part with the A reference controls, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the B and C reference controls, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended. The total HPLC analysis time should be less than 30 hours.

Results and discussion

Positive control results:

Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.51 mM for the cysteine and 0.50 mM for the lysine peptide.

A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions from the peptide stock solutions. Calibration standard points were analyzed by linear regression.

Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9954 for cysteine peptide and r2 = 0.9999 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. All validity criteria were within acceptable limits and therefore the study can be considered valid.

In vitro / in chemico

Results

Key result

Run / experiment:

other: 3

Parameter:

other: average percent peptide depletion

Value:

1.63

Vehicle controls validity:

valid

Remarks:

ultrapure water

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “L-Glutamic acid potassium salt monohydrate” showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method according to OECD 442C.

Executive summary:

In the course of this study the skin sensitization potential of the test item “L-Glutamic acid potassium salt monohydrate” was studied using the Direct Peptide Reactivity Assay (DPRA) in accordance with OECD 442C.

 

For the test item and the positive control substance, in order to derive a prediction two independent tests were evaluated, one with cysteine and one with lysine peptides. The results of two valid runs is used for the classification of the test item.

 

Peptide depletion resulting from the positive control cinnamaldehyde was within the expected percentage range both with cysteine and lysine peptides.

 

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range and the CV % for the nine reference controls B and C in acetonitrile were also acceptable. Moreover, the mean back-calculated peptide concentrations of the three reference controls C in the ultrapure water replicates were within the expected molarity concentration range for cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the study.

 

The mean cysteine peptide depletion value was 3.26 % ± 3.82 % while the lysine peptide depletion valueof the test itemwas 0.00 % ± 0.11 %.

 

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “L-Glutamic acid potassium salt monohydrate” showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method according to OECD 442C.