L-aspartic acid, sodium salt

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:

2022

Report date:

2022

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

Constituent 1

Reference substance name:

sodium 2-amino-3-carboxypropanoate

Cas Number:

323194-76-9

Molecular formula:

C4H7NO4.xNa

IUPAC Name:

sodium 2-amino-3-carboxypropanoate

Test material form:

solid: particulate/powder

Details on test material:

Name: L-aspartic acid, sodium salt monohydrate 1
Batch/Lot number: Z201125/ VG29848563
CAS number: 323194-76-9
Anhydrous substance name: L-aspartic acid, sodium salt, CAS: 17090-93-6
Appearance: Solid, white, crystalline powder
Purity: 98%
Expiry date: 24 November 2022
Storage conditions: Room temperature (15-25 ºC), protected from humidity (tight closed
container)

Specific details on test material used for the study:

Name: L-aspartic acid, sodium salt monohydrate
Lot No.: Z201125 / VG29848563
Expiry date: November 24, 2022
CAS number: 323194-76-9
Molecular weight: 155.1 g/mol*
Purity: min. 98 %
Appearance: white crystalline powder
Storage: at room temperature, protected from humidity
Safety precautions: According to the SDS

In chemico test system

Details on the study design:

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: 7 µL
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 – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Table 2. Gradient flow conditions
Time Flow A phase (%) B phase (%)
0 min 0.35 mL / min 90 10
10 min 75 25
11 min 10 90
13 min 10 90
13.5 min 90 10
20 min gradient ends

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 = 0.9997 for both cysteine and lysine peptides, covering the concentration range from 0.0167 mM to 0.534 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:

0.75

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:

In the course of this study the skin sensitisation potential of the test item L-aspartic acid, sodium salt monohydrate was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test item in order to derive a prediction, two independent valid tests were evaluated, one with cysteine and one with lysine peptides.

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 % values for the nine reference controls B and C in acetonitrile were also acceptable. For each peptide, all validity criteria were met, confirming the validity of the study.

The mean cysteine peptide depletion value was 1.51 % ± 1.86 % while the lysine peptide depletion value of the test item was 0.00 % ± 0.14 %. The overall mean peptide depletion of the test item was 0.75 %.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item L-aspartic acid, sodium salt monohydrate was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitiser under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

In the course of this study the skin sensitisation potential of the test item
“L-aspartic acid, sodium salt monohydrate” was studied using the Direct Peptide Reactivity Assay (DPRA).

 

For the test item in order to derive a prediction two independent valid tests were evaluated, one with cysteine and one with lysine peptides.

 

The standard calibration curve has r²=0.9997 for both cysteine and lysine peptides, covering the concentration range from 0.0167-0.534 mM. The mean back-calculated peptide concentrations of the reference control A replicates were within the expected molarity concentration range for the cysteine run (0.51 mM) and for the lysine run (0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2 % and 0.3 %  for the cysteine and lysine runs respectively.

 

Peptide depletion resulting from the positive control cinnamaldehyde was within the expected percentage range both with cysteine and lysine peptides. Peptide depletion resulted from the positive control cinnamaldehyde was 69.90 %  ± 0.23 % with cysteine peptide and the lysine peptide depletion value was 43.90 % ± 3.20 %. For each peptide, all validity criteria were met, confirming the validity of the study.

 

For the test item the mean cysteine peptide depletion value was 1.51 % ± 1.86 % while the mean lysine peptide depletion value was 0.00 % ± 0.14 %.

The overall percent peptide depletion was calculated for the test item. No co-elution was observed with the peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers. The mean peptide depletion of the test item was 0.75 %, which is under the 6.38 % threshold of the applicable prediction model and classified as negative with no or minimal reactivity.

 

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item L-aspartic acid, sodium salt monohydrate was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitiser under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.