trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In line with Article 13(1) and section 8.3 of Annex VII to the REACH Regulation, the information requirement for skin sensitisation have been fulfilled by using a tiered approach, starting from the non-animal test methods e.g. in a Weight-of-Evidence approach.

This test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues

Test material

Test material information

1

Reference substance name:

trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

EC Number:

948-778-9

Molecular formula:

C14O8H19N2Na3

IUPAC Name:

trisodium (2S)-2,6-bis(3-carboxylatopropanamido)hexanoate

Test material form:

liquid

Details on test material:

Active ingredient (%): 48.9% dry substance
Purity (%): 95.50 %
Stability: 5 years
Sterilization: None
Solubility: Soluble in water
Storage: Room Temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
23ADB/50
- Expiration date of the lot/batch:
21-11-2023

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature
- Stability under test conditions:
5 years

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage.
Since no molecular weight can be derived the test item was tested without any prior dilutions.

Controls
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
 
Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples

HPLC System
HPLC/DAD Agilent Infinity 1260 II with Chromeleon 7.2 SR5
Detection 220 nm signal for quantitation
258 nm signal used as indicator for co-elution
Analytical Column Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µ m, Agilent Art. Nr. 861753-902
Pre-Column Phenomenex, AJO-4286, 4.0 x 2.0 mm.
Column Temperature 30 °C
Sample Temperature 20-25 °C
Run Time 20 minutes
Injection Volume 4 µL

HPLC Mobile Phase
These HPLC conditions are described in Eurofins Munich working instruction EFM_DPRA_01 [16].
HPLC Mobile Phase A: 0.1% ( v/v) trifluoroacetic acid in water
HPLC Mobile Phase B: 0.085% ( v/v) trifluoroacetic acid in acetonitrile

 
Peptides
20.69 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.36 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
18.97 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (35.13 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose Groups
Reference Control C (solvent control) 0.5 mM
Test Item undiluted
Positive Control 100 mM stock solution


Pre-Experiments
Solubility of the test item was not determined prior to the main experiment; the substance was tested in its original form.
 

Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.

Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure.
 
Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range indicated in the following table (see Table 3).
Table 3: Peptide Concentrations for the Standard Curve
STD1 STD2 STD3 STD4 STD5 STD6 STD7
Cys and Lys Peptide [mM] 0.534 0.267 0.134 0.067 0.033 0.017 0.000

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days, all test chemical solutions were freshly prepared for both assays on each day.
The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine

Parameter:

other: mean depletion of cysteine

Value:

37.93

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Lysine

Parameter:

other: mean depletion of lysine

Value:

37.93

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed moderate reactivity towards both peptides. The test item is considered as “sensitiser”.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

Thein chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

In the present study Trisodium (2S)-2,6-bis(3-carboxylatopropanamido) hexanoate_Disuccinyl lysine sodium salt wastested in its original form.Since no molecular weight could be derived the test item was tested without any prior dilutions.

The test item solutions were testedby incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use. For the undilutedsolution of thetestitem no turbidityor precipitation was observed when diluted with the cysteine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation.No precipitation, turbidity or phase separation was observed for any of the samples.

For the undiluted solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity or phase separation was regarded as not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitisingpotential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the correspondingreference control C (RC C_acetonitrile).

The 100 mM stock solution of the testitem showedmoderatereactivitytowards the synthetic peptides. The mean depletion of both peptides was> 6.38% (37.93%). Based on the prediction model 1 thetest item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

The controls confirmed the validity of the study for both, the cysteine and lysine run