Disodium L-tyrosinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 April 2017 - 18 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n °154

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium L-3-(4-hydroxyphenyl)alaninate hydrate

Molecular formula:

C9H9NNa2O3 • xH2O

IUPAC Name:

Disodium L-3-(4-hydroxyphenyl)alaninate hydrate

Test material form:

solid

In chemico test system

Details on the study design:

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

Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 260515HS DVWV1115
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 120514HSDW W0517
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 19.56 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (36.19 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 20.54 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 (38.982 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
Three samples of the test substance in acetonitrile were incubated with each peptide for 24h at room temperature in the dark. The incubation tubes were sealed. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Sample preparation:
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).

HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 220 nm (Agilent, 1200 Series, with Chemstation, Rev. B.04.01) 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 (0.1% ( v/v) trifluoroacetic acid in water) and 50% phase B (0.085% ( v/v) trifluoroacetic acid in acetonitrile) 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.

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C)) * 100

Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r2 > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Evaluation of results:
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

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 64.08%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine

Parameter:

other: mean peptide depletion [%]

Value:

24.25

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Lysine

Parameter:

other: mean peptide depletion [%]

Value:

0.55

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: Yes
- Acceptance criteria met for positive control: Yes

OTHER EFFECTS:
For the cysteine and lysine peptide experiment no precipitation, turbidity or phase separation was observed for the samples of the test item. Precipitation was observed for the samples of the positive control.

Any other information on results incl. tables

Table 2: Depletion of the Cysteine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1441.9841	0.1695	68.28	68.80	0.51	0.74
	1417.9863	0.1668	68.81
	1395.7390	0.1644	39.30
Test Substance	3516.7920	0.3973	20.52	24.25	3.23	13.33
	3276.0418	0.3708	25.96
	3262.4739	0.3693	26.27

Table 3: Depletion of the Lysine Peptide

Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1712.7127	0.1998	60.59	59.36	1.37	2.31
	1755.7267	0.2049	59.60
	1830.2459	0.2136	57.89
Test Substance	4276.6836	0.5001	0.57	0.55	0.08	13.84
	4280.6788	0.5005	0.47
	4274.1211	0.4998	0.63

Table 4: Categorization of the Test Item according to prediction model 1

Prediction Model	Prediction Model 1, (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)
	Peptide Depletion [%]	Reactivity Category	Prediction
Test Substance	12.4	Low Reactivity	Sensitiser
Positive Control	64.08	High Reactivity	Sensitiser

Applicant's summary and conclusion

Interpretation of results:

other: peptide depletion

Remarks:

The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, the test item showed low reactivity towards cysteine or lysine, respectively under the given conditions.

Executive summary:

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, the direct peptide binding potential of the test item was determined. The test substance was solved in water, based on the results of the pre-experiments. Based on a molecular weight of 225.15 g/mol a 100 mM stock solution was prepared. Test item solutions were tested by 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. After the 24 h ± 2 h incubation period but prior to the HPLC analysis the cysteine and lysine peptide samples were inspected for precipitation, turbidity or phase separation. For the cysteine and lysine peptide experiment no precipitation, turbidity or phase separation was observed for the samples of the test item. Precipitation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria of the depletion range of the positive control were fulfilled, the observed precipitations were regarded as insignificant. No co-elution of test item with the peptide peaks was observed. Sensitising potential 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 corresponding reference control C (RC C). The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was between 6.38% and 22.62% (12.40 %). Based on the prediction model 1 (Cysteine 1:10/Lysine 1:50 prediction model) the test item may be considered as sensitiser.