Acetylcysteine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-01-24 to 2018-03-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

February 04, 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:

January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Acetylcysteine

EC Number:

210-498-3

EC Name:

Acetylcysteine

Cas Number:

616-91-1

Molecular formula:

C5H9NO3S

IUPAC Name:

N-acetylcysteine

In chemico test system

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Controls:
Positive control (PC) : Cinnamic aldehyde in acetonitrile (100 mM stock solution)
Sigma Aldrich; Cat. No.: W228613; ≥95%; CAS No.: 104-55-2; Lot. No.: MKBX1392V

Co-elution control: set up in parallel to sample preparation but without respective peptide solution
Reference controls (RCs): 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: see "Any other information on materials"

Test System
Peptides with > 95 % purity, synthesized by JPT Peptide Technologies GmbH, were used.
Cys-Peptide (Cysteine): Lot. No.: 111016HS_MHeW1017
Lys-Peptide (Lysine): Lot. No.: 120514HSDW_W1117

20.15 mg (experiment 1) and 19.65 mg (experiment 2) cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.18 mL: experiment 1; 38.10 mL: experiment 2) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
19.32 mg (experiment 1) and 20.42 mg (experiment 2) 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 (36.69 mL: experiment 1; 38.78 mL: experiment 2) to reach a concentration of 0.667 mM

Solubility of test item
The test item was completely soluble in acetonitrile, therefore, acetonitrile was chosen as suitable vehicle for the main experiments.

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 - 400xg) 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 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017/ 0.00 mM peptide.

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 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.

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 67.89% (experiment 1) and 69.46% (experiment 2). No co-elution of the test item with any of the peptide peaks was observed.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1st experiment

Parameter:

other: mean peptide depletion of both peptides [%]

Value:

6.28

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 2st experiment

Parameter:

other: peptide depletion of both peptides (%)

Value:

2.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: All acceptance criteria are fulfiled
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 1: Depletion of the Cysteine Peptide (experiment 1)

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	1371.9458	0.1446	72.07	72.27	0.27	0.38
	1367.3362	0.1441	72.17
	1346.7943	0.1419	72.58
Test Item	4922.7578	0.5198	0.00	0.00	0.00	-
	4953.7280	0.5231	0.00
	4937.0298	0.5214	0.00

Table 2: Depletion of the Cysteine Peptide (experiment 2)

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	3.7100	0.1138	77.40	77.79	0.34	0.44
	3.6200	0.1110	77.95
	3.6080	0.1106	78.02
Test Item	17.3900	0.5340	0.00	0.00	0.00	-
	16.7550	0.5145	0.00
	16.7130	0.5132	0.00

Table 3: Depletion of the Lysine Peptide (experiment 1)

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	1489.3650	0.1777	64.50	63.51	0.86	1.35
	1548.7262	0.1848	63.08
	1554.0549	0.1854	62.95
Test Item	3723.8833	0.4440	11.23	12.57	1.20	9.52
	3652.0933	0.4354	12.94
	3627.1846	0.4324	13.54

Table 4: Depletion of the Lysine Peptide (experiment 2)

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	1548.8514	0.1877	62.22	61.12	1.28	2.09
	1581.2108	0.1916	61.43
	1651.4639	0.2002	59.72
Test Item	3939.2124	0.4789	3.91	4.32	0.76	17.62
	3941.6335	0.4792	3.85
	3886.3860	0.4725	5.20

Table 5: Categorization of the Test Item (experiment 1)

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	6.28	Minimal Reactivity	no sensitiser	0.00	Minimal Reactivity	no sensitiser
Positive Control	67.89	High Reactivity	sensitiser	72.27	Moderate Reactivity	sensitiser

Table 6: Categorization of the Test Item (experiment 2)

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	2.16	Minimal Reactivity	no sensitiser	0.00	Minimal Reactivity	no sensitiser
Positive Control	69.46	High Reactivity	sensitiser	77.79	Moderate Reactivity	sensitiser

Applicant's summary and conclusion

Interpretation of results:

other: no peptide binding

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item shows minimal reactivity in the DPRA under the test conditions chosen.

Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item N-Acetyl-L-cysteine towards cysteine (Cys-) and lysine (Lys-) containing peptides. The test substance was dissolved in acetonitrile and a stock solution of 100 mM was prepared. The 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. All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide or lysine peptide solution in both independent experiments. 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 in both independent experiments.

Phase separation was observed for the co-elution of the positive control in both independent experiments and for the positive control in the second experiment.

Since the acceptance for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity or phase separation were regarded as insignificant.

Experiment 1:

No co-elution of test item with the peptide peaks was observed. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (6.28%). Based on the prediction model 1 the test item can be considered as non-sensitiser. According to the evaluation criteria in the guideline, for test items with a combined cysteine/lysine peptide depletion between 3% and 10% a second run should be considered for both peptides. Therefore, no prediction can be made.

Experiment 2:

No co-elution of test item with the peptide peaks was observed. The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (2.16%).

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 67.89% (experiment 1) and 69.46% (experiment 2).

In this study under the given conditions the test item showed minimal reactivity towards both peptides.