Propionamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-01-14 to 2019-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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 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 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:

Propionamide

EC Number:

201-172-1

EC Name:

Propionamide

Cas Number:

79-05-0

Molecular formula:

C3H7NO

IUPAC Name:

propanamide

Test material form:

solid

In chemico test system

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide stock solutions: Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides were freshly prepared just before their incubation with the test item. The stock solutions contained 100 mM peptide.

INCUBATION
- Incubation conditions: The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C.
- Precipitation noted: no

PREPARATION OF THE HPLC
- See tables below

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm signal for quantification; 258 nm signal used as indicator for co-elution

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.

Prediction model:
The mean percent cysteine and percent lysine depletion value was calculated for each test item. Prediction models are depicted below.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

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

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.03 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

0.21 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

no or minimal reactivity [in chemico]

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

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	4.7360 4.8660 4.6910	0.1551 0.1593 0.1537	69.56 68.73 69.85	69.38	0.58	0.84
Test item	15.5650 15.5010 15.5250	0.5050 0.5029 0.5037	0.00 0.39 0.23	0.21	0.19	94.37

  

Table 2: Depletion of the Lysine Peptide

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	4.4950 4.6220 4.7210	0.1644 0.1690 0.1726	67.20 66.27 65.55	66.34	0.83	1.25
Test Item	13.7810 13.7280 13.6890	0.5029 0.5010 0.4996	0.00 0.00 0.10	0.03	0.06	173.21

 

Table 3: Categorization of the Test Item

Prediction Model	Prediction Model 1			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	0.12	Minimal Reactivity	Negative	0.21	Minimal Reactivity	Negative
Positive Control	67.86	High Reactivity	Positive	69.38	Moderate Reactivity	Positive

 

Based on the results of the peptide depletion, categorization according to the prediction model might be performed. Since no-co-elution was observed, prediction model 1 based on the combination of cysteine and lysine depletion should be considered.

Applicant's summary and conclusion

Interpretation of results:

other: no peptide binding

Remarks:

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

Conclusions:

The test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay according to OECD guideline 442C. The test item is considered as "non-sensitiser".

Executive summary:

In this study the test item was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 73.1 g/mol a 100 mM stock solution 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 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 100 mM stock 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 phase separation was regarded as not relevant.
No co-elution of the 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 acetonitrile).
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% (0.12%). Based on the prediction model 1 the test item can be considered as non-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%.
In conclusion the test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay and the test item is considered as "non-sensitiser".