[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08-08-2019 to 10-10-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal

Cas Number:

1226911-73-4

Molecular formula:

C14H18O

IUPAC Name:

(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: approximately 4ºC, in the dark and under nitrogen
- Other: colourless liquid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

- Preparation of the test chemical solutions:
100 mM stock solutions of the test item were prepared in acetonitrile for both analytical occasions.
Triplicate solutions each of test item and the positive control stocks were diluted with the Cysteine peptide to prepare final solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. Also triplicate solutions each of test item and the positive control stocks were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control.

- Preparation of the positive controls, reference controls and co-elution controls:
Positive controls: The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Triplicate solutions each of test item and the positive control stocks were diluted with the Cysteine peptide to prepare final solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. Also triplicate solutions each of test item and the positive control stocks were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control.

Reference controls: Stability controls (Reference Control B, n=6) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM. Reference Control A and Reference Control B were prepared with buffer and acetonitrile.

Co-elution controls: For the co-elution controls, blank buffer solutions were used in place of the Cysteine and Lysine stock solutions.

INCUBATION
- Incubation conditions: The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection on the analysis run. Prior to injection of the samples the appearance of the samples in the vials was assessed and documented again.

- Precipitation noted: Precipitation not observed.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.
Calibration curve is available in the full study report. The calibration curve met the acceptability criteria: standard calibration curve having an r2 > 0.99.

- Verification of the suitability of the HPLC for test chemical and control substances:
The mean peptide concentration of Reference controls B was 0.491 mM and 0.507 mM for both Cysteine and Lysine. they both were within the acceptance criteria of 0.45-0.55 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN, acetonitrile) used to dissolve the test item did not impact the Percent SPCC nor SPCL Depletion.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: The relative peptide concentration is measured by high-performance liquid chromatograph (HPLC) with gradient elution and spectrophotometric detection at 220 nm.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

1.41 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

2.18 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No phase separation neither precipitation occurred after the incubation period for both Cysteine and Lysine samples.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for reference controls A to C: All criteria met.
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Any other information on results incl. tables

Table 1: Acceptability of the DPRA

	Peptide	Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <14.9%)	SD < 14.9%
	Lysine	r^2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <14.9%)	SD < 11.6%
Achieved results	Cysteine	r^2>0.999	71.5 (SD 0.21%, n=3)	B: 0.491 mM (CV=0.56%, n=6)	SD 0.71%
	Lysine	r^2>0.999	61.0 (SD 2.76%, n=3)	B: 0.507 mM (CV=1.68%, n=6)	SD 2.93%

CV: Coefficient of Variation

Table 2: Depletion of peptide in the presence of the test item

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide with test item (µV.sec)	Mean depletion (%)
Cysteine	B: 903730 (n=6)	890990 (n=3)	1.41
Lysine	B: 721990 (n=6)	706290 (n=3)	2.18

Table 3: Results of the DPRA with the test item

	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test Item	1.41%	0.71%	2.18%	2.93%	1.79%	Negative: No or minimal reactivity

SD: Standard Deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Test item was negative in the DPRA and was classified in the "no or minimal reactivity class when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Conclusions:

The test item gave a negative in DPRA and was classified in the "Negative: No or minimal reactivity class" using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C, 18 June 2019) was to assess the reactivity and sensitising potential of the test item, ST 08 C 19.  Solutions of this test item were incubated with two synthetic peptides (containing respectively a cysteine and a lysine amino acid) for approximate 24 hours and then the concentration of each peptide was measured relative to a series of peptide control solutions.

Solutions of the test item in acetonitrile were successfully analysed by the validated DPRA analytical method (analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides.  For each peptide analysis all acceptance criteria were met (linearity of standard response, reference and stability control concentrations, positive control depletion and test item reproducibility).  There was no co-elution of test item with either peptide. 

 

Under the conditions of this study, there was no or minimal depletion (mean depletion of 1.79%) of either the Cysteine or Lysine peptides in the presence of the test item, the DPRA assay categorises this test item as negative and hence it is predicted not to be a skin sensitiser.