1-[3-(Hydroxymethyl)pyridin-2-yl)-4-methyl-2-phenylpiperazine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 January 2018 - 30 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

{2-[(2R)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol; {2-[(2S)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol

EC Number:

700-296-8

Cas Number:

61337-89-1

Molecular formula:

C17-H21-N3-O

IUPAC Name:

{2-[(2R)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol; {2-[(2S)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol

Test material form:

solid: particulate/powder

Details on test material:

Physical appearance: white powder
Storage conditions: at room temperature

In chemico test system

Details on the study design:

TEST ITEM PREPERATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN). Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 48.59 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1715 μL ACN to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to
ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler, in the dark, and incubated at 25 ± 2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see Table 3 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC mean percentage

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 5.0%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 79.3% ± 5.3%.

Remarks on result:

other: SD: 1.5%

Results 2

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL mean percentage

Vehicle controls validity:

not applicable

Negative controls validity:

not valid

Remarks:

CV between reference controls: 38.5%

Positive controls validity:

valid

Remarks:

mean percentage SPCL: 68.1% ± 3.5%

Remarks on result:

other: Co-elution of the test item with SPCL, Therefore the depletion could not be determined

Other effects / acceptance of results:

Co-elution occured between the test item and SPCL.
See table 4 and 5 in " any other information on results incl. table" for results and acceptability of DPRA

Any other information on results incl. tables

Table 4: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.997	>0.99	0.998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.507 ± 0.007	0.50 ± 0.05	0.496 ± 0.001
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.505 ± 0.008	0.50 ± 0.05	0.742 ± 0.293
CV (%) for RC samples B and C	<15.0	5.0	<15.0	38.5
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	79.3	40.2-69.0	68.1
SD of peptide depletion cinnamic aldehyde (%)	<14.9	5.3	<11.6	3.5
SD of peptide depletion for the test item (%)	<14.9	1.5	<11.6	NA

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable

Table 5: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD		Cysteine 1:10 prediction model
HYDROPIP-NR	1.2%	±1.5%	Co-elution	Negative: No or minimal reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

HYDROPIP-NR was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.

Executive summary:

A Direct Peptide Reactivity Assay (DPRA) was performed according to OECD TG 422C and in compliance with GLP. HYDROPIP-NR was dissolved in acetonitrile at 100 mM. Upon preparation as well as after incubation of the test item with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) no precipitate was observed. In the cysteine reactivity assay the test item showed 1.2% SPCC depletion. Co-elution was observed in the lysine reactivity assay and therefore SPCL depletion could not be calculated. Based on the results, HYDROPIP-NR was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model and was considered to be negative in the DPRA.