3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 December 2019 to 08 June 2020

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: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This assay assesses the covalent binding potential of the test item to proteins. The DPRA is proposed to address the molecular initiating event of the skin sensitisation AOP, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either Cysteine or Lysine. Cysteine and Lysine percent peptide depletion values are then used to categorise a substance in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers.

Test material

Test material information

Constituent 1

Reference substance name:

3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

Molecular formula:

C9H7BrClN3

IUPAC Name:

3-bromo-1-(3-chloropyrid-2-yl)-5-methyl-1H-pyrazole

Test material form:

solid: particulate/powder

Details on test material:

Batch/Lot Number: 628-044-00
Analysed Concentration: 974.9 ± 3.3 g/kg
Physical State: Beige Solid
Date of Manufacture: 15 October 2019
Date of Expiry: 15 October 2021

Specific details on test material used for the study:

Storage Conditions:
Storage Temperature: Room temperature (15 to 30 °C).
Storage Condition: Cool and dry conditions.
Storage Container: In original container as supplied by the Sponsor.
Storage Location: Test Item Control Office, JRF.

In chemico test system

Details on the study design:

- Synthetic heptapeptides containing either Lysine (Ac-RFAAKAA-COOH) or Cysteine (Ac-RFAACAA-COOH) are used as the test system for the direct peptide reactivity assay.
- Synthetic heptapeptides used in this study were obtained from RS Synthesis, Louisville, KY 40270, USA. - Batch number of Cysteine and Lysine peptide was P181203-LC180433 and P170906-LC107617, respectively, used in the study.

INSTRUMENTS AND EQUIPMENT
Micropipettes: Brand, Eppendorf AG.
Refrigerator: LG Electronics Inc.
Digital Balance: Ohaus (Capable of measuring 10 mg to 210 g).
Microbalance: Rodwag (Capable of measuring 1 mg to 5 g).
pH meter: Thermo Scientific.
Cyclomixer: Remi Electrotechnik.
Millipore Water.
System :Millipore.
High Performance Liquid.
Chromatography (HPLC): Shimadzu.
Ultralow Temperature Freezer: SANYO.

SOLVENTS AND CHEMICALS
Acetonitrile: Finar (HPLC #292761121FS).
Trifluoro acetic acid: SDFCL (HPLC #F17A/0517/0905/53).
Sodium phosphate, monobasic dihydrate: Merk (# DL6D663605).
Sodium phosphate, dibasic heptahydrate: Sigma (BioReagent #SLBL6644V).
Ammonium acetate: SDFCL (HPLC #2400161214).
Ammonium hydroxide: Sigma-VETEC (AR #MMBB4397V).


SOLVENT CONTROL: Yes.
Acetonitrile
CAS Number: 75-05-8
Molecular Weight: 41.05 g/mol
Manufactured by: Finar
Lot N°: 292761121FS
Date of receipt: September 24, 2019
Date of Expiry: May 2024
Appearance: Colourless Liquid
Purity: 99.1%
Storage : Room Temperature

POSITIVE CONTROL USED: Yes.
Cinnamaldehyde was used as positive control (PC) at a concentration of 100 mM in acetonitrile.
Name: Cinnamaldehyde
CAS Number: 104-55-2
Density: 1.049 g/mL
Manufactured by: Sigma-Aldrich
Lot N°: MKBT8955V
Date of receipt: February 12, 2016
Date of Expiry: February 2020
Appearance: Yellow Liquid
Purity: 99.1%
Storage: Room Temperature
Note: 38.10 µL of cinnamaldehyde was dissolved in 2961.90 µL of acetonitrile for the preparation of 100 mM solution (3 mL) for both Cysteine and Lysine peptides.


VEHICLE
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was found to be soluble in acetonitrile. Precipitation was not observed in acetonitrile. Hence, acetonitrile was selected as the vehicle for the study.

Results and discussion

Positive control results:

Cinnamaldehyde was used as positive control in each assay with Cysteine and Lysine peptides. Value of the mean percent peptide depletion value of the positive control, viz., cinnamaldehyde was 76% for Cysteine peptide and 64% for Lysine peptide (TABLE 5). The relative Coefficient of Variability (RCV) for the positive control replicate was 6.18% for percent Cysteine depletion and 1.94% for percent Lysine depletion.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Actual % Depletion (Cysteine)

Parameter:

other: DPRA Prediction

Value:

2

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA Prediction: Non-Sensitizer (Negative).

Results 2

Key result

Run / experiment:

other: Actual % Depletion (Lysine)

Parameter:

other: DPRA Prediction

Value:

1

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA Prediction: Non-Sensitizer (Negative).

Results 3

Key result

Run / experiment:

other: % Mean Depletion (Cysteine and Lysine)

Parameter:

other: DPRA Prediction

Value:

1.2

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA Prediction: Non-Sensitizer (Negative).

Other effects / acceptance of results:

- Acceptance criteria met for positive control:: Yes.

Any other information on results incl. tables

Reference Controls

The relative coefficient of variability (RCV) of peptide peak areas for the Reference Control B in acetonitrile was 0.16% for Cysteine peptide and 1.49% for Lysine peptide

 

Mean Reference Control Concentrations:

Peptide	Mean Reference Control A (mM)	Mean Reference Control B (mM)	Expected Concentration (mM)
Cysteine	0.52	0.50	0.45-0.55
Lysine	0.52	0.50	0.45-0.55

Cysteine Peptide Stability in Acetonitrile

The stability of Cysteine peptide in acetonitrile was checked at 0, 24, and 48 h. The relative coefficient of variability (RCV) for the stability of Cysteine peptide in acetonitrile was 2.39% against set standard of <15%. This showed that Cysteine peptide was stable in acetonitrile.

 

Data of Precipitation for Cysteine and Lysine Peptide:

Test Item Name	Precipitation/Phase Separation (Yes/No)
	Cysteine	Lysine
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole-Replicate 1	No	No
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole-Replicate 2	No	No
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole-Replicate 3	No	No
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole- Co-elution control	No	No

Standard Curve

A standard calibration curve was generated on each day of the HPLC analysis and the value of r2 obtained was 1.00000 for Cysteine and 0.99999 for Lysine containing peptides. This showed that system was suitable for assay.

 

Percent Peptide Depletion

 

% Mean Depletion of Peptides with Test Item:

Test Item Name	Actual % Depletion (Cysteine)	Actual % Depletion (Lysine)	% Mean Depletion (Cysteine and Lysine)	Maximum Standard Deviation (Cysteine)	Maximum Standard Deviation(Lysine)	DPRA Prediction
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	2	1	1.2	0.25	0.28	Non-Sensitizer (Negative)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

From the results of this study, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was predicted to be a Non-Skin Sensitiser in the DPRA assay.

Executive summary:

This study was conducted to evaluate the skin sensitisation potential of 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole, using the synthetic heptapeptides. The method followed was as per OECD Test Guideline No. 442C.

3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was found to be soluble in acetonitrile at 100 mM.

Therefore, acetonitrile was selected as the vehicle for this study.

Synthetic heptapeptides containing either Lysine (Ac-RFAAKAA-COOH) or Cysteine (Ac-RFAACAACOOH), were used as the test system in this assay. Cysteine and lysine containing peptides were incubated with a positive control and the test item for ≈27 hours at 22.5-30 ºC (in the dark), separately. Relative peptide concentration was measured by the high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers (OECD, 2015).

HPLC system suitability was determined by the standard calibration curve and the value of r2 obtained was 1.00000 for cysteine and 0.99999 for lysine peptides, against set standard of r2 > 0.99 as per the guideline.

The mean peptide concentration of Reference Control A and B is as mentioned in the table below:

 

The mean peptide concentration of Reference Control A and B were mentioned below:

Peptide	Mean Reference Control A (mM)	Mean Reference Control B (mM)	Expected Concentration (mM)
Cysteine	0.52	0.50	0.45-0.55
Lysine	0.50	0.50	0.45-0.55

The Relative Coefficient of Variability (RCV) for the Reference Control B was 0.16 for cysteine peptide and 1.49 for lysine peptide.

 

The percent peptide depletion obtained for Cysteine and Lysine is as tabulated below:

Sample Name and Date	Percent Peptide Depletion (Cysteine)				Percent Peptide Depletion (Lysine)
	% Depletion	SD	RCV	Expected Depletion	% Depletion	SD	RCV	Expected Depletion
Cinnamaldehyde (Positive control)	76	24455.42	6.18	60.8-100	64	10974.35	1.94	40.2-69
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	2	4137.29	0.25	-	1	4401.97	0.28	-

Percentage peptide depletion values of 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole for Cysteine and Lysine were 2% and 1%, respectively. The mean percent depletion for 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was 1.5%.

The relative coefficient of variability (RCV) for the stability of Cysteine peptide in acetonitrile was 2.39% against the set standard of <15% as per the guideline, indicating that Cysteine is stable in acetonitrile. In the case of Lysine peptide, the area obtained in the Co-elution chromatograph was 2389, i.e., only 0.15% of the mean peptide peak area of Reference Control B, which was <10%. Hence it was not considered as Co-elution interference (EURL ECVAM, 2013).

From the results of this study, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was predicted to be a Non-Skin Sensitiser.