1H-Pyrazole-5-carboxylic acid, 3-bromo-1-(3-chloro-2-pyridinyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Remarks:

direct peptide reactivity assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2020 to 15 May 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:

June 18, 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:

-

EC Number:

481-150-8

EC Name:

-

Cas Number:

500011-86-9

Molecular formula:

C9H5BrClN3O2

IUPAC Name:

3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

Identification: 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid
CAS No.: 500011-86-9
Batch: 628-067-1117
Test item Content: 964.8 ± 1.5 g/kg
Production date: November 17, 2019
Expiry date: November 17, 2021
Supplier: ADAMA Makhteshim Ltd.
Appearance: White solid

Specific details on test material used for the study:

Storage Condition (at JRF):
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:

DETAILS OF TEST SYSTEM
- 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.

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
Milli Q Water: Prepared by using Millipore water purification system, (Elix-10 and Milli-Q gradient).
Acetonitrile: Finar (HPLC #292761121FS).
Trifluoro acetic acid: SDFCL (HPLC #F17A/0517/0905/53).
Sodium phosphate, monobasic dihydrat: Merk (# DL6D663605).
Sodium phosphate, dibasic heptahydrate: Sigma (BioReagent #SLBL6644V).
Ammonium acetat: Qualigens (HPLC #3293830219).
Isopropanol: Qualigens (#3126601218).

SOLVENT CONTROL: yes.
Isopropanol
CAS Number: 67-63-0.
Manufactured by: Qualigens.
Lot No: 3126601218.
Date of receipt: January 01, 2019.
Date of Expiry: December 2023.
Appearance: Clear Colourless Liquid.
Purity: 99.93%.
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-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was found to be soluble in isopropanol at 100 mM. Precipitation was not observed in isopropanol. Hence, isopropanol was selected as the vehicle for the experiment.

Results and discussion

Positive control results:

Cinnamaldehyde was used as the 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 90% for Cysteine peptide and 66% for Lysine peptide. The relative Coefficient of Variability (RCV) for the positive control replicate was 1.79% for percent Cysteine depletion against the guideline limit of <14.9 % and 4.30% for percent Lysine depletion against the guideline limit of <11.6 %.

In vitro / in chemico

Results

Key result

Run / experiment:

other: % Mean Depletion (Cysteine and Lysine)

Parameter:

other: DPRA Prediction

Value:

3

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA Prediction: No or Minimal Reactivity (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 A was 0.64 % for Cysteine peptide and 0.18 % for Lysine peptide. For Reference Control B relative coefficient of variability (RCV) of peptide peak areas was 0.62% for Cysteine peptide and 0.40 % for Lysine peptide Lysine, respectively.

 

Mean Reference Control Concentrations:

Peptide	Mean Reference Control A (mM)	Mean Reference Control B (mM)	Mean Reference Control C (mM)	Expected Concentration (mM)
Cysteine	0.50	0.50	0.50	0.45-0.55
Lysine	0.50	0.50	0.51	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 1.51% against the 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-Pyridinyl)-1HPyrazole-5-Carboxylic acid-Replicate 1	No	No
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1HPyrazole-5-Carboxylic acid-Replicate 2	No	No
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1HPyrazole-5-Carboxylic acid-Replicate 3	No	No
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1HPyrazole-5-Carboxylic acid- 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 0.99997 for Cysteine and 0.99998 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-Pyridinyl)-1HPyrazole-5-Carboxylic acid	3	3	3	0.07	0.62	No or Minimal Reactivity (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-Pyridinyl)-1HPyrazole-5-Carboxylic acid was identified as a Negative in the DPRA assay.

Executive summary:

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

3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was found to be soluble in isopropanol at 100 mM. Therefore, isopropanol 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 24 ± 2 hours at 22.5-30 ºC (in the dark), separately. Relative peptide concentration was measured, using 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, 2019).

HPLC system suitability was determined by the standard calibration curve and the value of r2 obtained was 0.99997 for cysteine and 0.99998 for lysine peptides, against the set standard of r2 > 0.99, as per the guideline.

 

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

Peptide	Mean Reference Control A (mM)	Mean Reference Control B (mM)	Mean Reference Control C (mM)	Expected Concentration (mM)
Cysteine	0.50	0.50	0.50	0.45-0.55
Lysine	0.50	0.50	0.51	0.45-0.55

The relative coefficient of variability (RCV) of peptide peak areas for the Reference Control A was 0.64 % for Cysteine peptide and 0.18 % for Lysine peptide. For Reference Control B relative coefficient of variability (RCV) of peptide peak areas was 0.62% for Cysteine peptide and 0.40 % for Lysine peptide Lysine, respectively. The Relative Coefficient of Variability (RCV) for the Reference Control C was 0.11% for cysteine peptide and 2.33% 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)	90	3312.01	1.79	60.8-100	66	24472.88	4.30	40.2-69
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1HPyrazole-5-Carboxylic acid	3	1172.48	0.07	-	3	10062.57	0.62	-

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

The relative coefficient of variability (RCV) for the stability of Cysteine peptide in acetonitrile was 1.51% against the set standard of <15% as per the guideline, indicating that Cysteine is stable in acetonitrile. The relative coefficient of variability (RCV) for the stability of Lysine peptide in acetonitrile was 0.40% against

the set standard of <15% as per the guideline, indicating that Lysine peptide was stable in acetonitrile over the analysis time.

From the results of this study, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid is predicted by DPRA to be a non-sensitiser (Negative).

 

From the results of the study “In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid using Synthetic Heptapeptides”, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was identified as a Negative in the DPRA assay.