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

Endpoint summary

Description of key information

1. Skin sensitisation according to OECD 442D:

In the KeratinoSens assay “In Vitro Skin Sensitisation: Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid Using KeratinoSensTM (HaCat) Cell line” 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid met all the evaluation criteria to be concluded as a non-sensitiser. Negative and positive controls met the acceptance criteria for the controls and were correctly identified as non-sensitiser and sensitiser, respectively. All criteria for a valid study were met.

2. Skin sensitisation according to OECD 442C:

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.

3. Skin sensitisation according to OECD 442E:

From the results of the study “In Vitro Skin Sensitisation: "Human Cell Line Activation Test (h-CLAT) of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid”, under the specified experimental conditions 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was identified as a sentisiser in the h-CLAT assay.

 

From the results of these studies under the specified experimental conditions 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid is predicted to be a sensitiser to skin, category 1, H317 according to EC regulation 1272/2008.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

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.

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 %.

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.

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)

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 January 2020 to

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC No. 440/2008, L 112, method B60: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method

Version / remarks:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The test system used for the in vitro KeratinoSensTM assay was KeratinoSensTM (HaCaT) cell line, an immortalised adherent human keratinocyte cell line. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The KeratinoSensTM test method has been validated by EURL ECVAM and considered scientifically valid to support the discrimination of skin sensitisers and non-sensitisers for the purpose of hazard identification.

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.

Details on the study design:

DETAILS OF TEST SYSTEM
- The genetically modified HaCaT cell line (KeratinoSensTM) obtained from Givaudan Schweiz AG.
- The KeratinoSensTM cell line contains a stable insertion of the luciferase gene under the transcriptional control of a constitutive SV40 promoter fused with an antioxidant response element (ARE) of the human AKR1C2 gene.
- Source: Mutagenicity section, Jai Research Foundation, India.
- Cell line Lot No.: JRF/HaCaT/2016/01.

VEHICLE
- Dimethyl sulfoxide (DMSO).
- 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was found to be soluble in dimethyl sulfoxide, at the concentration of 200 mM. Hence, dimethyl sulfoxide was selected as the vehicle for this study.

NEGATIVE (SOLVENT) CONTROL: yes
DMSO was used as the concurrent negative control for each set of experiments.
Name: DMSO.
CAS Number: 67-68-5.
Manufactured by: Qualigens.
Lot N°: 3491020519.
Retest Date: May 2024.
Appearance (Colour): Colourless Liquid.
Assay: 99.95%.
Storage: Room temperature.

POSITIVE CONTROL USED: yes.
Trans-cinnamaldehyde was used as a positive control for which stock concentrations from 0.4 to 6.4 mM were prepared in DMSO, which were further diluted to achieve final concentration of positive control ranging from 4 to 64 µM.
CAS Number: 14371-10-9.
Manufactured by: Sigma Aldrich.
Batch N°:STBF4119V.
Date of Receipt: 06 October 2015.
Date of Expiry: 05 October 2020.
Appearance (Colour): Light yellow liquid.
Purity: 99.4%.
Storage: 2–8 °C.
Density: 1.05 g/mL.

NUMBER OF REPLICATES, APPLICATION DOSE AND EXPOSURE TIME
- KeratinosensTM (HaCaT) cells were exposed to -Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid at concentrations: 0.98 µM to 2000 µM.
- KeratinosensTM (HaCaT) cells were exposed to Positive control (Trans-cinnamaldehyde) at concentrations: 4 to 64 µM.
- KeratinosensTM (HaCaT) cells were exposed to Negative control (DMSO).
- Cells were incubated for 48 ± 2 hours in 5 ± 1% CO2 at 37 ± 1 °C. After incubation, cells were analysed for luciferase activity.
- Cell viability of the concurrently treated cells was also evaluated using the MTT test with a separate set of plates.
- For each test item and positive control, one experiment consisting of at least two independent repetitions, each containing three replicates of each concentration.
- Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells.

Positive control results:

Luciferase activity induction obtained with the positive control, trans-cinnamaldehyde, was found to be >1.5 (the threshold value) at concentrations of 32 µM, and 64 µM in both repetitions. The induction for positive control at concentrations of 32 µM and 64 µM was found to be 1.77, and 2.85 µM in experiment 2 and 1.57 and 2.19 µM in experiment 4, respectively. The EC1.5 for positive control was 20.11 in experiment 2 and 28.15 in experiment 4. The average gene induction for positive control at 64 µM was found to be 2.85 and 2.19 for experiment 2 and 4, respectively (which was within the guideline acceptable limits of 2 and 8). A clear dose response, with increasing gene induction at increasing dose, was observed for trans-cinnamaldehyde in experiment 2 and experiment 4.

Key result

Run / experiment:

other: Imax

Parameter:

other: Luciferase gene induction

Remarks:

Imax: maximal fold gene-induction (Imax) value observed at any concentration of the test item and positive control.

Value:

1.5

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

KeratinoSensTM Prediction: Non-Sensitiser

Key result

Run / experiment:

other: EC1.5

Parameter:

other: Luciferase gene induction

Remarks:

EC1.5: value representing the concentration for which a gene induction above the 1.5-fold threshold.

Value:

2 000

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

KeratinoSensTM Prediction: Non-Sensitiser

Key result

Run / experiment:

other: IC50

Parameter:

other: Cellular viability

Remarks:

IC50: concentration value for 50% reduction of cellular viability.

Value:

2 000

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

KeratinoSensTM Prediction: Non-Sensitiser

Key result

Run / experiment:

other: IC30

Parameter:

other: Cellular viability

Remarks:

IC30: concentration value for 30% reduction of cellular viability.

Value:

1 126.98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

KeratinoSensTM Prediction: Non-Sensitiser

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes.
- Acceptance criteria met for positive control: yes.
- Acceptance criteria met for variability between replicate measurements: yes.

Negative Control

The coefficient of variation observed for the negative control, during experiment 7 and 8 for 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was 11.46% and 12.14%, respectively, which was below 20%. Since variation amongst the replicates was less than 20%, results of this run were considered as valid.

 

In experiment 7, variability in the negative control was 49.43, 41.95, and 31.94 % for six wells of each plate, but this was considered to be due to one well. Since the data from one well was ˃25% the average of the other five wells, it was considered as an outlier. After removal of the outlier, the variation in the negative control was 17.11, 9.81, and 6.63%, which was within the acceptable range.

 

Luciferase Activity

The maximal average fold induction of luciferase activity (Imax), following the treatment with 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid, was 1.89 at a concentration of 2000 µM in experiment 8, which is greater than the 1.5-fold increase required for a positive result. However, the cellular viability was 46.31% at the same concentration of 2000 µM in experiment 8. Therefore, the induction of luciferase activity above 1.5 fold was not considered as positive response.

 

In Vitro Skin Sensitisation: Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid using KeratinoSensTM (HaCat) Cell line.

Mean Summary of Luciferase Activity Induction and Cellular Viability Values:

I_max(Maximal average fold induction of luciferase activity):

Test Item	Rep2	Rep4	Average
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid	1.12	1.89*	1.50
trans-Cinnamaldehyde	5.86	29.14	17.50

EC_1.5(Concentration for which induction of luciferase activity is above the 1.5-fold threshold:

Test Item	Rep2	Rep4	GeoMean
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid	>2000µM	>2000µM	>2000µM
trans-Cinnamaldehyde	6.26 µM	6.17 µM	6.22 µM

IC₅₀(Concentration value for 50% reduction of cellular viability):

Test Item	Rep2	Rep4	GeoMean
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid	>2000µM	1837.05µM	>2000µM

IC₃₀(Concentration value for 30% reduction of cellular viability):

Test Item	Rep2	Rep4	GeoMean
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid	1287.98µM	986.10µM	1126.98µM

Key: * = As the viability at the highest concentration was less than 70%, the induction ≥1.5 fold will not be considered as positive response,

Rep = Repetition/Experiment – each comprising 3 plates.

 

Note: Values shown for each repetition are the mean values of 3 replicates.

IC50 and IC30 cannot be determined for positive control.

In Vitro Skin Sensitisation: Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene Test of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid using KeratinoSensTM (HaCat) Cell line.

 

Summary of Mean Luciferase Activity Induction Values (Mean of Two Repetitions /Experiments):

3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid (µM)	Fold Luciferase Activity Induction
	Mean	SD
0.98	1.05	0.09
1.95	1.12	0.12
3.91	1.08	0.07
7.81	0.99	0.06
15.63	0.89	0.04
31.25	0.87	0.29
62.5	0.93	0.30
125	0.91	0.26
250	0.88	0.25
500	1.14	0.08
1000	1.25	0.19
2000	1.21	0.96

Cytotoxicity Assessment

Cytotoxicity was observed in the higher concentrations in this study. The cellular viability was 64.33% at a concentration of 2000 µM in experiment 7; 68.90% and 46.32% at a concentration of 1000 and 2000 µM, respectively, in experiment 8, with induction of luciferase activity above 1.5 fold.

 

Interpretation

For 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid, eight experiments were conducted and out of these, two valid experiments (experiment 07 and 08) were considered for the final evaluation. The reason for conducting repeat experiments was due to the failure of the negative control and positive control to meet the acceptance criteria in experiment 01 to 06. Results of valid experiments are presented in this report.

The mean results are summarised below:

Test Item Name	Luciferase gene induction		Cellular viability		KeratinoSens^TM Prediction
	Imax	EC1.5	IC50	IC30
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid	1.50*	>2000µM	>2000µM	1126.98 µM	Non-Sensitiser
Positive Control (Trans-Cinnamaldehyde)	17.50	6.22	-	-	Sensitiser

Results of this KeratinoSensTM assay indicate that 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid met all the evaluation criteria to predict that it is a non-sensitiser in this test. The negative and positive controls met the acceptance criteria and were correctly identified as the non-sensitiser and sensitiser, respectively. This showed the suitability of the test system and procedures used.

Interpretation of results:

GHS criteria not met

Conclusions:

From the results of this KeratinoSensTM assay, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid is predicted to be a non-sensitiser to skin according to EC regulation 1272/2008 (CLP).

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 by assessing keratinocyte activation, using a Keratinocyte-Based ARE-Nrf2 Luciferase Reporter Gene method, as recommended by the OECD Test guideline 442D.

3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was found to be soluble at 200 mM in dimethyl sulfoxide (DMSO). Therefore, DMSO was selected as a vehicle. The test item was tested in two independent experiments. KeratinosensTM (HaCaT) cells were exposed to 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid at the test concentration from 0.98 µM to 2000 µM, to the positive control (trans-cinnamaldehyde) at concentrations of 4 to 64 µM or to the negative control (DMSO). Cells were incubated for 48 ± 2 hours in 5 ± 1% CO2 at 37 ± 1 °C. After incubation, cells were analysed for luciferase activity. Cell viability of the concurrently treated cells was also evaluated using the MTT test with a separate set of plates. The value of Imax and EC1.5 was calculated based on luciferase activity, i.e., luminescence measured (reading of three plates) while the value of IC50 and IC30 was calculated based on the results of cytotoxicity (OD values, reading of one plate).

Test Item Name	Luciferase gene induction		Cellular viability		KeratinoSens^TM Prediction
	Imax	EC1.5	IC50	IC30
3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid	1.50*	>2000µM	>2000µM	1126.98 µM	Non-Sensitiser
Positive Control (Trans-Cinnamaldehyde)	17.50	6.22	-	-	Sensitiser

* = As the viability at the highest concentration was less than 70%, the induction ≥1.5 fold was not considered as positive response.

 

The maximal average fold induction of luciferase activity (Imax), following the treatment with 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid, was 1.89 at a concentration of 2000 µM in experiment 8, which is greater than the 1.5-fold increase required for a positive result. However, the cellular viability was 46.31% at the same concentration of 2000 µM in experiment 8. Response was less than 1.5 fold where the cell viability was above 70% at tested concentrations below 1000 µM. Therefore, the induction of luciferase activity above 1.5 fold was not considered as positive response.

 

3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid met all the evaluation criteria to be predicted as a non-sensitiser in the KeratinoSens assay. Negative and positive controls met the acceptance criteria for the controls and were correctly identified as non-sensitiser and sensitiser, respectively. All criteria for a valid study were met, as described in the study plan.

 

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

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E, In Vitro Skin Sensitisation: Human Cell Line Activation test (h-CLAT Test Method)

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B7:In Vitro Skin Sensitisation: Human Cell Line Activation test (h-CLAT Test Method).

Version / remarks:

July 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The test system used for the in vitro human cell line activation test (h-CLAT assay) was the THP 1 cell line which is used as a surrogate of dendritic cells (DC). Upon contact with sensitizers, upregulation of cell surface markers (i.e., CD86 and CD54) on these cells can be measured, which may mimic DC activation. This cell line is the recommended test system by OECD 442E for the assay.

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.

Details on the study design:

DETAILS OF TEST SYSTEM
- Test system: THP-1, a human monocytic leukemia cell line, procured from American Type Culture Collection (ATCC), maintained at the Mutagenicity section, Jai Research Foundation as specified in OECD 442E.
- The doubling time of THP-1 cells was found to be approximately 24 h.
- Cells of passage number 23 (DRF experiments-1 and 2), 07 (Reactivity check), 8 [Expression study (experiment -1)], 9 [Expression study (experiment -2)], 10 [Expression study (experiments-3)], 13 [Expression study (experiment -4)], 14 [Expression study (experiment -5)] were used for the study.
- Batch number of the cell line used was 63176297 for the study.

CULTURE MEDIUM
The medium used for culturing cells as well as during the assay consisted of RPMI-1640 with
2 mM L-glutamine and 25 mM HEPES supplemented with 10% v/v Fetal Bovine Serum (FBS), 0.05 mM 2-Mercaptoethanol and appropriate antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin).


POSITIVE CONTROL
2,4-Dinitrochlorobenzene was used as a positive control during CD86/CD54 expression measurement, tested at a single concentration of 4.0 µg/mL in DMSO.
Name: 2, 4-Dinitrochlorobenzene
CAS Number: 97-00-7
Molecular Weight: 202.55 g/mol
Manufactured by: Sigma Aldrich
Batch N°: STBD7009V
Date of receipt: September 21, 2015
Date of expiry: September 20, 2020
Appearance (color): Brown Crystalline chunk(s)
Purity: 99.9%
Storage: Room temperature

NEGATIVE CONTROL
Concurrent negative controls, consisting of solvent and complete medium alone, without test item was included in each assay.
Medium Control: Complete medium.
DMSO Control: DMSO diluted to 0.4% in complete medium, which was tested at a final concentration of 0.2% in the plate.

SOLVENTS AND CHEMICALS
RPMI-1640 (1X): Gibco (Lot # 2085340, 2120429)
Fetal Bovine Serum: Gibco (Lot # 1841109)
Penicillin Streptomycin: Gibco (Lot # 214554)
2-Mercaptoethanol: Gibco (Lot # 1929845)
DPBS (1X): Gibco (Lot # 2085310)
Dimethyl Sulfoxide: Sigma (Lot # SHBG5286V) and Qualigens (Lot # 3491020519)
Globulin Cohn Fraction II, III
Human, lyophilized powder : Sigma (Lot# 017K7650V)
Bovine Serum Albumin : HiMedia (Lot #0000345510)
Phosphate buffered Saline Tablets: Sigma (Lot #SLBT3740, SLBW3999)
FITC mouse anti-human CD86
Antibody (Clone: Fun-1): BD Pharmingen (Lot #7348597)
Monoclonal mouse anti-human
CD54, ICAM-1 antibody/FITC (Clone 6.5 B5): Dako (Lot #20051521)
Isotype reagent mouse IgG1/
FITC antibody: Dako (Lot #20058363)
Propidium iodide: Sigma (Lot #MKCC9922)
Tryphan blue solution: Sigma (Lot #RNBF6596)
FACSFlow Sheath fluid: BD (Lot #1903002839)
FACS Clean: BD (Lot #1908002830)
FACSuite CS&T Research Beads: BD (Lot #91487)
Dettol (Disinfectant Solution): Reckitt Benckiser (Lot #GL991)

CULTURE VESSELS
- Disposable tissue cultures flasks of 75 cm2 culture area with canted neck (Nunc) was used for culturing cells and treatment was performed in 96-well flat-bottom transparent plates (for Dose Range Finding assay) and 24-well culture plates (for CD86/CD54 Expression Measurement).

VEHICLE
- DMSO was selected as a vehicle for the study and the highest stock concentration selected was 500 mg/mL.

Positive control results:

The acceptance criteria for the positive control were met:
The RFI values of CD86 and CD54 were greater than 150% and ≥200%, respectively, and the cell viability was greater than 50%, in both experiments.

Key result

Run / experiment:

other: h-CLAT assay: Observed EC200 for CD54 (µg/mL)

Parameter:

other: Calculated Effective Concentration (EC) value.

Value:

296

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes.

Dose

Two independent experiments of the Dose Range Finding (DRF) assay were performed (with concentrations ranging from 1.95 µg/mL to 1000 µg/mL) to determine CV75 value of the test item (the concentration showing 75% of THP-1 cell survival, i.e., 25% cytotoxicity).

In DRF experiment 1, any significant cell death (i.e., <75% viability) was not observed at any concentration up to 1000 µg/mL.

On the basis of results of both DRF experiments, CV75 of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was found to be >1000 µg/mL.

 

CD86/CD54 Expression Measurement (Expression Study)

Five independent experiments were performed to determine the upregulation of CD86/CD54 markers at sub-cytotoxic concentrations of the test item (with concentrations ranging from 279 µg/mL to 1000 µg/mL). The results of two valid experiments (i.e, experiments-4 and 5), which met the acceptance criteria are presented here.

 

Negative (Solvent) Control 

Medium Control

The MFI ratio of the medium control for CD86 to isotype control and for CD54 to isotype control was found to be 248.63% and 115.01% respectively, in experiment-4, and 201.50% and 108.25% respectively, in experiment-5.

The MFI ratio of the medium control for CD86 and CD54 to isotype control was found to be >105% in both experiments.

The cell viability of the medium control was found to be 99.0% and 98.9% in experiments-4 and 5, respectively. The cell viability of the medium control was >90% in both experiments.

 

DMSO Control

RFI values and the MFI ratio of CD86 and CD54 markers to isotype control for experiment-4 and experiment-5:

Parameters	Expt.4	Expt.5	Acceptance Value
RFI CD86	77.82%	143.51%	<150%
RFI CD54	91.97%	128.57%	<200%
MFI ratio CD86 to isotype control	218.12%	245.97%	>105%
MFI ratio CD54 to isotype control	114.09%	110.63%	>105%
Cell viability	99.0%	99.0%	>90%

The acceptance criteria were met for DMSO control:

·        the RFI values of CD86 and CD54 were less than 150% and 200%, respectively, in both experiments; 

·        the MFI ratio for CD86 and CD54 markers to isotype control was greater than 105% in both experiments;

·        the cell viability was greater than 90% in both experiments.

 

Positive Control

 

RFI values of CD86 and CD54 markers and cell viability:

Parameters	Expt.4	Expt.5	Acceptance  Value
RFI CD86	465.25%	479.76%	≥150%
RFI CD54	724.60%	1282.83%	≥200%
Cell viability	83.3%	89.8%	≥50%

The acceptance criteria for the positive control were met:

The RFI values of CD86 and CD54 were greater than 150% and ≥200%, respectively, and the cell viability was greater than 50%, in both experiments.

 

The test item produced a positive response for CD54 marker (i.e., RFI CD54 ≥200%, with cell viability ≥50%) in 3/8 and 6/8 tested concentrations in experiment 4 and 5, respectively. The cell viability of the test item was found to be >50% at all tested concentrations in both experiments, which met the assay acceptance criteria.

The individual values of the cell viability, MFI, and RFI of the test item obtained in the individual experiments of CD86/CD54 Expression Measurement are provided in the following table.

 

CD86/CD54 Expression Measurements: Cell Viability, MFI and RFI Values of 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid:

Conc. (µg/mL)	Expt.4
	IgG1			% Cell Viability	MFI			% RFI
	Total Count	THP-1 cells | Count (Number of Gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)		IgG1	CD86	CD54	CD86	CD54
1000	10378	9643	9514	98.7 %	93.4	201	109	101.89	123.81
833	10370	9621	9490	98.6 %	94.8	193	117	92.99	176.19
694	10310	9735	9632	98.9 %	93.9	199	109	99.53	119.84
579	10427	9604	9475	98.7 %	97.8	205	121	101.52	184.13
482	10388	9694	9584	98.9 %	95.6	192	122	91.29	209.52
402	10458	9645	9514	98.6 %	98.1	193	131	89.87	261.11
335	10489	9651	9508	98.5 %	96.4	179	125	78.22	226.98
279	10382	9676	9568	98.9 %	99.5	188	114	83.81	115.08

Conc. (µg/mL)	Expt.5
	IgG1			% Cell Viability	MFI			% RFI
	Total Count	THP-1 cells | Count (Number of Gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)		IgG1	CD86	CD54	CD86	CD54
1000	10413	9646	9575	99.3 %	109	224	130	84.62	212.12
833	10454	9630	9541	99.1 %	110	214	143	76.53	333.33
694	10421	9580	9498	99.1 %	105	229	122	91.24	171.72
579	10483	9537	9443	99.0 %	103	211	123	79.47	202.02
482	10544	9477	9379	99.0 %	102	226	124	91.24	222.22
402	10460	9619	9527	99.0 %	101	244	121	105.22	202.02
335	10503	9499	9412	99.1 %	98.8	239	121	103.16	224.24
279	10573	9464	9355	98.8 %	100	232	119	97.13	191.92

Shaded values indicate %RFI ≥150% (CD86) or ≥200% (CD54) with cell viability ≥50%

 

 Based on results of experiments-4 and 5, the prediction obtained for the test item was P12 and P12, respectively, and the Effective Concentrations (EC) values were calculated. The mean values and assay predictions for the test item is summarised below:

Observed CV75 (µg/mL)	Observed EC150 for CD86 (µg/mL)^	Observed EC200 for CD54 (µg/mL)*	Expt. 4 Prediction	Expt. 5 Prediction	Final Prediction
>1000	-	296	Positive (P2)	Positive (P2)	Positive

Key: * = Higher EC200 value of test item

 

Individual results of CV75, EC150, and EC200, in different experiments, are provided in the following table.

 

Individual CV75, EC150 and EC200 values of all experiments:

Sr. N^o	Parameters	Expt.4	Expt.5	Average values (in µg/mL)
1	CV75 (in µg/mL)	>1000*	>1000*	>1000
2	EC150 (in µg/mL)	-	-	-
3	EC200 (in µg/mL)	296	206	296^#

Key: * = CV75 values are from DRF Experiments 1 and 2, respectively, # = Higher EC200 value out of the two experiments.

 

Interpretation

In both experiment 4 and 5, a positive response was obtained for CD54 (P2). The final prediction for the test item was “positive”.

The final EC200 value was determined as the median value of the ECs calculated from the two acceptable independent runs. The EC200 value reported were the higher EC value from the two valid experiments that met the criteria for positivity.

Results of the present study indicate that, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid met all the evaluation criteria to be concluded as the “positive” in h-CLAT assay. The negative and the positive controls met the specified acceptance criteria for controls. This showed the suitability of the test system and procedures used in this test facility.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

From results of this h-CLAT assay, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was concluded as “positive” in an in vitro skin sensitisation assay and may be predicted to be a skin sensitiser skin according to EC regulation 1272/2008 (CLP).

Executive summary:

This study was conducted to evaluate the skin sensitization potential of

3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid based on the human cell line activation test (h-CLAT) method, as recommended by the OECD 442E.

3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was found to be soluble in dimethyl sulfoxide (DMSO) at the final concentration of 500 mg/mL. Hence, DMSO was selected as the vehicle for this study.

Two Dose Range Finding (DRF) assays were performed to determine CV75 value of the test item. For DRF assays, THP-1 cells were exposed to the test item at ten concentrations from 1.95 µg/mL to 1000 µg/mL and incubated for 24 ± 0.5 h at 37 ± 1 °C under 5 ± 1% CO2. After incubation, cells were washed, stained with propidium iodide, and analysed on flow cytometer, to determine cell viability (for the calculation of CV75 value). On the basis of CV75 value of the test item, i.e., >1000 µg/mL, concentrations were selected for CD86/CD54 expression measurement.

CD86/CD54 expression measurement (expression study), was performed to determine the expression of CD86 and CD54 cell surface markers at the sub-cytotoxic concentrations of the test item. For the assay, THP-1 cells were exposed to the test item at eight concentrations between 279 µg/mL and 1000 µg/mL and incubated for 24 h at 37 ± 1 °C under 5 ± 1% CO2. After incubation, cells were washed, blocked, stained with specific antibodies and propidium iodide, and analysed on the flow cytometer. Based on results of Expression study, RFI values of the test item were calculated and used in a prediction model to support the discrimination between the sensitiser and non-sensitiser.

The test item produced a positive response for CD54 marker (i.e., RFI CD54 ≥200%, with cell viability ≥50%) in 3/8 and 6/8 tested concentrations in experiments-4 and 5, respectively. The cell viability of the test item was found to be >50% at all tested concentrations in both experiments, which met the assay acceptance criteria. Based on results of experiments-4 and 5, the predictions obtained for the test item were P2 and P2, respectively. Based on the calculated RFI CD54 values, the higher EC200 value of the test item was 296 µg/mL.

Observed CV75 (µg/mL)	Observed EC150 for CD86 (µg/mL)	Observed EC200 for CD54 (µg/mL)*	Expt. 4 Prediction	Expt. 5 Prediction	Final Prediction
>1000	-	296	Positive (P2)	Positive (P2)	Positive

Key: * = Higher EC200 value of test item

 

The RFI values of the positive control, i.e., 2, 4-Dinitrochlorobenzene were greater than 150% for CD86 marker, and greater than 200% for CD54 marker in both experiments-4 and 5, and met the assay acceptance criteria. (RFI CD86 ≥150%, RFI CD54 ≥200%). The cell viability of the positive control was >50% in both experiments. All observed values of the negative (solvent) control were within the acceptable range of the test guideline. All criteria for a valid study were met, as described in the study plan.

From the results of this h-CLAT assay, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid was concluded as “positive” in an in vitro skin sensitization assay and may be predicted to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on three in vitro skin sensitisation tests according to OECD 442C, OECD 442D and OECD 442E 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid is determined to be a sensitising substance to skin.