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

Endpoint summary

Description of key information

1. Skin corrosion according to OECD 431:

The mean percent relative viability in tissues treated with the test item was 97.67% after 3-minute exposure and 94.69% after 60-minute exposure. No significant reduction in the percent cell viability was observed after 3-minute and 60-minute exposure in treated tissues when compared with that of the concurrent negative control. The difference between viability of treated tissues was less than 3%, i.e., % CV.

All criteria for a valid study were met. From the results of this study, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was concluded to be non-corrosive in the in vitro skin corrosion test, using reconstructed human epidermis (RhE) tissues.

2. Skin irritation according to OECD 439:

The mean percent cell viability in tissues treated with the test item showed no significant reduction in the percent cell viability observed in that 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole treated tissues, when compared with that of the concurrent negative control. The mean percent relative viability in tissues treated with the test item was 93.3 % after 42-minute exposure. From the results of this study, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was concluded to be not an irritant in the in vitro skin irritation test, using reconstructed human epidermis (RhE) tissues.

3. Eye irritation according to OECD 492:

In a reconstructed Human Cornea-like Epithelium (RhCE) Test according to OECD 492 the mean per cent cell viability in tissues treated with 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole was found to be 32.33% of the concurrent negative control (100%) which is below the established tissue viability cut-off value of 50%. Therefore the study is not sufficient to give reliable results on eye corrosion and classification according to regulation 1272/2008 (CLP) is not possible.

Although the pH value of 6.75 (in a 1% w/w solution) is neither critical nor relevant for classification, the registrant decided due to safety reasons to classify 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole as eye damaging category 1 (H318).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17. February 2020 to 08 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU method B40 bis, “In Vitro Skin Corrosion:Human Skin Model Test”

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage Condition:
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.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

other: human epidermis (RHE/S/17)

Justification for test system used:

This study addresses the human health endpoint skin corrosion. It makes use of reconstructed human epidermis (RhE) (human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RHE) is recommended by the OECD and other regulatory authorities. The SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is a recommended model for conducting in vitro skin corrosion studies. The results of the study are believed to be predictive for the potential of inducing skin corrosivity in humans.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model.
- Tissue batch number: Lot N° 20-RHE-011.
- Production date: 27 January 2020.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at 37 ± 1 °C, in 5±1% CO2 in a 95% humidified atmosphere for 60 minute exposure.
- Positive control tissues: 40 µL/0.5 cm2 of 8N KOH was applied for an exposure period of 60 minutes at 37 ± 1 °C in 5 ± 1% CO2 in a humidified incubator.
- Negative control tissues: 40 µL/0.5 cm2 of sterile distilled water was applied for 3 minutes at room temperature and 60 minutes at 37±1 °C in 5±1% CO2 in a 95% humidified atmosphere.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 20 mg ± 3 mg of test item/0.5 cm2.

VEHICLE
- Concentration: 20 µL sterile distilled water

NEGATIVE CONTROL
- Concentration: 40 µL/0.5 cm2 of sterile distilled water

POSITIVE CONTROL
- Concentration: 40 µL/0.5 cm2 of 8N KOH

Duration of treatment / exposure:

3 minutes at room temperature and 60 minutes at 37 ± 1 °C

Number of replicates:

three replicates/time point

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean percent viability (3 minutes exposure)

Value:

97.67

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean percent viability (60 minutes exposure)

Value:

94.69

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

no indication of corrosion

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No visible damage.
- Direct-MTT reduction: No interaction.
- Colour interference with MTT: No interaction.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
OD values (corrected ODs) of the negative controls in all tissues were between 1.537 and 1.918 i.e., within the test guideline optical density requirement of ≥ 0.8 and ≤ 3.0 (the acceptance criteria for SkinEthicTM RHE model). Results of the negative control met the OECD 431 guideline acceptance range for the prediction model SkinEthicTM RHE.
- Acceptance criteria met for positive control:
The mean percent viability of the positive control was 0.34%, which met OECD 431 acceptance criteria, i.e., <15% viability.
- the observed results were within the acceptance/historical range of SkinEthic Laboratories/OECD TG acceptance criteria.

Pre-Tests

 

Colour Interference Test:

No significant difference in the absorbance was observed between the negative control (distilled water) and the test item. Therefore results of the colour interference test shows that interference was not observed due to test item. The results of the colour interference test are provided in the below-mentioned table:

Treatment	Optical Density (nm)	Interaction
Negative Control (distilled water)	0.041	No
	0.041
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	0.045	No
	0.044

Direct MTT Reduction Test:

The test item did not produce any direct MTT reduction (in the absence of tissues) when compared with that of the concurrent negative control (distilled water). Results of the direct MTT reduction test are summarised below:

Treatment	Interaction
Negative Control (distilled water)	No
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	No

Main Study:

The mean percent viability of tissues treated with 3-Bromo-1-(3-Chloro-2-Pyridinyl)-1H-Pyrazole-5-Carboxylic acid, negative control and positive control are summarised below:

Treatment	Viability
	3 Minutes Exposure	60 Minutes Exposure
Negative control (Sterile distilled water)	100%	100%
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	97.67%	94.69%
Positive control(8N KOH)	-	0.34%

Based on the results of this study, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole is classified as non-corrosive, as per the “United Nations Globally Harmonized System of Classification and Labelling of Chemicals”.

Interpretation of results:

GHS criteria not met

Conclusions:

From the results of this study, under the specified experimental conditions, it is concluded that 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole is predicted to be non-corrosive to the skin as indicated in OECD 431, using reconstructed human epidermis (RhE) tissues. The substance is not classified as skin corrosive according to EC regulation 1272/2008 (CLP).

Executive summary:

This study was performed to evaluate the corrosive potential of 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole, using reconstructed human epidermis (RhE) tissue, in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Tissues were exposed to 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole or sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37 ± 1 °C in 5 ± 1% CO₂using three replicates/time point. Positive control tissues were exposed for 60 minutes at 37 ± 1 °C in 5 ± 1% CO₂. Killed tissues were also exposed for 60 minutes at 37 ± 1 °C in 5 ± 1% CO₂.

The mean percent relative viability in tissues treated with the test item was 97.67% after 3-minute exposure and 94.69% after 60-minute exposure. No significant reduction in the percent cell viability was observed after 3-minute and 60-minute exposure in treated tissues when compared with that of the concurrent negative control.

The difference between the viability of the treated tissues was less than 3%, i.e., % CV.

Treatment	Viability
	3 Minutes Exposure	60 Minutes Exposure
Negative control (Sterile distilled water)	100%	100%
to 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	97.67%	94.69%
Positive control(8N KOH)	-	0.34%

All Optical Density (OD) values (corrected OD) for the negative control replicates were between 1.537 and 1.918, against a guideline requirement of ≥ 0.8 and ≤ 3.0 (the acceptance criteria for SkinEthicTM RHE model).

The positive control showed 0.34% cell viability, against a guideline requirement of <15%, when compared with that of the concurrent negative control, demonstrating the efficiency of the SkinEthicTM RHE model.

All criteria for a valid study were met. From the results of this study, under the specified experimental conditions, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole was concluded to be non-corrosive in the in vitro skin corrosion test, using reconstructed human epidermis (RhE) tissues.

 

The substance is not classified as skin corrosive according to EC regulation 1272/2008 (CLP).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2020 to 08 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

July 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Storage Condition:
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

Test system:

human skin model

Remarks:

reconstructed human epidermis

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

This study addresses the human health endpoint skin irritation. It makes use of reconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal keratinocytes) which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis. The use of reconstructed human epidermis (RhE) is also recommended by the OECD and other regulatory authorities. SkinEthicTM RHE model has been validated and is part of OECD validated reference methods (VRMs) and is also a recommended model for conducting in vitro skin irritation studies.The results of the study are believed to be of value in predicting the potential of inducing skin irritation by the test item in humans.

Vehicle:

water

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RHE model / Human Epidermis (RHE/S/17).
- Tissue batch number(s): 20-RHE-011, Lot N° 20-RHE-011.
- Procured from: SkinEthic Laboratories, Episkin 4, Rue Alexander Fleming, 69366 Lyon Cedex 07, France.
- Origin: Foreskin.
- Suggested Expiration Date: 27 January 2020.

NEGATIVE CONTROL: Yes.
tissues were treated with sterile Dulbecco’s Phosphate Buffered Saline (DPBS,
Lot N°: RNBH6544)

POSITIVE CONTROL: Yes.
Tissues were treated with sterile sodium dodecyl sulfate (5% aq.):
CAS Number: 151-21-3.
Purity: 100%.
Lot N°: 0000000192.
Date of receip : May 06, 2016.
Date of Expiry: May 05, 2021.
Appearance: White powder.
Storage: Room temperature.
1g sodium dodecyl sulfate was dissolved in 20 mL sterile distilled water (5% aq.) for the preparation
of 5% sodium dodecyl sulfate (5% aq.).

SOLVENTS AND CHEMICALS
MTT: Sigma (MKCB8895).
Isopropanol: Qualigens (3591580819).
Dulbecco's phosphatebuffered saline-10X: Sigma (RNBH6544).
SkinEthic Maintenance Medium : SkinEthic Laboratories (20 SMM 003).
SkinEthic Growth Medium : SkinEthic Laboratories (20 SGM 008).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature of Pre-incubation: 37±1 °C in 5 CO2 in a 95% humidified incubator.
- Temperature used during treatment / exposure: at room temperature.
- Temperature of post-treatment incubation: 37±1°C in 5 CO2 in a humidified incubator.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing 25 times in a constant soft stream of 1 mL DPBS from 5–8 cm distance from the insert.
- Observable damage in the tissue due to washing: No.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL.
- Incubation time: 180 minutes at 37 ± 1°C in 5% CO2 in a 95% humidified incubator.

NUMBER OF REPLICATE TISSUES:
- N. of replicates : Three replicates of test item (3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1HPyrazole); three replicates of negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)); three replicates of positive control (Sodium dodecyl sulfate (5% aq.)).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied : 16 mg of test item/0.5 cm2, 16 μL/0.5 cm2 of sterile Dulbecco’s phosphate buffered saline (DPBS) as negative control; 16 μL/0.5 cm2 of 5% sodium dodecyl sulphate (5% aq.) as positive control.

VEHICLE
- Amounts applied: 10 µL of sterile distilled water

NEGATIVE CONTROL
- Amounts applied: 16 µL/0.5 cm2 sterile Dulbecco’s phosphate buffered saline (DPBS)

POSITIVE CONTROL
- Amounts applied: 16 µL/0.5 cm2 of 5% sodium dodecyl sulphate (5% aq.)

Duration of treatment / exposure:

42 minutes.

Duration of post-treatment incubation (if applicable):

42 hours.

Number of replicates:

Three replicates/test item/negative control/positive control.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean Percent Viability/ 42 minutes exposure

Value:

93.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: No interaction.
- Colour interference with MTT: Was not observed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The results of the negative control met the acceptance range of the OECD test guideline 439 for the prediction model SkinEthicTM RHE. Test guideline requirement of optical density is ≥ 0.8 and ≤ 3.0 (the acceptance criteria for SkinEthicTM RHE model, as per the OECD TG 439). The standard deviation value of negative controls in all tissues was 0.81%, which met the acceptance range of the OECD test guideline 439 for the prediction model SkinEthicTM RHE.
The NC data meet the acceptance criteria if the mean OD value of the 3 tissues is ≥ 0.8 at 570 ± 30 nm with an upper acceptance limit ≤ 3 according to the guideline requirements. The standard deviation value is considered valid if it is ≤ 18%.

- Acceptance criteria met for positive control:
The mean % viability of the positive control was 0.9%, which met the acceptance criteria of the OECD test guideline 439, i.e. <40% viability.
The PC data meet the acceptance criteria if the mean viability, expressed as % of the NC, is < 40 % and the Standard Deviation value is ≤ 18 %.

- Acceptance criteria met for variability between replicate measurements:
The results met the acceptance range of the OECD test guideline 439. Standard deviation of each intra-batch mean (3 Replicate/Tissue and 3 Tissue/Run) should not be > 18%.

Pre-Tests

 

Colour Interference Test:

Treatment	Optical Density (nm)	Interaction
Negative Control (distilled water)	0.041	No
	0.041
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	0.043	No
	0.044

Direct MTT Reduction Test:

Treatment	Interaction
Negative Control (Maintenance Medium)	No
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	No

Main Study:

Treatment	Mean Percent Viability
	42 Minutes Exposure
Negative control (Dulbecco’s Phosphate Buffered Saline (DPBS))	100
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	93.3
Positive control (Sodium dodecyl sulfate (5% aq.))	0.9

 

Data Summary of Percent Viability:

Treatment	Tissue Replicate	OD at 570 nm	Blank Corrected OD	Mean of Corrected OD	Mean OD of Three Tissues	% Viability/ Tissue	Mean % Viability	SD of % Viability	CV of % Viability
Negative Control (Dulbecco’s Phosphate Buffered Saline (DPBS))	1	2.431	2.388	2.364	2.343	100	100	0.019	0.81
		2.399	2.356
		2.392	2.349
	2	2.369	2.326	2.338
		2.391	2.348
		2.382	2.339
	3	2.367	2.324	2.326
		2.359	2.316
		2.380	2.337
Test item 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole	1	2.391	2.348	2.268	2.185	96.8	93.3	3.075	3.30
		2.289	2.246
		2.254	2.211
	2	2.128	2.085	2.136		91.2
		2.228	2.185
		2.180	2.137
	3	2.193	2.150	2.152		91.8
		2.198	2.155
		2.195	2.152
Positive control (Sodium dodecyl sulfate (5% aq.))	1	0.066	0.023	0.022	0.022	0.9	0.9	0.000	0.00
		0.065	0.022
		0.065	0.022
	2	0.066	0.023	0.022		0.9
		0.065	0.022
		0.064	0.021
	3	0.066	0.023	0.022		0.9
		0.064	0.021
		0.065	0.022

Key: OD. = Optical Density, SD = Standard Deviation, CV = Coefficient of Variation, NA = Not Applicable

Note: For Negative control, SD and CV of % viability was calculated using corrected OD at 570 nm and for the test item and positive control SD and CV of % viability was calculated using % viability/tissue.

Based on the results of this study, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole is classified under “No Category (Non Irritant)”, as per the “United Nations Globally Harmonized System of Classification and Labelling of Chemicals”.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole is predicted not to be a skin irritant. The substance is not classified as skin irritating according to EC regulation 1272/2008 (CLP).

Executive summary:

This study was performed to evaluate the skin irritation potential of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole, using a reconstructed human epidermis (RHE) tissue, in accordance with the United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS).

Tissues were exposed to the negative control (Dulbecco’s Phosphate Buffered Saline (DPBS)), positive control (sodium dodecyl sulfate, 5% aqueous (SDS)) and 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole in triplicate for 42 minutes, at room temperature.

The mean percent cell viability in tissues treated with the test item is shown below. There was no significant reduction in the percent cell viability observed 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole treated tissues, when compared with that of the concurrent negative control.

Treatment	Mean Percent Viability
	(42 Minutes Exposure)
Negative control (Dulbecco’s Phosphate Buffered Saline (DPBS))	100
3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole	93.3
Positive control (Sodium dodecyl sulfate (5% aq.))	0.9

The negative and positive controls met the acceptance range for the OECD guideline and the efficiency of the test system was demonstrated. All criteria for a valid study were met as described in the OECD 439 guideline.

Based on the results of this study, 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole is predicted not to be a skin irritant according to EC regulation 1272/2008 (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2020 to 20 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Species:

human

Strain:

other: Reconstructed human cornea epithelium (RhCE)

Details on test animals or tissues and environmental conditions:

Test System: Reconstructed human cornea epithelium (RhCE).
Model: SkinEthicTM HCE.
Source: EPISKIN– 4 Rue Alexander Fleming, 69366 Lyon Cedex 07 – France.
Lot No.: 20-HCE-012.

Vehicle:

other: Dubelcco’s Phosphate Buffer Saline (DPBS)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 30 μL DPBS + 30 mg 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole.

NEGATIVE CONTROL:
Name: Dubelcco’s Phosphate Buffer Saline (DPBS).
- Amounts applied: 30 μL DPBS.
Batch Nº: 1918956.
Expiry Date: September 30, 2020.
Manufactured by: Gibco.

POSITIVE CONTROL:
Reference Substance Name: Methyl acetate.
- Amounts applied: 10 μL DPBS + 30 μL methyl acetate (undiluted).
CAS No: 79-20-9.
Analysed Purity: 99.5% .
Batch No: STBG5814V.
Supplied by: Sigma.
Manufactured by: Sigma.
Date of Receipt: May 08, 2017.
Date of Expiry : May 07, 2022.
Physical Appearance: Colourless liquid.
Storage Condition (at JRF): Room temperature.

Duration of treatment / exposure:

Tissues were exposed to test item, negative and positive control for 4 h.

Duration of post- treatment incubation (in vitro):

For post incubation, the MTT test was performed. Tissues were placed in MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37 ± 2°C in 5 ± 1% CO2 and saturated with humidity.

Number of animals or in vitro replicates:

Two replicates in each group (test item, positive control, negative control).

Details on study design:

NEGATIVE CONTROL USED: Yes.
- Name: Dubelcco’s Phosphate Buffer Saline.
- Batch Nº: 1918956.

POSITIVE CONTROL USED: Yes.
- Reference Substance Name: Methyl acetate.
- Analysed Purity: 99.5%.
- Batch No: STBG5814V.

NUMBER OF REPLICATES , APPLICATION DOSE AND EXPOSURE TIME
- Test material: 30 mg 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole + 30 μL DPBS.
- Positive control: 10 μL DPBS + 30 μL methyl acetate (undiluted).
- Negative control: 30 μL DPBS.
- two replicates in each group (test item, positive/negative control).

Irritation parameter:

other: optical density

Run / experiment:

Mean Percent Viability

Value:

32.33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

According OECD 492 "No prediction can be made".

Other effects / acceptance of results:

Validity of the Test:
- Negative control OD values were found to be within the range of > 1.0 to ≤ 2.5.
- Mean tissue viability values for positive control was found < 20%.
- Variation within the replicates was acceptable (< 20%).
Therefore, the experiment is considered valid.

Pre-Tests

Direct MTT Reduction Test

The test item did not produce direct MTT reduction when compared with that of the concurrent negative control (distilled water). Results of direct MTT reduction test are summarised in the table mentioned below:

Treatment	Interaction
Negative Control (distilled water)	No
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1HPyrazole	No

Colour Interference Test

Any difference in the absorbance, due to colour interference, was not observed visibly for the test item. Results of direct MTT reduction test are summarised in the table mentioned below:

Treatment	Interaction
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1HPyrazole	No

Negative Control

The individual optical density, corrected optical density, mean of corrected optical density, viability, and mean percent viability, are summarised in the following table.

The OD values (corrected mean ODs) of the negative control was 1.678 and 1.751. Therefore, results of the negative control met the acceptance range of the OECD test guideline 492 for the prediction model SkinEthicTM HCE. Test guideline requirement of optical density is > 1.0 and ≤ 2.5 (the acceptance criteria for SkinEthicTM HCE model, as per the OECD TG 492). Variation between the two negative controls tissues was < 20%, which met the acceptance range of the OECD test guideline 492 for the prediction model SkinEthicTM HCE. As per the certificate of analysis for the batch of tissues used in the study (Batch# 20-HCE-012), average optical density observed was 2.0 in QC testing at SkinEthic Laboratories. Therefore, the observed results were within the acceptable range of the SkinEthic Laboratories/OECD TG acceptance criteria.

Positive Control

The individual optical density, corrected optical density, mean of corrected optical density, viability, and mean percent viability, are summarised in the following table.

The mean % viability of the positive control was 18.51%, which met the acceptance criteria of the OECD test guideline 492, i.e., <20% viability.

 

Main Study

The individual optical density, corrected optical density, mean of corrected optical density, viability and mean per cent viability for test item are summarised in the following table.

The mean per cent viability of the 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole treated tissues, and the control tissues, is tabulated below:

Treatment	Mean Percent Viability
Negative control (DPBS)	100.00
Positive control (methyl acetate)	18.51
3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole	32.33

The mean per cent cell viability in tissues treated with 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole was found to be 32.33% in the concurrent negative control (100%) which is below the established tissue viability cut-off value of 50%.

 

Therefore, based on these results, 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole is according OECD 492 assessed as “No prediction can be made”.

 

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Based on the mean viability score of 32.33%, determined under the specified experimental conditions using SkinEthicTM HCE RhCE, the classification for 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1Hpyrazole is “No prediction can be made” (UN GHS) and that further testing may be required in order to classify and/or determine if the test item is an ocular irritant. In accordance with the provisions of regulation 1272/2008 a prediction of classification can not be made.

Executive summary:

Study type: Reconstructed Human Cornea-like Epithelium (RhCE) Test, OECD 492.

Study title: In Vitro Eye Irritation Test of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole Using Reconstructed Human Cornea-Like Epithelium.

This study was performed to evaluate the ocular irritation potential of 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole, as measured by the potential to induce cytotoxicity in a reconstructed human cornea-like epithelium (RhCE) tissue construct which closely mimics the properties of the human corneal epithelium. The RhCE test can identify test items not requiring classification, according to the UN GHS (No category).

RhCE tissues (two tissues per set) were treated with 30 μL of either Dulbecco’s Phosphate Buffered Saline (DPBS) (Set 1 - control), 30 μL of undiluted methyl acetate (Set 2 - positive control), and 30 μL of Dulbecco’s Phosphate Buffered Saline (DPBS) along with 30 mg 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole (Set 3 – test group). The tissues were incubated for 4 h ± 6 minutes. At the end of the exposure period, residue was removed, and the tissue were kept for 30 minutes in the maintenance medium at room temperature, followed by an incubation period of 18 h ± 30 minutes.

Tissue viability was measured, following the treatment and a post-treatment incubation period, by enzymatic conversion of the vital dye MTT into a blue MTT formazan salt, measured after extraction from tissues. The optical density (OD) of the extracted formazan was determined using a microplate reader at 570 nm. The viability of the RhCE tissue was determined in comparison to tissues treated with the negative control substance (% viability) and was then used to predict the ocular hazard potential of the test chemical.

The mean per cent cell viability in tissues is shown below. The mean per cent cell viability in tissues treated with 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole was found to be 32.33% of the concurrent negative control (100%) which is below the established tissue viability cut-off value of 50%.

Treatment	Mean Percent Viability
Negative control (Dulbecco’s Phosphate Buffered Saline)	100.00
Positive control (methyl acetate)	18.51
3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole	32.33

The negative and positive controls met the acceptance criteria, as described in the study plan, and confirmed the reliability of the test procedure.

Based on the mean viability score of 32.33%, determined under the specified experimental conditions using reconstructed human cornea-like epithelium (RhCE) from EPISKIN, the classification for 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole is “No prediction can be made” (UN GHS) and that further testing may be required in order to classify the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

1. From results of the study “Skin corrosion – In vitro; OECD 431 and EC Method B40: Skin Corrosion test with Reconstructed Human Epidermis (RHE) Tissues, 3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazole is predicted to be non-corrosive to the skin under the specified conditions of this study.

2. From results of the study “Skin irritation – In Vitro; OECD 439 (June 2019), EC method B46 (July 2012): Skin Irritation test with Reconstructed Human Epidermis (RHE) Tissues”,3-Bromo-1-(3-Chloro-2-Pyridyl)-5-Methyl-1H-Pyrazoleis predicted not to be a skin irritant under the specified conditions of this study.

 

3. From the results of the study “Reconstructed Human Cornea-like Epithelium (RhCE) Test, OECD guideline 492: In Vitro Eye Irritation Test with 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole Using Reconstructed Human Cornea-Like Epithelium”, a conclusive assessment of classification is not possible. Due to safety reasons the registrant classifies 3-bromo-1-(3-chloro-2-pyridyl)-5-methyl-1H-pyrazole as eye damaging, category 1 (H318 "Causes serious eye damage").