3-methylpyrazole

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-09-28 - 2022-12-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

no guideline available

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: endocrine disruptive properties

Test material

Test material information

Constituent 1

Reference substance name:

3-methylpyrazole

EC Number:

215-925-7

EC Name:

3-methylpyrazole

Cas Number:

1453-58-3

Molecular formula:

C4H6N2

IUPAC Name:

3-methyl-1H-pyrazole

Test material form:

liquid

Test animals

Details on test animals or test system and environmental conditions:

Alcohol Dehydrogenase Activity Assay Kit (ADH-Kit)
Identification Alcohol Dehydrogenase Activity Assay Kit
Manufacturer Sigma-Aldrich, St. Louis, MO 63103, USA
Cat-Nr. MAK053
vivo Science ID KIT188
Batch number 7G27K07870
Expiry date to be used within 2 month of reconstitution
Method Colorimetric Enzymatic Assay
Intended use Measuring ADH activity using ethanol as substrate in enzyme reaction, which results in colorimetric product proportional to the enzymatic activity

Administration / exposure

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 other: µM

Doses / concentrations 2

Dose / conc.:

50 other: µM

Doses / concentrations 3

Dose / conc.:

10 other: µM

Doses / concentrations 4

Dose / conc.:

1 other: µM

Details on study design:

see details under "any other information"

Examinations

Examinations:

Reduction of ADH activity by 3-Methylpyrazole (3-MP)

Positive control:

ADH positive control as provided with the SIGMA test kit

Results and discussion

Details on results:

ADH Activity Inhibition with 4-Methylpyrazole (Control Item):
For the highest ADH dilution (1:500), two values had to be excluded for the 10 µM Inhibitor concentration and for the control without inhibitor on the control item plate. As between the 10 µM and 1 µM concentrations only minor difference can be observed, similar as in the preliminary proof of principle study, the excluded values were seen as uncritical deviation from the system suitability criteria for the further analysis.
The assay conducted for the control item displayed a high inhibition rate up to 93% for the samples treated with 100 µM 4-MP. The additional concentration of 50 µM showed an inhibition rate from approximately 50% (44% up to 56%) of the ADH activity in all three ADH dilutions. The lower concentrations of the control item (10 µM and 1 µM) induce a minor reduction range (7%-9%) for the ADHhigh and ADHmed dilutions. The slight ADH activity increase (<1%) in the 1:250 diluted ADH sample treated with concentration of 1 µM displays the natural variation of the assay and is therefore not regarded as critical.
For the lowest ADH content (ADHlow 1:500) the control item displayed the most prominent inhibition effect across all inhibitor concentrations ranging from 14% (1 µM) up to 93% (100µM).

Across the three ADH dilutions (ADHhigh 1:100, ADHmed 1:250 and ADHlow 1:500) the inhibition rate behaves antiproportional to the amount of ADH in the samples. The higher the ADH dilution and therefore decreased amount of ADH, the inhibition rate is intensified with exception for the 1:250 diluted ADH sample treated with the control item concentration of 1 µM.

ADH Activity Inhibition with 3-Methylpyrazole (Test item):
For the test item plate the ADH Activity [nmole/min/mL] was calculated from the equation from the Kits manual. The inhibition rate, given in percentage for each value, of the selected dilutions of ADHHigh (1:100), ADHmed (1:250) and ADHlow (1:500) were set up against the control without inhibitor, which was set as 100%.
For the test item plate no deviation from the system suitability criteria occurred.
The assay conducted for the test item 3-MP displayed a high inhibition rate from 87 up to 95% for the samples treated with the highest concentration (100 µM). The additional concentration of 50 µM showed an inhibition rate from 65% (ADHhigh) up to 74% (ADHlow 1:500). In comparison with the control the values for the test item concentration of 10 µM inhibit the ADH Activity from 22 % up to 28%. The lowest inhibitor concentration displays a minor reduction range from 3-8% for the three selected ADH dilutions.

Across the ADH dilutions (ADHhigh 1:100 and ADHmed 1:250) the inhibition rate behaves antiproportional to the amount of ADH in the samples. The higher the ADH dilution and therefore decreased amount of ADH, the inhibition rate is intensified with exception for highest diluted ADH sample (1:500) treated with the test item concentration of 1 µM. This discrepancy displays the natural variation of the assay and is therefore not regarded as critical.

Any other information on results incl. tables

see attached documents

Applicant's summary and conclusion

Conclusions:

In order of the subsequent assessment of the inhibitory activity of 3-MP on ADH, the test item was tested at four different concentrations (100 µM, 50 µM, 10 µM, and 1 µM) against the selected ADH dilutions 1:500, 1:250 and 1:100 and was compared to the well-known ADH-inhibitor 4-MP.
The colorimetric assay displayed for both, test item and control item, a similar strong inhibitory effect in combination with the highest inhibitor concentration (100 µM). The additional concentration of 50 µM of 3-MP as well as 4-MP, added to obtain a more clearly defined critical dose, showed an approximately 50% inhibiting effect of the ADH activity across all three ADH dilutions for the 4-MP, whereas 3-MP displayed an inhibitory activity of approximately 70%. This trend can also be observed in attenuated form at the 10 µM Inhibitor concentration. For the lowest inhibitor concentration (1 µM) the inhibitory activity of 3-MP is overall slightly reduced compared to 4-MP.
Based on the natural variation of the assay, and the effect in the three higher inhibitor concentrations, it becomes apparent that 3-MP, compared to 4-MP a known ADH Inhibitor, has the ability to inhibit ADH in vitro.

Executive summary:

The aim of this study, was the determination of the reduction of ADH activity by 3-Methylpyrazole (3-MP) in order to evaluate further the suspected potential endocrine disruptive properties of this substance and the underlying mode of action since alcohol dehydrogenase is a key enzyme in the biosynthesis of retinoic acid which is essential for vertebrate embryo development including kidney and cardiovascular development.

Since the inhibitory activity of 3-MP to ADH is unknown, the current study tested the inhibitory activity of 3-MP to ADH with 4-MP, a structurally similar substance with well-known inhibitory activity to ADH, as a positive control. In the prior proof of principle study, the three most appropriate dilutions of ADH for its use in an ADH inhibition assay with a commercial kit were identified and were applied in this study. 

In alliance with the results of the proof of principle study, in which only minor differences in the inhibition rate at the 10 µM and 1 µM concentrations were observed, an additional concentration of 50 µM of 3-MP as well as 4-MP was tested after agreement with the sponsor. The additional concentration of 50 µM of 3-MP as well as 4-MP was tested alongside the concentrations of 100 µM, 10 µM and 1 µM required by the ECHA decision, to obtain a more clearly defined critical dose.

The assay conducted for the control item displayed a high inhibition rate up to 93% for the samples treated with 100 µM 4-MP. The additional concentration of 50 µM showed an inhibition rate from approximately 50% (44% up to 56%) of the ADH activity in all three ADH dilutions. The lower concentrations of the control item (10 µM and 1 µM) induce a minor reduction range (7%-9%) for the ADH_high and ADH_med dilutions. The slight ADH activity increase (<1%) in the 1:250 diluted ADH sample treated with concentration of 1 µM displays the natural variation of the assay and is therefore not regarded as critical.

For the lowest ADH content (ADH_low 1:500) the control item displayed the most prominent inhibition effect across all inhibitor concentrations ranging from 14% (1 µM) up to 93% (100µM).

Across the three ADH dilutions (ADH_high 1:100, ADH_med 1:250 and ADH_low 1:500) the inhibition rate behaves antiproportional to the amount of ADH in the samples. The higher the ADH dilution and therefore decreased amount of ADH, the inhibition rate is intensified with exception for the 1:250 diluted ADH sample treated with the control item concentration of 1 µM.

The assay conducted for the test item 3-MP displayed a high inhibition rate from 87 up to 95% for the samples treated with the highest concentration (100 µM). The additional concentration of 50 µM showed an inhibition rate from 65% (ADH_high) up to 74% (ADH_low 1:500). In comparison with the control the values for the test item concentration of 10 µM inhibit the ADH Activity from 22 % up to 28%. The lowest inhibitor concentration displays a minor reduction range from 3-8% for the three selected ADH dilutions.

Across the ADH dilutions (ADH_high 1:100 and ADH_med 1:250) the inhibition rate behaves antiproportional to the amount of ADH in the samples. The higher the ADH dilution and therefore decreased amount of ADH, the inhibition rate is intensified with exception for highest diluted ADH sample (1:500) treated with the test item concentration of 1 µM. This discrepancy displays the natural variation of the assay and is therefore not regarded as critical.