1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 15 August 2016 and 20 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

EC Number:

611-033-0

Cas Number:

536759-91-8

Molecular formula:

C22H20N4O6

IUPAC Name:

ethyl 1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-1H,4H,5H,6H,7H-pyrazolo[3,4-c]pyridine-3-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

powder; stored at room temperature in the dark

Specific details on test material used for the study:

Identification: BMS-589152-01
Batch Number: AAG8999N
Purity: 100.1% W/W
Physical state/Appearance: Beige powder
Expiry Date: 27 December 2017
Storage conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the solvent control and the 1.0 mg/L test vessel at 0 and 72 hours from fresh media and at 24 and 96 hours from old media for quantitative analysis. The 0, 24 and 72-Hour samples were stored frozen prior to analysis.
Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours (old media) were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

yes

Remarks:

dimethylformamide

Details on test solutions:

Based on the results of a previous Acute Toxicity to Daphnia Magna Test conducted on this test item (Harlan Study Number 417401006), a "Limit test" was conducted at a nominal concentration of 1.0 mg/L to confirm that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure were observed.

A nominal amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give a 10 mg/mL stock solution. An aliquot (2200 µL) of this solvent stock solution was dispersed in 22 liters of test water with the aid of magnetic stirring for approximately 10 minutes to give a 1.0 mg/L solvent stock solution. After the stirring period the preparation was filtered (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to ensure any undissolved test item was removed. Due to the light sensitive nature of the test item, all preparation was conducted under non-actinic light or in darkness.
The stock solution was inverted several times and the test media mixed with a flat bladed stirrer for 1 minute to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout (Oncorhynchus mykiss). Fish were obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and maintained in house since 19 May 2016. Fish were maintained in a glass fiber tank with a "single pass" water renewal system. Fish were acclimatized to test conditions from 08 August 2016 to 15 August 2016. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
The water temperature was controlled at 14 °C with a dissolved oxygen content of greater than or equal to 9.0 mg O2/L. These parameters were recorded daily. The stock fish were fed commercial trout pellets which was discontinued approximately 24 hours prior to the start of the definitive test. There was 1 mortality in the 7 days prior to the start of the test and the fish had a mean standard length of 5.4 cm (sd = 0.4) and a mean weight of 1.34 g (sd = 0.38) at the end of the definitive test. Based on the mean weight value this gave a loading rate of 0.47 g bodyweight/liter.
The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test conditions

Hardness:

approximately 140 mg/L as CaCO3

Test temperature:

approximately 14 °C in a temperature controlled room

pH:

7.3-8.4

Dissolved oxygen:

8.3-10.3 mg O2/L

Nominal and measured concentrations:

Analysis of the freshly prepared test preparations at 0 and 72 hours showed measured test concentrations of 0.67 and 0.49 mg/L were obtained respectively. There was no significant change in the measured concentrations of the old or expired media at 24 and 96 hours and so the results are based on the mean measured concentration of the freshly prepared media, determined to be 0.58 mg/L.

Details on test conditions:

Test Water
The test water used for the definitive test was the same as that used to maintain the stock fish.
Laboratory tap water was dechlorinated by passage through an activated carbon filter (Purite Series 500) and partly softened (Elga Nimbus 1248D Duplex Water Softener) giving water with a total hardness of approximately 140 mg/L as CaCO3. After dechlorination and softening the water was passed through a series of computer controlled plate heat exchangers to achieve the required temperature.

Exposure Conditions
In the definitive test, 25-30 liter glass exposure vessels containing 20 liters of test media were used for each control and test concentration. At the start of the test seven fish were placed in each test vessel at random, in the test preparations. The test vessels were then covered to reduce evaporation and maintained at approximately 14 °C in a temperature controlled room in darkness for a period of 96 hours. The test vessels were aerated via narrow bore glass tubes. The fish were not individually identified and received no food during exposure.
The solvent control group (containing 100 µl/L of dimethylformamide) was maintained under identical conditions but not exposed to the test item.
A semi-static test regime was employed in the test involving a daily renewal of the test preparations to prevent the build-up of nitrogenous waste products.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 0.58 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

0.58 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

There were no mortalities in 7 fish exposed to a test concentration of 0.58 mg/L for a period of 96 hours.

Sub-Lethal Effects
There were no sub-lethal effects of exposure observed in the test.

Validation Criteria
The test was considered to be valid given that none of the solvent control fish died or showed signs of stress during the test and that the oxygen concentration at the end of the test was ≥60% of ASV in the solvent control and test vessels.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Inspection of the mortality data gave the following results:

Time (h)	LC50(mg/L)
3	> 0.58
6	> 0.58
24	> 0.58
48	> 0.58
72	> 0.58
96	> 0.58

Water Quality Criteria

Temperature was maintained at approximately 14 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

 Observations on Test Item Solubility

The test item preparations were observed to be clear colorless solutions throughout the test.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater fish rainbow trout (Oncorhynchus mykiss) has been investigated and based on the mean measured concentrations of the freshly prepared media gave a 96-Hour LC50 of greater than 0.58 mg/L. The No Observed Effect Concentration was 0.58 mg/L.
This study showed that there were no toxic effects at saturation (i.e. the functional limit of solubility).

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout (Oncorhynchus mykiss). The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as Method C.1 of Commission Regulation (EC) No. 440/2008.

 Methods

Due to the poorly water soluble nature of the test item, and based on a previous Acute Toxicity to Daphnia Magna Study conducted on this test item (Harlan Study Number 41401006), the test item was prepared using a solvent spike method of preparation at a nominal concentration of 1.0 mg/L.

The test was run for a period of 96 hours at a temperature of approximately 14 ºC under semi-static test conditions. An initial solvent stock solution was prepared by dissolving 100 mg of test item in a final volume of 10mL of dimethylformamide to give a10 mg/mL solvent stock solution. An aliquot (2200µL) of this solvent stock solution was dispersed in 22 liters of test water with the aid of magnetic stirring for approximately10 minutes to give a1.0 mg/L stock solution. After the stirring period the preparation was filtered (0.2 µm Sartorius Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to ensure any undissolved test item was removed. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 1, 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours.

Results…….

Analysis of the freshly prepared test preparations at 0 and 72 hours showed measured test concentrations of 0.67 and 0.49 mg/L were obtained respectively. There was no significant change in the measured concentrations of the old or expired media at 24 and 96 hours and so the results are based on the mean measured concentration of the freshly prepared test preparations.

Exposure of rainbow trout (Oncorhynchus mykiss) to the test item gave LC₅₀values based on the mean measured concentration of the freshly prepared test concentrations of greater than 0.58 mg/L. The No Observed Effect Concentration was 0.58 mg/L.

This study showed that there were no toxic effects at saturation (i.e. the functional limit of solubility).