1H-Pyrazolo[3,4-b]pyridine-3-carboxamide, 5-fluoro-1-[(2-fluorophenyl)methyl]-

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-12 to 2019-04-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted April 13, 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"

Version / remarks:

December 15, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

EC Number:

811-886-0

Cas Number:

1361232-73-6

Molecular formula:

C14 H10 F2 N4 O

IUPAC Name:

5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridine-3-carboxamide

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control were collected at the end of the test (after 48 hours) by pouring together the contents of the test beakers of each treatment.
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the control and the dilution solvent were taken at each sampling test start and test end without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

The test item is not well soluble in test medium. To avoid physical effects of undissolved test item on the daphnids, no concentrations above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 101.6 mg test item in 1016 mL test water. The stock suspension was stirred for 24 hours at room temperature in the dark to dissolve as much test item as possible. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 µm cellulose acetate filter). The solution with dissolved test item was used as the test medium of the highest test concentration and to prepare the desired 1:2, 1:4, 1:8 and 1:16 dilutions.
The test media were prepared just before introduction of the daphnids (= start of the test).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Species: Daphnia magna (Straus), clone 5
Age at Test Start: From 3.25 to 19.25 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate

Test temperature:

19.8 to 20.2 °C at test start; 19.6 to 19.9 °C at test end

pH:

7.8 to 7.9 at test start; 7.8 at test end; and thus the pH-value did not vary by more than 1.5 units

Dissolved oxygen:

7.6 to 9.2 mg/L at test start; 8.9 to 9.0 mg/L at test end

Nominal and measured concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L as the highest test concentration and the dilutions of 1:2, 1:4, 1:8 and 1:16 of were tested. Additionally, a control was tested in parallel.
The mean measured concentrations are 4.23, 1.89, 1.03, 0.519 and 0.265 mg test item/L

Details on test conditions:

Test Environment: Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature: 19.8 to 20.2 °C at test start; 19.6 to 19.9 °C at test end
pH-Values: 7.8 to 7.9 at test start; 7.8 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 7.6 to 9.2 mg/L at test start; 8.9 to 9.0 mg/L at test end
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 230 to 730 lux (measured once during the test).

Reference substance (positive control):

yes

Remarks:

For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 4.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 4.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

> 4.23 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and in all concentrations of the test item.

Results with reference substance (positive control):

In the most recent test with the reference item potassium dichromate the EC50 after 24 hours was determined to be 1.58 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).

Reported statistics and error estimates:

No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.9 mg O2/L in the control and test vessels at the end of the test.

Conclusions:

The toxic effect of the test item Fluorpyridoamid to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be at least 4.23 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than 4.23 mg test item/L. These values could not be quantified due to the absence of toxicity of Fluorpyridoamid up to 4.23 mg test item/L, the concentration representing the limit of solubility.
The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to mean measured concentrations, since the test item concentrations were within ± 20 % of measured initial concentrations during the test.

Executive summary:

Title:

Fluorpyridoamid: Acute Toxicity to Daphnia magna in a Static 48-hour Immobilisation Test

Purpose:

The purpose of this study was to determine the influence of the test item Fluorpyridoamid on the mobility of Daphnia magna.

For this purpose, young daphnids (< 24 hours old) were exposed in a static test to various concentrations under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours. The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species. The purpose of the analytical part of this study was to verify the concentrations of the test item in the test medium.

Guidelines

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures", December 15, 2000

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

Materials and Methods

Test Item:

Fluorpyridoamid

Test Species:

Female Daphnia magna, clone 5; 3.25 to 19.25 hours old

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design:

This study encompassed 6 treatment groups (5 dose rates of the test item and a control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours.

Endpoints:

Number of immobile organisms after 24 and 48 hours

Test Concentrations:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L as the highest test concentration and the dilutions of 1:2, 1:4, 1:8 and 1:16 of were tested. Additionally, a control was tested in parallel.

The mean measured concentrations are 4.23, 1.89, 1.03, 0.519 and 0.265 mg test item/L.

Test Conditions:

Water temperature: 19.6 to 20.2 °C; pH value: 7.8 to 7.9; dissolved oxygen concentration: 7.6 to 9.2 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 230 to 730 lux; and thus were within the ranges requested by guideline OECD 202

Results

Biological test results:

After 48 hours of exposure no immobilisation of the test animals was observed in the control and up to and including the mean measured test item concentration of 4.23 mg test item/L, the highest concentration tested. NOEC and LOEC were determined directly from the raw data. The 48-hour NOEC was determined to be at least 4.23 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than 4.23 mg test item/L. These values could not be quantified due to the absence of toxicity of Fluorpyridoamid up to 4.23 mg test item/L, the concentration representing the limit of solubility.

Analytical test results:

The quantification of the test item Fluorpyridoamid in the test samples was performed using liquid chromatography with UV.

The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (test item stock solution and its dilutions) and the duplicate control samples from all sampling times. The mean measured values are

4.23 mg test item/L in the filtrate

1.89 mg test item/L in the 1:2 dilution of filtrate

1.03 mg test item/L in the 1:4 dilution of filtrate

0.519 mg test item/L in the 1:8 dilution of filtrate and

0.265 mg test item/L in the 1:16 dilution of filtrate.

Conclusion:

The toxic effect of the test item Fluorpyridoamid to Daphnia magna was assessed in a static concentration-response test. The 48-hour NOEC was determined to be at least 4.23 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than 4.23 mg test item/L. These values could not be quantified due to the absence of toxicity of Fluorpyridoamid up to 4.23 mg test item/L, the concentration representing the limit of solubility.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to mean measured concentrations, since the test item concentrations were within ± 20 % of measured initial concentrations during the test.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.