4,6-Pyrimidinediamine, 2-[5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl]-5-[(1E)-2-phenyldiazenyl]-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Apr - 20 Aug 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-{5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-[(1E)-2-phenyldiazen-1-yl]pyrimidine-4,6-diamine

EC Number:

811-933-5

Cas Number:

1350653-29-0

Molecular formula:

C23 H17 F2 N9

IUPAC Name:

2-{5-fluoro-1-[(2-fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5-[(1E)-2-phenyldiazen-1-yl]pyrimidine-4,6-diamine

Test material form:

solid

Sampling and analysis

Analytical monitoring:

yes

Remarks:

HPLC-MS/MS

Details on sampling:

Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from 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 (containing algae) were taken at the end of the test (after the 72 hours test period) by pouring together the contents of the test beakers of each treatment.
The samples were diluted by a factor of two with acetonitrile.
Additional samples of the control and of the dilution solvent were taken at each sampling without any sample treatment.

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to nominal concentrations of 100, 33.3, 11.1, 3.70 and 1.23 mg test item/L. Additionally, a control was tested in parallel. The nominal concentrations of the two highest test concentrations correspond to the following geometric mean measured concentrations of 0.208, 0.0773 µg test item/L. The lower concentrations were below limit of quantification (LOQ) of 0.05 µg test item/L.
In the control, test water was used without addition of the test item.
The test item was not well soluble in test water. In this study, only the inhibitory effects of dissolved test item was tested and thus no concentrations above the solubility limit of the test item in test water were tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 100 mg test item in 1000 mL test water for preparing the test concentrations. 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 1:3, 1:9, 1:27 and 1:81 dilutions. The test media were prepared just before introduction of the algae (= start of the test).

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG
Origin: The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
Breeding Conditions: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

22 to 23°C

pH:

pH 8.0 at test start and 9.1 to 9.4 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 and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to nominal concentrations of 100, 33.3, 11.1, 3.70 and 1.23 mg test item/L. Additionally, a control was tested in parallel. The nominal concentrations of the two highest test concentrations correspond to the following geometric mean measured concentrations of 0.208, 0.0773 µg test item/L. The lower concentrations were below limit of quantification (LOQ) of 0.05 µg test item/L.

Details on test conditions:

Water Temperature: The temperature was measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.
Water temperature was: 22.1 to 23.3 °C
pH-Values: The pH was measured in all test item concentrations and the control at the start and the end of the test. The pH in the control was: 8.0 at test start and 9.3 at test end.
The pH at different test item concentrations was:8.0 at test start and 9.1 to 9.4 at test end.
Light Regime: Continuous illumination
Light Intensity: The light intensity was measured once during the test at 6 positions distributed over the experimental area at the surface of the test media.
Mean light intensity: 6475 lux (range: 5820 to 7040 lux)
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.

Reference substance (positive control):

yes

Remarks:

reference item potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The 72-hour EC50 and the 72-hour EC10 for the parameters growth rate and yield could not be calculated due to the low effects of the test item. Therefore the 72-hour EC50 and the 72-hour EC10 was determined to be higher than the limit of water solubility of the test item, represented by the filtrate of nominal 100 mg test item/L (0.208 µg/L), the highest concentration tested.
The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L (0.208 µg/L) for the parameters growth rate and yield and the associated 72-hour LOEC was determined to be higher than the filtrate of nominal 100 mg test item/L (0.208 µg/L) for both parameters.
The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing up to a nominal test concentration of the filtrate of 100 mg test item/L (0.208 µg/L) and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Results with reference substance (positive control):

Parameter Yield Growth rate Biomass
(0-72 h) [mg test item/L] [mg test item/L] [mg test item/L]
72-hour EC50 0.445 0.941 0.494
95 % conf. interval 0.437 - 0.453 0.910 - 0.973 0.485 - 0.504

72-hour EC20 0.256 0.604 0.279
95 % conf. interval 0.249 - 0.263 0.568 - 0.636 0.270 - 0.287

72-hour EC10 0.192 0.479 0.207
95 % conf. interval 0.184 - 0.199 0.440 - 0.514 0198 - 0.215

72-hour NOEC < 0.094 0.094 < 0.094
72-hour LOEC ≤ 0.094 0.21 ≤ 0.094
n.d. = not determinable, values refer to nominal test concentrations

Reported statistics and error estimates:

Based on the calculated cell densities, the 72 hours ErC50 and the 72 hour EyC50 could not be calculated by a statistical analysis due to the lack of effects and were therefore determined directly from the raw data.
For the determination of the 72 hour LOEC and the 72 hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Dunnetts t-test.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Cell Density Increase: 208.4-fold increase within 72 hours; Coefficient of Variation of Sectional (Daily) Growth Rates: 14.5%; Coefficient of Variation of Average Growth: 2.8 %

Conclusions:

The influence of Fluoroazopyrimidine on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg (0.208 µg/L) 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 72-hour LOEC and the 72-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (0.208 µg/L). These values could not be quantified due to the absence of toxicity of Fluoroazopyrimidine up to the filtrate of nominal 100 mg test item/L (0.208 µg/L), the concentration representing the limit of solubility.
The initial concentrations and the maintenance of the exposure concentrations during the test were determined in the analytical part. All reported results refer to nominal test concentration since the filtrate of 100 mg test item/L was used. Furthermore the results are presented using geometric mean measured concentration since a decrease of test item was observed.

Executive summary:

Title: Fluoroazopyrimidine: Toxicity to Pseudokirchneriella subcapitata in an Algal Growth Inhibition Test

Purpose: The purpose of this test was to determine the inhibitory effect of the test item Fluoroazopyrimidine on the growth of the freshwater green algae Pseudokirchneriella subcapitata.

For this purpose, exponentially growing cultures of this unicellular green algal species were exposed to various concentrations of the test item under defined conditions. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.The test method of application and the test system are recommended by the test guidelines and Pseudokirchneriella subcapitata is one of 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/Recommendations:

OECD Guidelines for the Testing of Chemicals, Section 2, No. 201: "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", adopted March 23, 2006, corrected July 28, 2011

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: Fluoroazopyrimidine

Test Species: Pseudokirchneriella subcapitata, Strain No. 61.81 SAG formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).

Cultivated in the laboratories of ibacon; original source: "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissen-schaften, Universität Göttingen", 37073 Göttingen, Germany.

Test Design: This study encompassed 6 treatment groups (5 dose rates of the test item and a control) with three replicates per test concentration and six replicates for the control. At test start 50 mL of the test media were inoculated with nominal 5000 algal cells per mL test medium and defined volumes of the algal suspensions were sampled after 24, 48 and 72 for determination of cell densities by spectrophotometrical measurement.

Endpoints: Yield and growth rate of the algae

Test Concentration: Due to the low limit of water solubility of the test item, a filtrate of nominal 100 mg/L and dilutions of 1:3, 1:9, 1:27 and 1:81 of this filtrate were tested, corresponding to nominal concentrations of 100, 33.3, 11.1, 3.70 and 1.23 mg test item/L. Additionally, a control was tested in parallel.

Test Conditions:

Water temperature: 22.1 to 23.3 °C;

pH values in the control at test start: 8.0

pH values in the control at test end: 9.3

pH values in the test item treatments at test start: 8.0,

pH values in the test item treatments at test end: 9.1 to 9.4;

continuous illumination; mean light intensity: 6475 lux (5820 to 7040 lux).

Results

Biological Results:

Biological Results:

The 72-hour EC50 and the 72-hour EC10 for the parameters growth rate and yield could not be calculated due to the low effects of the test item. Therefore the 72-hour EC50 and the 72-hour EC10 was determined to be higher than the limit of water solubility of the test item, represented by the filtrate of nominal 100 mg test item/L (0.208 µg/L), the highest concentration tested.

The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L (0.208 µg/L) for the parameters growth rate and yield and the associated 72-hour LOEC was determined to be higher than the filtrate of nominal 100 mg test item/L (0.208 µg/L) for both parameters.

Analytical Results:

The quantification of the test item Fluoroazopyrimidine in the test samples was performed using liquid chromatography with MS/MS detection. The concentrations of the test item were determined in the duplicate test media samples from all test concentrations (filtrate of the test item stock solution and its dilutions) and the duplicate control samples from all sampling times. The geometric mean measured values for the two highest concentrations are 0.208 µg test item/L in the filtrate and 0.0773 µg test item/L in the 1:3 dilution of filtrate. The dilutions of 1:9, 1:27 and 1:81 were below the limit of quantification (LOQ) which was 0.05 µg test item/L.

Conclusion:

The influence of Fluoroazopyrimidine on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour NOEC was determined to be at least the filtrate of nominal 100 mg (0.208 µg/L) 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 72-hour LOEC and the 72-hour EC50 were clearly higher than the filtrate of nominal 100 mg test item/L (0.208 µg/L). These values could not be quantified due to the absence of toxicity of Fluoroazopyrimidine up to the filtrate of nominal 100 mg test item/L (0.208 µg/L), the concentration representing the limit of solubility.

The initial concentrations and the maintenance of the exposure concentrations during the test were determined in the analytical part. All reported results refer to nominal test concentration since the filtrate of 100 mg test item/L was used. Furthermore the results are presented using geometric mean measured concentration since a decrease of test item was observed.

This study is classified acceptable and satisfies the guideline requirements for growth inhibition algae studies.