2-Pyrimidinamine, 4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-N-[2-methoxy-4-(4-morpholinyl)-5-nitrophenyl]-

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:

26 Mar 2021 to 26 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD series on testing and assessment number 23 (2nd edition),

Version / remarks:

Adopted February 08, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-N-[2-methoxy-4-(morpholin-4-yl)-5-nitrophenyl]pyrimidin-2-amine

Cas Number:

1903009-55-1

Molecular formula:

C27H30N8O4

IUPAC Name:

4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]-N-[2-methoxy-4-(morpholin-4-yl)-5-nitrophenyl]pyrimidin-2-amine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch (Lot) Number: I20HD2581
- Purity/Composition: 99%
- Purity test date : 2020-09-01
- Expiry date: 2022-08-15 (retest date)
- Physical Description: yellow powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of test item in the solvent/vehicle: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
No treatment

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0, t=24 and t=72 hours
- Volume: 2.0 mL from the approximate center of the test solutions.
- Sample storage conditions before analysis: Samples were stored in a freezer (set to maintain -20°C) until analysis at the analytical laboratory of the Test Facility

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10% of the SS prepared at a loading rate of 100 mg/L, but without algae (abiotic control) and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (set to maintain -20°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of the test solutions started with a loading rate of 100 mg/L applying a fifteen-minute period of ultrasonic waves followed by a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 1E+04 cells/mL.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all test solutions were clear and colorless at the end of the preparation procedure.

During the final test, a pre-incubation step of the test vessels was included in the experimental set-up due to the adsorbing properties of the test item to glass. Therefore, after preparation, each test vessel was pre-incubated with the respective test concentration for 24 hours.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before start of the test)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

between 21 and 22°C

pH:

8.0 - 8.1

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Conductivity:

Not relevant

Nominal and measured concentrations:

nominal test concentrations final test: 0 (blank), Solutions containing 1.0, 3.2, 10, 10 (without algae), 32 and 100% of the Saturated Solution prepared at a loading rate of 100 mg/L

measured test concentration final test t= 0 h: measured test concentration final test t= 24 h: measured test concentration final test t= 72 h:
- actual exposure concentrations: 0.0050, 0.0050, 0.0059, 0.0066 and 0.0093 mg/L (time weighted average concentrations, TWA)
- LOQ: Limit of quantification, 0.01 mg/L; half of the LOQ value was used to calculate the TWA

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): with aluminium caps, perforated for ventilation
- Material, size, headspace, fill volume: 100 mL, containing 50 mL of test solutions
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 1E+04 cells/mL
- Control end cells density: 107 x 1E+04 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water
- Culture medium different from test medium: Yes, (M1 versus M2). Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2). The pre-culture was maintained under the same conditions as used in the test.
- Intervals of water quality measurement: Temperature was measured continuously in a temperature control vessel, and pH was measured at the beginning and end of of the test, for all test concentrations and the control.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: continuous ilumination
- Light intensity and quality: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank
- Effect calculated parameters: specific growth rate and yield.

TEST CONCENTRATIONS (range finding study)
- Spacing factor for test concentrations: 10 (range finding study); 3.2 (final limit test)
- Range finding study: test concentrations: 0.10, 1.0, 10 and 100% of the SS prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.009 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.009 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- 72-h EC50 (yield) > 0.0093 mg/L
- 72-h EC20 (yield) > 0.0093 mg/L
- 72-h EC20 (growth rate) > 0.0093 mg/L
- 72-h EC10 (growth rate) > 0.0093 mg/L
- 72-h EC10 (yield) > 0.0093 mg/L
- 72-h NOEC (yield) >= 0.0093 mg/L

Results with reference substance (positive control):

Experiment performed: Apr 2021
The batch of Raphidocelis subcapitata tested, showed expected sensitivity to Potassium dichromate based on the historical range of reference tests performed by the Test Facility in the last ten years.
The raw data from this study are kept in the Charles River Den Bosch archives. The test described above was performed non-GLP.

Reported statistics and error estimates:

Statistical analysis of the data was not needed as the effects recorded showed a stimulation rather than an inhibition of growth.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of T003903 tested.
The 72h-EC50 for growth rate inhibition (ERC50) and for yield inhibition (EYC50) were beyond the range tested, i.e. exceeded a TWA concentration of 0.0093 mg/L, being considered the maximum soluble concentration in test medium.
The 72h-NOEC for growth rate inhibition and for yield inhibition appears to be higher than or equal to a TWA concentration of 0.0093 mg/L, being considered the maximum soluble concentration in test medium.
Due to the very low solubility of T003903 in test medium, concentration levels that might be toxic for algae could not be reached.