N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

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:

08 Apr 2022 to 02 Jun 2022

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 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

1

Reference substance name:

N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

Cas Number:

1903009-56-2

Molecular formula:

C27H32N8O2

IUPAC Name:

N-[4-[4-[(dimethylamino)methyl]-3-phenyl-1H-pyrazol-1-yl]pyrimidin-2-yl]-6-methoxy-4-(morpholin-4-yl)benzene-1,3-diamine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: I20ID2980
- Purity, including information on contaminants, isomers, etc.: 99.3%
- Expiration date of the lot/batch: 17 September 2022 (retest date)
- Purity test date: 2020-10-26 (certificate of analysis release date)
- Physical appearance: beige powder
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

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 h, t=24 h and t=72 h.
- Volume: 2.0 mL from the approximate centre of the test vessels.
- Storage Samples were stored in a freezer (≤-15°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 of the limit concentration 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 (≤-15°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:

Details on test solutions
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Preparation of test solutions started with a loading rate of 100 mg/L applying 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. All test solutions were clear and colorless at the end of the preparation procedure. 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 final test solutions were clear and colourless

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: Algal 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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 medium at a cell density of 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured 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:

24 mg CaCO3/L

Test temperature:

Between 22 and 23 °C

pH:

At t=0: 8.2-8.3
At t=72h: 7.6-7.7

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Conductivity:

Not relevant

Nominal and measured concentrations:

nominal test concentrations final test: 0 (blank), 100% of a S.S. prepared at 100 mg/L with algae and 100% of a S.S. prepared at 100 mg/L without algae
measured test concentration final test t= 0 h: n.d., 0.375, 0.401 mg/L
measured test concentration final test t= 24 h: measured test concentration final test t= 72 h:
The LOQ is 0.1 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- 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: 10,000 cells/mL
- Control end cells density: 211.8 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 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 the algal stock culture were inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning an d the end of the test.

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 84 to 88 μE/m²/s.
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, 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.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10 (range finding study); none (final limit test)
- Range finding study
- Test concentrations: 1.00, 10 and 100 % of the SS (range finding study); 100% of the SS (final limit test)
- 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.:

>= 0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.12 mg/L

95% CI:

>= 0.12 - <= 0.13

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.013 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

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

Results with reference substance (positive control):

Experimental performed in March 2022
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 at the limit concentration showed stimulation rather than inhibition of growth.
The ECx-values could not be determined because the observed effects were below 10%.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, JNJ 73848281-AAA (T003904) inhibited growth rate of this freshwater algae species significantly at nominal concentrations of 0.080 mg/L and higher, and inhibited yield significantly at nominal concentrations of 0.032 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ErC50) was beyond the range tested.
The 72h-EC50 for yield inhibition (EyC50) was 0.12 mg/L with a 95% confidence interval ranging from 0.12 to 0.13 mg/L.
The 72h-NOEC for growth rate inhibition was 0.032 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 0.013 mg/L based on statistical significance.