4-chloro-N-[2-methoxy-4-(morpholin-4-yl)-5-nitrophenyl]pyrimidin-2-amine

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:

23 APR 2021 to 16 JUN 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-N-[2-methoxy-4-(morpholin-4-yl)-5-nitrophenyl]pyrimidin-2-amine

Cas Number:

2270944-70-0

Molecular formula:

C15H16ClN5O4

IUPAC Name:

4-chloro-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
- Source and lot/batch number of test material: I20CD1048
- Expiration date of the lot/batch: 2022-0-22 (retest date)
- Purity test date: 2020-05-11 (certificate of analysis release date)
- Physical appearance: Light yellow to dark orange solid

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 the test material in the solvent/vehicle and the exposure medium: not indicated

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 and t=72 h.
- Volume 1.5 mL from the approximate center of the test solutions.
- Storage 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 but without algae (abiotic control) and samples for analysis were taken at the start and at the end of the test period.
Additionally, reserve samples of 1.5 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)
The batch of JNJ-73848242-AAA (T003901) tested was a light yellow to dark orange solid with a purity of 100.7% and was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions started with a loading rate of 100 mg/L applying fifteen-minutes of ultrasonic waves followed by a one-day period of magnetic stirring to ensure maximum dissolution of the test item in test 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 104 cells/mL.

Any residual volumes were discarded.

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: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture 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 immediately before use.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 4 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:

maintained between 21-22°C

pH:

t=0h/t=72h: control: 8.2/7.9, 0.0034 mg/L: 8.2/7.9, 0.011 mg/L: 8.1/7.9, 0.035 mg/L: 8.1/7.9, 0.12 mg/L: 8.1/7.9, 0.36 mg/L: 8.2/7.9

Dissolved oxygen:

not reported

Salinity:

not relevant

Conductivity:

not relevant

Nominal and measured concentrations:

- nominal (mg/L): 0-1.0-3.2-10-32 and 100% of the SS prepared at a loading rate of 100 mg/L
- measured (mg/L): 0-0.0034-0.011-0.035-0.12 and 0.36 mg/L (geometric mean)

Samples taken from all concentrations and the control were analyzed. The measured concentrations at the start of the test were 0.0040, 0.013, 0.040, 0.13 and 0.41 mg/L, for solutions containing 1.0, 3.2, 10, 32 and 100% of the SS, respectively. During the exposure period, the concentrations decreased slightly and were at 72-86% relative to the initial concentrations at the end of the test.
The concentrations measured in the samples taken from the solution with algae were comparable with the concentrations measured in the samples without algae. Hence, the presence of the algae did not affect the concentration of the test item in test medium throughout the test.

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: 1 x 10,000 cells/mL
- Control end cells density: 127 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

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: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 83 to 84 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: 3.2

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.:

> 0.36 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.035 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- EC50 yield: > 0.36 mg/L, NOEC yield: 0.035 mg/L
- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: Due to the low solubility of JNJ-73848242-AAA (T003901) in test medium, concentration levels that might be toxic for algae could not be reached.

Results with reference substance (positive control):

expected sensitivity of test species

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s test) and a test for homogeneity of the variances (Levene’s test).

Calculation of ECx-values was based on Probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding geometric mean concentrations of the test item.

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:

The objective of the study was to evaluate JNJ-73848242-AAA (T003901) for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield. A final test was performed based on the results of a range-finding test. The study met the validity criteria prescribed by the study plan. In conclusion, under the conditions of the present study with Raphidocelis subcapitata, JNJ73848242-AAA (T003901) inhibited growth rate and yield of this freshwater algae species significantly at geometric mean concentrations of 0.12 mg/L and higher. The 72h-EC50 for growth rate inhibition (ERC50) and yield was beyond the range tested. The 72h-NOEC for growth rate inhibition and yield inhibition was 0.035 mg/L based on statistical significance. Due to the low solubility of JNJ-73848242-AAA (T003901) in test medium, concentration levels that might be toxic for algae could not be reached. The results of the test can be considered reliable without restrictions.