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

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:

From 2021-03-26 to 2021-04-09

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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

Adopted April 13, 2004

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on the Use of the Harmonised System for the Classification of Chemicals which are Hazardous for the Aquatic Environment, OECD series on testing and assessment number 27

Version / remarks:

Adopted July 23, 2001

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: 97.1%
- 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:

Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0 and t=48 hours. 2.0 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 were pooled at each concentration before sampling. Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not 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 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 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.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Source: In-house laboratory culture with a known history.
- Age at study initiation (mean and range, SD): Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old.
- Method of breeding: Approximately 250 new-born daphnids, i.e. less than 3 days old, were placed into 5 liters of medium in an all-glass culture vessel.
- Feeding during cultivation: Daily, a suspension of freshwater algae
- Feeding during test: no

ACCLIMATION
- Acclimation period: not relevant

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L

Test temperature:

18-22°C, constant within ±1°C

pH:

At t-0h: 7.8 - 8
At t-48h: 8.0

Dissolved oxygen:

At t-0h: 8.6 - 8.7
At t-48h: 9.1 - 9.2

Salinity:

Not relevant

Nominal and measured concentrations:

combined limit/range-finding study
- nominal concentrations: 0.10, 1.0, 10 and 100% of the SS, prepared at a loading rate of 100 mg/L
- measured: control: < LOQ, geometric mean of highest test concentration: 2.3 µg/L

Samples taken from the highest test concentration were analyzed. The measured concentration at the start of the test was 3.1 µg/L. During the exposure period, the concentration decreased to 55% relative to initial at the end of the test. The measured concentrations were below the limit of quantification (LOQ). The LOQ of the analytical method was determined to be 0.01 mg/L.

As the test concentrations were above the limit of detection (LOD of 0.0019 µg/L) and furthermore within the calibration range of 0.1-20 µg/L, it was decided to report the measured concentrations and use them to express effect parameters. As measured concentrations were below LOQ, they should be considered as indicative values.

Details on test conditions:

TEST SYSTEM
- Test vessel: beaker
- Material, size, headspace, fill volume: 60 mL, glass beaker filled with 50 mL
- Aeration: not during the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Test medium: adjusted ISO medium

OTHER TEST CONDITIONS
- Photoperiod: 16h light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility was defined as those organisms which are not able to swim within 15 seconds after gentle agitation of the test container (even if they can still move their antennae) and was monitored after 24 and 48 hours of continuous treatment with the test substance.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study
- Test concentrations: 0.10, 1.0, 10 and 100% of a SS prepared at 100 mg/L (setup: combined range finder/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

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Details on results:

- Immobility of control: none
- Other adverse effects control: no
- Abnormal responses: none
- EC50 (24h): > 2.3 µg/L
- 5% immobility was observed at the highest test concentration

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: no
- Limit test:
- EC50/LC50: 48-h EC50 = 0.48 mg/L. The sensitivity of the daphnids was within the range determined with the historical data collected at Charles River Laboratoire Den Bosch.

Reported statistics and error estimates:

No EC50 could be calculated because the test item proved to be not acutely toxic to Daphnia magna (EC50 > maximum soluble concentration tested).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of T003903 to Daphnia was determined in a 48 hour static test according to OECD guideline 202. In conclusion, under the conditions of the present study with Daphnia magna, immobility of 5% was recorded at the highest concentration of JNJ-73848268-AAA (T003903) tested. However, effects <10% were considered biologically not relevant. The 48h-EC50 for Daphnia magna exposed to JNJ-73848268-AAA (T003903) was beyond the range of concentrations tested, i.e. exceeded a geometric mean measured concentration of 2.3 µg/L, being considered the maximum soluble concentration in test medium. Due to the very low solubility of JNJ-73848268-AAA (T003903) in test medium, concentration levels that might be toxic for Daphnia magna could not be reached.