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

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-03-01 to 2021-04-06

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 301 F (Ready Biodegradability: Manometric Respirometry Test)

Version / remarks:

July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634. "Water Quality - Preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium"

Version / remarks:

2018

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: 2022-09-17 (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
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent vehicle: solubility in water: not available

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Remarks:

The source of test organisms was activated sludge freshly obtained from a municipal sewage treatment plant: 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands, receiving predominantly domestic sewage

Details on inoculum:

The freshly obtained sludge was kept under continuous aeration until further treatment. Before use, the sludge was coarsely sieved (1 mm). After treatment, the concentration of suspended solids (SS) was determined to be 4.2 g/L in the concentrated sludge as used for the test. The sludge was used as inoculum at a concentration of 7.2 mL per liter of mineral medium, leading to a final concentration SS of 30.0 mg/L.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

28.7 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

80.6 mg/L

Based on:

ThOD

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: test water prepared according to test guidelines.
* mineral stock solution A: 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4.2H2O in 1 L Milli-Q water
* mineral stock solution B: 0.5 g NH4Cl dissolved in 1 L Milli-Q water
* mineral stock solution C: 36.4 g CaCl2.2H2O dissolved in 1 L Milli-Q water
* mineral stock solution D: 22.5 g MgSO4.7H2O dissolved in 1 L Milli-Q water
* mineral stock solution E: 0.25 g FeCl3.6H2O, 0.56 K2-EDTA dissolved in 1 L Milli-Q water
* Final test medium: 10 mL of solution A and and 1 mL of solutions B through D per L of test medium were added to 0.244 L distilled water.
- Additional substrate: no
- Test temperature: 21.7 - 22.2°C
- pH: 7.60-8.50; measured prior to testing in each test flask before addition of inoculum, and again in each test flask at the end of the incubation period.
- pH adjusted: no
- Suspended solids concentration: initially 30 mg dw/L
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: Soda lime pellets (Sigma-Aldrich, Steinheim, Germany).
- Number of culture flasks/concentration: 2
- The CO2 produced in each test bottle reacted with the CO2 absorbent in the rubber gaskets. As gaseous O2 was converted into gaseous CO2 that was absorbed, the gas pressure in the test system slowly decreased. This decrease in air pressure was measured by the respirometer heads and automatically converted into oxygen consumption (mg O2/L).
- Each vessel is connected with an oxygen-producing unit (sulphuric acid-copper solution in an electrolytic bottle) and a manometer.

SAMPLING
- Sampling frequency: every day during 28 days

CONTROL AND BLANK SYSTEM
- Biotic control: yes, two replicates with inoculum
- Toxicity control: yes
- Procedure control: yes, 1 replicate with reference substance and inoculum

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

Sodium acetate

Results and discussion

Test performance:

1. The procedural control item was biodegraded by at least 60 % (actual value: 73 %) within 14 days.
2. The difference of duplicate values for %-degradation of the test item at the end of the test was less than 20 (actual value: 0 %).
3. The total oxygen uptake in the blank at the end of the test did not exceed 60 mg O2/L (average actual value: 28.0 mg O2 per litre).
4. The pH value in the test item bottles at the end of the test was within the range 6.0-8.5 (7.6 for both vessels).
Since all validity criteria were satisfied the study was considered to be valid.

% Degradation

Key result

Parameter:

% degradation (O2 consumption)

Value:

0

Remarks on result:

other: in both test vessels

Details on results:

The relative biodegradation values calculated from the measurements performed during the test period revealed no biologically relevant biodegradation of JNJ-73848281-AAA (T003904), based on ThOD (0 % in both test vessels).
In the toxicity control, more than 25 % biodegradation occurred within 14 days (37 %, based on ThOD). Therefore, the test item was considered not to inhibit microbial activity.

BOD5 / COD results

Results with reference substance:

Functioning of the test system was checked by testing the procedural control item sodium acetate, which showed a normal biodegradation curve

Any other information on results incl. tables

Monitoring of Temperature and pH

The temperature recorded in a vessel with water in the same incubator varied between 21.7 and 22.2 °C and complied with the requirements as laid down in the Study Plan. The pH values of the different test media are presented in the table below.

 

pH Values of Different Test Media

Test medium:	Day 0:	Day 14:	Day 28:
Blank control (A)	7.7	-	7.6
Blank control (B)	7.7	-	7.6
Procedural control (A)	7.7	8.5	-
Procedural control (B)	7.7	8.5	-
JNJ-73848281-AAA (T003904) (A)	7.7	-	7.6
JNJ-73848281-AAA (T003904) (B)	7.7	-	7.6
Toxicity control	7.7	8.5	-

The pH of all test vessels at the start of the test (Day 0) was outside the range laid down in the Study Plan. It was 7.7, which is only 0.1 pH unit above the recommended range. Such a small deviation does not  impact the viability of the inoculum.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 28-d ready biodegradability test (OECD 301F, manometric respirometry test) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that JNJ-73848281-AAA (T003904)  was not readily biodegradable under the conditions of the test (initial concentrations 28.7 mg/L). The test substance showed 0% biodegradation (in both test bottles A and B, based on ThOD). The test substance did not inhibit microbial activity at the concentration used in the test. The results of the test can be considered reliable without restriction.