1-amino-N-[6-cyano-5-(trifluoromethyl)-3-pyridyl]cyclobutanecarboxamide

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:

FROM 13 SEP 2022 TO 13 OCT 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 301 B (Ready Biodegradability: CO2 Evolution 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

Constituent 1

Reference substance name:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Cas Number:

1950587-17-3

Molecular formula:

C12H11F3N4O

IUPAC Name:

1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: JNJ-63632283-AAA (T003665)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: 22BN0551
- Expiry date: 28 April 2023 (retest date)
- Physical Description: Off-white powder
- Purity: 98.3%
- Purity test date: 2022-05-05 (certificate of analysis release date)
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, non-adapted

Details on inoculum:

- Source: 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
- Storage conditions: sludge was kept under continuous aeration until further treatment.
- Preparation of inoculum for exposure: Before use, sludge was coarsely sieved (1 mm).
- Pretreatment: no
- Concentration of sludge: the concentration of suspended solids (SS) was determined to be 4.2 g/L in concentrated sludge as used for the test. Magnetically stirred sludge was used as inoculum at an amount of 2.4 mL per liter of mineral medium, leading to a SS concentration of 10 mg/L
- Water filtered: Tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

23 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

12 mg/L

Based on:

TOC

Remarks:

The organic carbon content was based on the molecular formula

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

CO2 produced in each test bottle reacted with barium hydroxide in the gas scrubbing bottle and precipitated out as barium carbonate. The amount of CO2 produced was determined by tit rating remaining Ba(OH)2 with 0.05 M standardized HCl

Details on study design:

TEST CONDITIONS
- Composition of medium: test water prepared according to test guidelines, analytical grade salts dissolved in tap-water purified by reverse osmosis (Milli-RO) and subsequently passed over activated carbon.
* mineral stock solution A: 8.5 g KH2PO4, 21.75 g K2HPO4, 67.20 g Na2HPO4.12H2O, 0.5 gNH4Cl dissolved in 1 L Milli-Q water, pH 7.4 ± 0.2
* mineral stock solution B: 22.50 g MgSO4.7H2O 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: 0.25 g FeCl3.6H2O dissolved in 1 L Milli-Q water
* Final test medium: 10 mL of solution A and 1 mL of solutions B, C and D per L of test medium, and Milli-RO water
- Additional substrate: no
- Test temperature: 22-23°C
- pH: 7.6, 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
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2-L all-glass brown coloured bottles
- Number of culture flasks/concentration:
* test substance and inoculum: 2 replicates
* inoculum blank: 2 replicates
* positive control: 1 replicate
* toxicity control: 1 replicate
- Method used to create aerobic conditions: A mixture of oxygen (~20%) and nitrogen (~80%) was passed through a bottle, containing 0,5 - 1 L 0,0125 M Ba(OH)2 solution to trap CO2. The synthetic air was sparged through the scrubbing solutions at a rate of ~1-2 bubbles per second (~30-100 mL/min). The initial suspension of unspiked test medium and inoculum was aerated with this CO2-free air overnight to purge the system of CO2 prior to testing. This CO2-free air was also used for aeration during the test.
- Measuring equipment: CO2-evolution was determined through titration of the remaining Ba(OH)2 with 0.05 M standardized HCl.
- Details of trap for CO2 and volatile organics if used: Three CO2-absorbers (bottles filled with 100 mL 0.0125 M Ba(OH)2) were connected in series to the exit air line of each test bottle.

SAMPLING
- Sampling frequency: every second or third day during the first 10 days, and thereafter at least every fifth day until the 28th day
- Sampling method: the absorber bottle closest to the incubation system was sampled each time, the second and third bottle were moved one position closer to the system and a new bottle was added at the end
- On the 28th day, pH of test suspensions was measured and 1 mL of concentrated HCl was added to each bottle. Bottles were aerated overnight to drive off CO2 present in the test suspension. The final titration was made on day 29.

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes, two replicates with only inoculum
- Toxicity control: yes, one replicate with test item, reference substance, and inoculum
- Procedure/positive control: yes, 1 replicate with reference item and inoculum

Reference substance

Reference substance:

benzoic acid, sodium salt

Remarks:

sodium benzoate

Results and discussion

Test performance:

1. The procedural control material was biodegraded by at least 60 % (actual result: 74 %) within 14 days.
2. Difference between duplicate values for %-degradation at the end of the test was less than 20 % (actual result: 1 %).
3. Total CO2 release in the blank at the end of the test did not exceed 40 mg/L (64 mg CO2 per 2 liters of medium, corresponding to 32 mg CO2/L).
4. Inorganic Carbon content (IC) of the test material (suspension) in mineral medium at the beginning of the test was less than 5 % of the Total Carbon content (TC). Since the test medium was prepared in tap-water purified by reverse osmosis (Milli- RO water (Millipore Corp., Bedford, Mass., USA, carbon levels <500 ppb)), IC was less than 5 % of TC (mainly coming from the test material, 12 mg TOC/L).
Since all validity criteria were satisfied the study was considered to be valid.

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

1

Sampling time:

29 d

Remarks on result:

other: for both test item vessels, based on ThCO2. CO2 measured on Day 29 is actually part of CO2 production of Day 28, since microbial activity was ended on Day 28 by addition of concentrated HCl.

Details on results:

Relative biodegradation values calculated from measurements performed during the test period revealed no biologically relevant biodegradation of JNJ-63632283-AAA (T003665) (1 % for both test item vessels, based on ThCO2).
In the toxicity control, more than 25 % biodegradation occurred within 14 days (33 %, based on ThCO2). Therefore, the test material 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 material sodium benzoate, which showed a normal biodegradation curve (74 % within 14 days)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

A 28 day ready biodegradability test (OECD 301B, modified Sturm test) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that JNJ-63632283-AAA (T003665) was not readily biodegradable under the conditions of the modified Sturm test.
The test substance showed only 1% biodegradation (in both test bottles A and B, based on ThCO2). 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.