(1R,2S,3S,4R)-3-((2-chloro-5-fluoropyrimidin-4-yl)amino)bicyclo[2.2.2]octane-2-carboxylic acid

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:

2018-01-23 to 2018-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ISO International Standard 10634 "Water Quality - Guidance for the preparation and treatment of poorly water-soluble organic compounds for the subsequent evaluation of their biodegradability in an aqueous medium" (1995).

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ISO DIS 9439 (Ultimate Aerobic Biodegradability - Method by Analysis of Released Carbon Dioxide)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(2S,3S)-3-[(2-chloro-5-fluoropyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-carboxylic acid

EC Number:

821-184-6

Cas Number:

1777721-60-4

Molecular formula:

C13H15ClFN3O2

IUPAC Name:

(2S,3S)-3-[(2-chloro-5-fluoropyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-63757096-AAA (T003689)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Analytical purity: 99.3% (GC)
- Source and lot/batch No.of test material: I16ED2695
- Expiration date of the lot/batch: 2018-05-28 (retest date)
- Purity test date: 2017-11-14 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility in water: 0.07 g/L

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source: Municipal sewage treatment plant receiving predominantly domestic sewage, 'Waterschap Aa en Maas', 's-Hertogenbosch, The Netherlands.
- Storage conditions: Sludge was kept under continuous aeration until further treatment
- Preparation of inoculum for exposure: Before use, the sludge was coarsely sieved (1 mm) and washed with mineral medium.
- Pretreatment: no
- Concentration of sludge: The concentration of suspended solids was determined to be 3.0 g/L in the concentrated sludge.
- 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

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

CO2 evolution

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
- Additional substrate: no
- Test temperature: 22-23°C
- pH: 7.5-7.9, 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
- Aeration of dilution water: The test solutions were continuously aerated and stirred during the test. On nominal day 23 there was a short breakdown in aeration for test item bottle B. Aeration was immediately restored. Evaluation: Such a short breakdown in aeration has no impact on the validity or outcome of the study.
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: 2-L glass brown coloured bottles
- Number of culture flasks/concentration: 2
* 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).
- 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 control: yes, 1 replicate with reference item and inoculum

Reference substance

Reference substance:

acetic acid, sodium salt

Results and discussion

Test performance:

- In the toxicity control more than 25 % degradation occurred within 14 days (41% based on ThCO2). Therefore, the test substance was assumed to be not inhibitory on microbial activity.
- The difference of duplicate values for %-degradation of the test item was always less than 20 (≤6%).
- The total CO2 release in the blank at the end of the test slightly exceeds 40 mg/L (81.1 mg CO2 per 2 litres of medium, corresponding to 40.6 mg CO2/L). Since the CO2 release in the blank was still well below 70 mg/L this does not constitute a breach of the validity criterion.
- The Inorganic Carbon content (IC) of the test item (suspension) in the 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 item, 12 mg TOC/L).

% Degradation

Key result

Parameter:

% degradation (CO2 evolution)

Value:

ca. 4

Sampling time:

28 d

Remarks on result:

other: mean of test bottle A and B

Details on results:

The criterion for ready biodegradability (at least 60% biodegradation within 10 days of biodegradation exceeding 10%) was not met.

BOD5 / COD results

Results with reference substance:

The positive control item was biodegraded by at least 60% (83%) within 14 days, confirming suitability of the activated sludge.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

A 28-d ready biodegradability test (OECD 301B, modified Sturm test) using unadapted activated sludge from a predominantly domestic waste water treatment plant indicated that T003689 was not readily biodegradable under the conditions of the test (initial concentration 23 mg/L). The test substance showed only 2% and 6% biodegradation (test bottle A and B, respectively, 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.