cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Jan 2019 to 05 Feb 2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

EC Number:

951-622-2

Molecular formula:

C12H15N5O2

IUPAC Name:

cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

Specific details on test material used for the study:

Batch (Lot) Number: GR13449
Expiry date: 29 February 2020
Physical Description: White solid
Purity/Composition: 100%
Storage Conditions: At room temperature

Sampling and analysis

Analytical monitoring:

not specified

Test solutions

Vehicle:

yes

Remarks:

Purified tap water

Details on test solutions:

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the amber coloured test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item – Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added, resulting in the required concentrations. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

Test organisms

Test organisms (species):

activated sludge, domestic

Details on inoculum:

Municipal sewage treatment plant: 'Waterschap Aa en Maas','s -Hertogenbosch, Heeswijk-Dinther, The Netherlands, receiving predominantly domestic sewage. The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. The concentration of the suspended solids was determined (3.0 g/L, as used for the test). The pH was 6.7 on the day of testing. The batch of sludge was used one day after collection. Therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Study design

Test type:

static

Water media type:

other: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot, The Netherlands) with the following composition: CaCl2.2H2O 211.5 mg/L MgSO4.7H2O 88.8mg/L NaHCO3 46.7 mg/L KCl 4.2 mg/L

Limit test:

no

Total exposure duration:

3 h

Test conditions

Test temperature:

Between 20 and 23.3°C

pH:

7.2 on the day of testing

Nominal and measured concentrations:

Final test Five concentrations: 100, 180, 320, 560 and 1000 mg/L

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 2

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Any other information on results incl. tables

Range finding test:

The combined limit/range-finding showed no inhibition of the respiration rate at 10 and 100 mg/L. A mean inhibition of the respiration rate of 16% was observed at 1000 mg/L.

Therefore, the expected EC50 was above 1000 mg/L. The inhibition of the respiration rate observed at 1000 mg/L was statistical significant. Therefor a full test was performed to determine the NOEC.

There was no oxygen uptake, but a slight oxygen release from abiotic processes.

Final Test:

The effects observed were slightly lower then what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination

of a NOEC and ECx values. No inhibition of the respiration rate was observed at any of the tested concentrations. Therefore, the NOEC was considered to be the highest concentration tested of 1000mg

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test PF-07085579 was not toxic to waste water bacteria (activated sludge) at or below a concentration of 1000 mg/L (NOEC).
The EC50 was above 1000 mg/L.

Executive summary:

The objective of the study was to evaluate  PF-07085579 for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the

no - observed effect concentration (NOEC). The influence of  PF-07085579 on the respiration rate of activated sludge was investigated after a contact time of 3 hours. The study procedures described in this report were based on the OECD guideline No. 209, 2010. The batch of  PF-07085579 tested was a white solid with a purity of 100%. No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore, weighed amounts were added to the test bottles containing Milli-RO  water. The test item – Milli-RO  water mixtures were magnetically stirred for a short period. Subsequently, synthetic medium, sludge and Milli  - RO  water were added, resulting in the required concentrations. Optimal contact between the test item and test medium was ensured by applying continuous aeration and stirring during the 3 hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

The final test was performed based on the result of a preceding combined limit/range-finding test. Five concentrations were tested, ranging from 100 to 1000 mg/L and increasing with a factor 1.8. Five replicates per concentration and six replicates for an untreated control group were tested. No inhibition of the respiration rate was observed at any of the tested concentrations. Therefore, the NOEC was considered to be the highest concentration tested. The batch of activated sludge was tested for sensitivity with the reference item 3,5-dichlorophenol, and showed normal sensitivity. The study met the acceptability criteria prescribed by the Study Plan and was considered valid.  PF-07085579 was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/L (NOEC). The study met the acceptability criteria prescribed by the Study Plan and was considered valid.  PF-07085579 was not toxic to waste water (activated sludge) bacteria at a concentration of 1000 mg/L (NOEC).