isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09-01-2019 to 15-02-2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

yes

Remarks:

None of the deviations were considered to have impacted the overall integrity of the study or the interpretation of the study results and conclusions.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

EC Number:

950-484-0

Molecular formula:

C9H17NO2*C4H6O4

IUPAC Name:

isopropyl (1s,3s)-3-(methylamino)cyclobutane-1-carboxylate succinate

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13335
Expiry date: 31 January 2020 (expiry date)
Physical Description: White solid
Purity/Composition: 97.1%
Storage Conditions: At room temperature

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Details on test solutions:

The batch of PF-07097547-24 tested was a white solid with a purity of 97.1%. No correction
was made for the purity/composition of the test item.
A stock solution of 10 g/L was prepared by adding the test item into Milli-RO water (tap
water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The batch of PF-07097547-24 tested was a white solid with a purity of 97.1%. No correction
was made for the purity/composition of the test item.
A stock solution of 10 g/L was prepared by adding the test item into Milli-RO water (tap
water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Post exposure observation period:

10 minutes

Test conditions

Test temperature:

19.2-22.5 C

pH:

5.1 to 7.6 before sludge addition; 7.8 to 8.4 following 3 hours exposure

Dissolved oxygen:

The aeration was adjusted in such a way that the dissolved
oxygen concentration at the start was above 60-70% saturation
(60% of air saturation is > 5 mg/L at 20°C) and to maintain the
sludge flocs in suspension.

Salinity:

N/A

Conductivity:

N/A

Nominal and measured concentrations:

Nominal concentrations: 100, 180, 320, 560 and 1000 mg/L

Details on test conditions:

see below

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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

Details on results:

The mean control oxygen uptake rate exceeded 20 mg oxygen per gram of activated
sludge (dry weight of suspended solids) per hour (23 mg oxygen per gram of activated
sludge per hour, in the final test).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30%
at the end of the definitive test (6%).

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total
respiration (7.9 mg/L in the final test)

Reported statistics and error estimates:

ECx
For the reference item, calculation of the EC50 value was based on a 3-parameter logistic
cumulative distribution function (CDF) using non-linear regression analysis, with the
percentages of respiration inhibition versus the logarithms of the corresponding
concentrations of the reference item.
For PF-07097547-24, no EC50 value could be calculated because the test item proved to be
non-toxic (EC50 > 1000 mg/L).

NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the
test concentrations compared with those obtained in the control revealed significant inhibition
of the respiration rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided,
smaller).
Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Combined Limit/Range-Finding Test

The combined limit/range-finding showed 12%, 8% and 29% inhibition of the respiration rate

at a concentration of 10, 100 and 1000 mg/L, respectively. Therefore, the expected EC50

was above a concentration of 1000 mg/L. Since the effect observed at 1000 mg/L was statistically

significant a final test was performed to determine the NOEC.

There was an oxygen release of 3% from abiotic processes. Since this value was considered to

be unreliable, a second abiotic control was performed . The second abiotic control showed no significant oxygen release or uptake from abiotic processes.

The temperature continuously measured in the temperature control vessels ranged between

20.2 and 23.0°C during the test, and was slightly outside the range prescribed by the Study

Plan (20 ± 2°C).

Except for the temperature, all test conditions and acceptability criteria prescribed by the

Study Plan were met.

Final Test

Inhibition of the Respiration Rate

The effects observed were lower then what was expected based on the results of the combined

limit/range-finding test. The results of final test allowed for reliable determination of a NOEC

and ECx  values.

No statistical significant inhibition of the respiration rate was observed at any of the tested

concentrations . Therefore, the NOEC was considered to be the highest concentration tested.

		mean respiration rate
treatment	concentration mg/L	mg O2/L h	mg O2/g h (1)	% inhibition of the respiration rate (mean)
control	0	35.11	23.40
T1	100	32.81	21.87	6.55
T2	180	37.45	24.97	-6.69
T3	320	35.35	23.57	-0.7
T4	560	38.74	25.83	-10.35
T5	1000	36.56	24.37	-4.13

1) The amount of suspended solids in the final test mixture was 1.5 g/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test PF-07097547-24 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-07097547-24 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-07097547-24 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-07097547-24 tested was a white solid with a purity of 97.1%. No correction

was made for the purity/composition of the test item.

The test started with the preparation of a stock solution of 10 g/L. Volumes of the clear and

colourless stock solution corresponding to the test concentration were then added to the test

media. 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 seven 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-07097547-24 was not toxic to waste water (activated sludge) bacteria at a concentration of

1000 mg/L (NOEC). The EC50 is >1000 mg/L