4-chloro-7H-pyrrolo[2,3-d]pyrimidine

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:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

EC Number:

628-079-2

Cas Number:

3680-69-1

Molecular formula:

C6H4ClN3

IUPAC Name:

4-chloro-7H-pyrrolo[2,3-d]pyrimidine

Test material form:

solid

Details on test material:

Batch (Lot) Number: GR13290
Expiry date: 31 January 2020 (expiry date)
Physical Description: White to off-white solid
Purity/Composition: 99.1%
Storage Conditions: At room temperature

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

no

Details on test solutions:

Test Item – Preparation and Test Concentrations
The batch of PF-01323624 tested was a white to off-white solid with a purity of 99.1%. 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 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 (see
Table 1 and Table 2). Optimal contact between the test item and test organisms was ensured
by applying continuous aeration and stirring.
Any residual volumes were discarded.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Source Municipal sewage treatment plant: 'Waterschap Aa en Maas',
's -Hertogenbosch, The Netherlands, receiving predominantly
domestic sewage.
Preparation of the sludge 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 7.8 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.
Medium Adjusted ISO-medium, formulated using RO water (tap water
purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot,
The Netherlands) with the following composition:

CaCl2.2H2 O 211.5 mg/L
MgSO4 .7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L
Rationale Recognized by international guidelines as the recommended
test system.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Remarks on exposure duration:

aeration and stirring took place.

Post exposure observation period:

10 minutes

Test conditions

Test temperature:

The temperature continuously measured in the temperature control vessels ranged between
19.2 and 22.5°C during the test, and was slightly outside the range prescribed by the Study
Plan (20 ± 2°C)

pH:

The pH in all test vessels, before addition of sludge, was between 7.4 and 7.6. After the
3 hour exposure period the pH was between 7.9 and 8.4.

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.

Nominal and measured concentrations:

Seven concentrations: 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L.

Details on test conditions:

Combined Limit/Range-Finding Test
In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested.
The highest concentration was tested in triplicate, lower concentrations consisted of one
replicate. In addition, a control (8 replicates) and an abiotic control (1 replicate) were tested.
Test procedure and conditions were similar to those applied in the final test with the
following exceptions:
• The pH of the sludge was 5.8 on the day of testing. The sludge was buffered to a pH of
7.3 using a 70 g/L sodium bicarbonate solution. Before the sludge was used, the
respiration rate of the sludge was determined to verify the quality of the sludge. The mean
respiration rate of the sludge was 21 mg O2
/g.h and within the validity criterion.
• A 1.0 g/L stock solution was prepared by dissolving 250.3 mg 3,5-dichlorophenol in
Milli-RO water and making up to a total volume of 250 mL. The pH as used for the test
was 7.8.

Final Test – Test Concentrations
PF-01323624 Seven concentrations: 1.0, 3.2, 10, 32, 100, 320 and 1000 mg/L.
Replicates 5 replicates per test group and 7 replicates for the control.
Control Test medium without test item, treated in the same way as the
test item solutions.

Test Procedure and Conditions
Contact time 3 hours, during which aeration and stirring took place.
Vessels All glass open bottles/vessels.
Milli-RO / Milli-RO water Tap water purified by reverse osmosis (Millipore Corp.,
Bedford, Mass., USA).
Synthetic medium 16 g peptone
(=sewage feed) 11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.
Air supply Clean, oil-free air.
Aeration 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.


Performance of the test
The synthetic medium (16 mL) made up to 50 mL with
Milli- RO and 200 mL test item solution were mixed (total
volume 250 mL). The pH was determined. Thereafter, 250 mL
activated sludge was added. This was the start of the test.
After the 3-hour contact time the oxygen consumption was
recorded for a period of approximately 10 minutes. During
measurement, the sample was not aerated but continuously
stirred on a magnetic stirrer.
The pH was determined in the remaining part of the reaction
mixture. This procedure was repeated for all test/reference item
concentrations and controls.
The medium temperature was recorded continuously in
temperature control vessels. The temperature control vessels
were identically prepared compared to the control vessels. A
temperature control vessel with a REES sensor was placed in
each fume cupboard of the climate room.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

5.7 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:

other: EC20

Effect conc.:

19 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 3

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

185 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Effect concentrations 4

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 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) in an hour (23 mg oxygen per gram of
activated sludge 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, see also Appendix 3).

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 the test item, calculation of the ECx values were based on probit analysis using linear
maximum likelihood regression, with the percentages of respiration inhibition versus the
logarithms of the corresponding concentrations of the test item.
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 25%, 56% and 79% inhibition of the respiration

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

was at a concentration between 10 and 100 mg/L.

There was an oxygen release of 22%, 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

A summarized overview of the study results is presented in the Table below.

The effects observed were in agreement with what was expected based on the results of the

combined limit/range-finding test. The results of the final test allowed for reliable

determination of a NOEC and ECx  values.

No statistically significant inhibition of the respiration rate of the sludge was recorded at

1.0 mg  PF-01323624 per liter (for more information on statistics see Appendix 4). At higher

concentrations the inhibitory effect of  PF-01323624 on aerobic waste water (activated sludge)

bacteria increased with increasing concentration, ranging from 11% inhibition at 3.2 mg/L to

75% at 1000 mg/L.

		Mean Respiration Rate
Treatment	Concnetration mg/L	mg O2/L h	mg O2/g h	% inhibition of the respiration rate (mean value
Control	0	35.11	23.40
T1	1.0	34.80	23.20	0.88
T2	3.2	31.26	2.084	10.96*
T3	10	32.57	21.71	7.24*
T4	32	23.68	15.79	32.55*
T5	100	21.29	14.19	39.36*
T6	320	15.71	10.47	55.24*
T7	1000	8.61	5.74	75.46*

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present test PF-01323624 was not toxic to waste
water bacteria (activated sludge) at a concentration of 1.0 mg/L (NOEC).
The EC10 was 5.7 mg/L (95% confidence interval: 2.7 – 9.9 mg/L).
The EC20 was 19 mg/L (95% confidence interval: 11 - 28 mg/L).
The EC50 was 185 mg/L (95% confidence interval: 138 - 254 mg/L).

Executive summary:

The objective of the study was to evaluate  PF-01323624 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-01323624 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-01323624 tested was a white to off-white solid with a purity of 99.1%. 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. Seven concentrations were tested, ranging from 1.0 to 1000 mg/L and increasing with a

factor 3.2. Five replicates per concentration and seven replicates for an untreated control

group were tested.

No statistically significant inhibition of the respiration rate of the sludge was recorded at

1.0 mg  PF-01323624 per liter. At higher concentrations the inhibitory effect of  PF-01323624

on aerobic waste water (activated sludge) bacteria increased with increasing concentration,

ranging from 11% inhibition at 3.2 mg/L to 75% at 1000 mg/L.

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-01323624 was not toxic to waste water (activated sludge) bacteria at a concentration of

1.0 mg/L (NOEC).

The effect parameters for the inhibition of the respiration rate are presented below.  

Parameter                     PF-01323624       Concentration (mg/L)

NOEC                                   1.0

EC10                                   5.7 (2.7 – 9.9 mg/L)

EC20                                   19 (11 - 28 mg/L)

EC50                                   185 (138 - 254 mg/L)

( ) Between brackets the 95% confidence intervals are given.