7-Cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb - Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

7-cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

EC Number:

807-111-0

Cas Number:

1211441-98-3

Molecular formula:

C23H30N8O

IUPAC Name:

7-cyclopentyl-N,N-dimethyl-2-{[5-(piperazin-1-yl)pyridin-2-yl]amino}-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

Test material form:

solid: bulk

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Singular samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing
the undissolved fraction was retained for possible analysis.
Frequency at t=0 h, t=24 h and t=72 h
Volume 2.0 ml
Storage
Samples were stored in a freezer until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at an intermediate substance concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

yes

Details on test solutions:

Preparation of test solutions started with an aqueous mixture of 100 mg/l that was magnetically stirred for two days to achieve maximum dissolution in test medium. The resulting dispersion was filtered through a 0.45 μm membrane filter (Whatman; RC55) to remove the undissolved particles. The lower test concentrations were prepared by subsequent dilutions of the filtrate in test medium. The final test solutions were all clear and colourless.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration.
Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 104 cells/ml.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Pseudokirchneriella subcapitata, strain: NIVA CHL 1

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21-24°C

pH:

6.0-9.0

Reference substance (positive control):

no

Remarks:

Controls: Test medium without test substance or other additives

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

>= 2.6 - <= 4.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield inhibition

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Growth rates were in the range of the controls at the TWA concentrations of 0.21 and 0.76 mg/l during the 72-hour test period, whereas the growth rate of algae exposed to 2.5 mg/l and higher were increasingly reduced. It should be noted that, due to mathematical reasons (lack of normal distribution
and heterogeneity of variance) the differences between control and treatments could not be determined at 72 hour of exposure. Therefore, the NOEC for growth rate inhibition could not be statistically determined. However, as only 1.3% effect was observed at 0.76 mg/l this could be safely set as the NOEC as an effect less than 10% is generally considered not biologically relevant. In addition, the EC10 could be used as an alternative measure.
A slight, biologically irrelevant, inhibition of yield was observed at TWA concentrations of 0.21 and 0.76 mg/l. Significant inhibition of more than 90% was observed at TWA concentrations of 2.5 mg/l and higher. Statistically significant inhibition of yield was found at TWA concentrations of 2.5 mg/l and
higher.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, LEE011-A4 reduced growth rate and inhibited the yield of this fresh water algae species significantly at TWA concentrations of 2.5 mg/l and higher.
The EC50 for growth rate inhibition (72h-ERC50) was 3.4 mg/l with a 95% confidence interval ranging from 2.6 to 4.5 mg/l.
The EC50 for yield inhibition (72h-EYC50) was 1.4 mg/l with a 95% confidence interval ranging from 1.3 to 1.6 mg/l.
The 72h-NOEC for both growth rate inhibition and yield inhibition was 0.76 mg/l.