4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid

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:

23 to 26 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP, guidance study was followed

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid

EC Number:

429-790-9

EC Name:

4-[2-(2-amino-4,7-dihydro-4-oxo-3H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid

Cas Number:

137281-39-1

Molecular formula:

Hill formula: C15 H14 N4 O3 CAS formula: C15 H14 N4 O3

IUPAC Name:

4-(2-{2-amino-4-oxo-3H,4H,7H-pyrrolo[2,3-d]pyrimidin-5-yl}ethyl)benzoic acid

Test material form:

solid: particulate/powder

Details on test material:

Nature of substance: Compound 202723
Storage: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

A single sample of the exposure solution for each treatment level and control was taken at both the beginning and end of the incubation period. Samples taken at the beginning were prior to division into replicates. Samples taken at the end were from a composite of the replicates.

Test solutions

Vehicle:

no

Details on test solutions:

A 500 mg/L primary stock solution of compound 202723 was prepared by placing 1 gram of test material (corrected for purity as compound 202723) in a 2 L volumetric flask and bringing to volume with the dilution water (Algal Assay Procedure (AAP) medium).

The resulting stock solution was observed to be cloudy and pink -brown in color with a large amount of undissolved particulates suspended in solution and settled on the bottom of the flask. Then the stock solution mixture was then subject to: 24 hours of mixing using a magnetic stir plate and Teflon®-coated stir bar; one hour settling period and 30 minutes centrifugation at 1000rpm.
At this point the primary stock solution was observed to be slightly cloudy with visible undissolved test substance at the bottom of the vessel. Exposure solutions were prepared from the water soluble portion of the 500 mg/L solution, which was removed from the vessel using a pipette.

The water soluble solution was used to prepare the nominal concentrations of 7.8, 16, 31, 63, 130, 250 and 500 mg/L exposure solutions.

Test organisms

Test organisms (species):

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

Details on test organisms:

The green alga used in this study was originally received from University of Texas (Austin Texas) and was maintained in culture in AAP medium at the performing laboratory. The inoculum used in this study was taken from a stock culture that had been transferred to fresh medium three days before testing

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not measured

Test temperature:

Temperature range of 23 to 24C (continuous temperature monitoring)

pH:

The pH of the test and control solutions ranged from 6.8 to 7.4 at test initiation. At test termination, the pH ranged from 7.2 to 7.7

Dissolved oxygen:

Not Measured

Salinity:

Not Applicable

Nominal and measured concentrations:

Nominal concentrations of 7.8, 16, 31, 63, 130, 250 and 500 mg compound 202723/L were prepared from the water-accommodated fraction (WAF) of a
500 mg compound 202723/L stock solution. The mean measured concentrations were 1.2, 2.4, 4.2, 9.3, 19, 35 and 81 mg compound 202723/L.

Details on test conditions:

Test vessels: sterile 250-mL Erlenmeyer flasks containing 100 mL of the appropriate test solution
Replicates: 3 replicates for each treatment level, 6 replicates for the control (100 mL of AAP medium without test substance) Inoculum: each vessel contained 10,000 cells/mL at test initiation
Abiotic sample: A single additional replicate was included that contained a mid-concentration of test substance but no algae. Temperature: 23 -24 °C
Duration: 72 hours
Light: continuous, 4400 - 5900 lux
Shaking: continuous
Endpoints: Cell counts at 24 -, 48 -, 72 -hours using a hemacytometer used to calculated yield and average growth rate.

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other:

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 81 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 80 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 80 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

At test termination, cells exposed to all treatment levels tested and the control were observed to be normal. Yield and average growth rate of Pseudokirchneriella subcapitata after 72 -hours of exposure to compound 202723 is presented in Table 1 below. Undissolved test substance was observed in both the 35 and 81 mg compound 202723/L treatment levels at the 24 hour observation interval and in the 81 mg compound 202723/L treatment level at each observation interval thereafter.

The cell density increase by 59.17 times in the control and the daily CV for growth rates was 22% and the CV for 0 - to 72 -hour growth rate was 5.0%. Therefore, the study was acceptable based on the growth and variability in the control.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Average growth rate for each replicate flask was calculated for the period from test initiation to each observation time. Yield was calculated as biomass (cell density) at each interval of the test minus the initial biomass (1.0 x 104 cells/mL) at the start of the test. Means and standard deviations for yield and growth rate were calculated from individual replicate values and are in Table 1 below.

Yield and growth rate in the treatment groups were compared to those in the control by Bonferroni's Adjusted t -Test. No signficant differences from control were detected so the NOEC was the highest concentration tested. Based on the lack of inhibition in the treatment groups (<10% inhibition), the EC10, EC20, and EC50 values were all greater than the highest concentration tested.

Any other information on results incl. tables

Table 1: Yield and average growth rate of Pseudokirchneriella subcapitata after 72 hours of exposure to compound 202723

	Yield (x10^4cells/mL)		Growth Rate (days-^1 )
Mean Measured Conc. (mg compound 20273/L)	Mean	SD	Mean	SD
Control	58.17	12.11	1.39	0.07
1.2	51.75	3.19	1.36	0.02
2.4	57.58	13.25	1.39	0.07
4.2	56.92	11.10	1.39	0.06
9.3	55.08	5.64	1.38	0.03
19	69.92	7.99	1.46	0.04
35	57.83	6.39	1.40	0.04
81	60.92	11.09	1.41	0.06

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

(Based on appropriate exponential growth (>16x) and appropriate variability in growth rate (5%) in the controls)

Conclusions:

The EC10, EC20, and EC50 values for yield growth rate are all greater than the highest concentration tested, 81 mg compound 202723/L. The NOEC values for yield and growth rate are 81 mg compound 202723/L.

Additional testing to further define EC values was not performed since the highest concentration tested (81 mg/L) represents the functional solubility limit of compound 202723 in AAP medium.

Executive summary:

Test Substance:              Compound 202723

Test Organism:               Pseudokeichneriella subcapitata, inoculum - three days since previous transder - performing laboratory culture

Dilution water:                 Algal Assay Procedure (AAP) medium

Test conditions:              72 hour duration, temperature range 23 -24oC, continuous illumination at 430 to 550 footcandles (4700 to 5900 lux), shaking rate of 100rpm

Nominal Test Concentrations: 7.8, 16, 31, 63, 130, 250 and 500mg/l prepared from the water accommodated fraction (WAF) of a 500mg/L stock solution.

Mean Measured Concentrations: 1.2, 2.4, 4.2, 9.3, 19, 35 and 81 mg/l

Results

	Based on Mean Measured Concentration (mg compound 202723/L)
Biological Parameter	EC10	EC20	EC50	NOEC
0-72HrYield	>81	>81	>81	81
0-72Hr Average Growth Rate	>81	>81	>81	81
	No effects were observed, therefore, EC values were empirically estimated as greater than the highest concentration tested