1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d] pyrimidin-5-yl)phenylsulphonyl]-4-methylpiperazine

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:

2005-10-13 to 2005-06-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one;2-hydroxypropane-1,2,3-tricarboxylic acid

Cas Number:

171599-83-0

Molecular formula:

C28H38N6O11S

IUPAC Name:

5-[2-ethoxy-5-(4-methylpiperazin-1-yl)sulfonylphenyl]-1-methyl-3-propyl-4H-pyrazolo[4,3-d]pyrimidin-7-one;2-hydroxypropane-1,2,3-tricarboxylic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot No.of test material: Sponsor and R207
- Expiration date of the lot/batch: 2005-11-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient
- Solubility and stability of the test substance in the solvent/vehicle: The 150 mg active ingredient / L stock solution was slightly cloudy after being sonicated for approximately 30 minutes and inverted at least 20 times to mix. Additionally, the 68 and 150 mg a.i./L test solutions which appeared slightly cloudy white with increasing intensity with increasing concentration. At test termination the 30, 68 and 150 mg a.i./L treatment levels had test material sticking to test chamber at water surface level, however, the remaining test solutions appeared clear and colorless.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: A primary stock solution was prepared by dissolving sildenafil citrate in freshwater algal medium at a nominal concentration of 150 mg active ingredient / L. The stock was sonicated approximately 30 minutes and inverted at least 20 times to mix and appeared slightly cloudy.
- Final dilution of a dissolved solid, stock liquid or gel: Aliquots of the primary stock were proportionally diluted with algal medium to prepare the
five additional test solutions at nominal concentrations of 2.8, 6.2, 14, 30 and 68 mg active ingredient / L.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Nominal test concentrations were 2.8, 6.2, 14, 68, and 150 mg active ingredient / L.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Nominal test concentrations were 2.8, 6.2, 14, 68, and 150 mg active ingredient / L. Additionally, there was a negative control with 0 mg active ingredient / L concentration.
- Sampling method: Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. At test initiation samples were collected from batch solutions of each treatment and control group prior to distribution into test chambers. At test termination, the replicates from each respective treatment and control group were pooled and then sampled. All samples were collected in glass vials.
- Sample storage conditions before analysis: All samples were analyzed immediately without storage.

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct weight addition
- Controls: A negative control was used
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water mixed with freshwater algal medium and then sterilized.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Only solvent in solution
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The 150 mg active ingredient / L stock solution was slightly cloudy after being sonicated for approximately 30 minutes and inverted at least 20 times to mix. Additionally, the 68 and 150 mg a.i./L test solutions which appeared slightly cloudy white with increasing intensity with increasing concentration. At test termination the 30, 68 and 150 mg a.i./L treatment levels had test material sticking to test chamber at water surface level, however, the remaining test solutions appeared clear and colorless.

Test organisms

Test organisms (species):

Scenedesmus capricornutum

Details on test organisms:

TEST ORGANISM
- Common name: Freshwater green algae
- Strain: scenedesmus capricornutum
- Source (laboratory, culture collection): Original algal cultures were obtained from the University of Toronto Culture Collection, and had been maintained in culture medium at Wildlife International, Ltd., Easton, Maryland
- Age of inoculum (at test initiation): >2 weeks
- Method of cultivation: Growing in culture medium

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: None

Study design

Test type:

static

Water media type:

other: Freshwater mixed with a specific mineral mixture known as algal medium

Limit test:

no

Total exposure duration:

3 d

Post exposure observation period:

None

Test conditions

Test temperature:

24.1 to 24.7°C, which is within the 23 ± 2°C range established for the test.

pH:

The pH of the test solutions at test initiation ranged from 6.0 to 8.1 and at exposure termination ranged from 7.8 to 9.2. This change in pH is typical for this strain of algae.

Nominal and measured concentrations:

Nominal concentrations selected for use in this study were 2.8, 6.2, 14, 30, 68 and 150 mg active ingredient /L

Measured concentrations of samples collected on Day 0 ranged from 90 to 106% of nominal concentrations. Measured concentrations of samples collected on Day 3 ranged from 12 to 56% of nominal concentrations. Mean measured test concentrations were 2.0, 4.6, 11, 21, 40 and 78 mg active ingredient /L representing 71, 74, 79, 70, 59 and 52% of nominal, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile 250-mL Erlenmeyer flasks plugged with foam stoppers
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 100 mL of test or control medium
- Initial cells density: 10,000 cells/mL
- Control end cells density:
- No. of organisms per vessel:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Stirring: 100 rpm continuous for the duration of the test

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used:
Compound Nominal Concentration
NH4Cl 15 mg/L
MgCl2•6H2O 12 mg/L
CaCl2•2H2O 18 mg/L
MgSO4•7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3•6H2O 0.08 mg/L
Na2EDTA•2H2O 0.1 mg/L
H3BO3 0.185 mg/L
MnCl2•4H2O 0.415 mg/L
ZnCl2 3 µg/L
CoCl2•6H2O 1.5 µg/L
CuCl2•2H2O 0.01 µg/L
Na2MoO4•2H2O 7 µg/L
NaHCO3 50 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Wildlife International, Ltd. Well Water
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine: 6.9 mg/L
- Alkalinity:
- Ca/mg ratio: 31.1 mg/LL
- Conductivity:
- Culture medium different from test medium: No
- Intervals of water quality measurement: Analysis was last performed on 2004-12-22, and the alkalinity was determined in the preceding 4 week period

OTHER TEST CONDITIONS
- Sterile test conditions: The solution was not sterile, but the flasks were sterilized prior to use.
- Adjustment of pH: Yes, using 10% HCl to pH = 8.0 +-0.1
- Light intensity and quality: The light intensity ranged from 6930 to 8590 lux, which was within the desired range of 8000 lux ± 20%.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The cell density, temperature, pH and light intensity were measured once a day for 72 hours
- Determination of cell concentrations: was done using a hemacytometer and a microscope. The samples then were diluted using an electron solution (Isoton), as needed, to maintain counting accuracy. A small amount of each sample was loaded onto a hemacytometer and 10 grids were counted. The mean number of cells per grid was calculated and this value was used to calculate the cell density of the sample. Using this technique, the minimum quantifiable cell density was 1,000 cells/mL.


TEST CONCENTRATIONS
- Test concentrations: Nominal concentrations selected for use in this study were 2.8, 6.2, 14, 30, 68 and 150 mg active ingredient/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 2

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Effect concentrations 4

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 4.4 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 43 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 78 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Effect concentrations 7

Key result

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

ca. 1 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area Under the Growth Curve

Effect concentrations 8

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 21 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area Under the Growth Curve

Effect concentrations 9

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 20 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Area Under the Growth Curve

Effect concentrations 10

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Growth rate, area under growth curve, and the cell density

Details on results:

After 72 hours of exposure, inhibition of cell density in the 2.0, 4.6, 11, 21, 40 and 78 mg a.i./L groups was -6.4, 11, 12, 52, 66 and 89%, respectively, relative to the negative control. Inhibition of area under the growth curve in the 2.0, 4.6, 11, 21, 40 and 78 mg active ingredient / L groups was -7.3, 9.2, 13, 53, 67 and 90%, respectively, relative to the negative control. Inhibition of growth rate in the 2.0, 4.6, 11, 21, 40 and 78 mg active ingredient / L treatment groups was -0.93, 1.9, 2.2, 13, 20 and 39%, respectively, relative to the negative control. Treatment related effects were apparent in the three highest test concentrations. Dunnett’s test indicated that there were statistically significant differences (p < 0.05) in cell density, area under the growth curve and growth rate between the negative control and the 21, 40 and 78 mg active ingredient / L treatment groups. Consequently, the 72-hour NOEC for cell density, area under the growth curve and growth rate was 11 mg active ingredient / L.

After 72 hours of exposure, there were no signs of adherence of cells to the test chambers or aggregation/flocculation of algae in the controls or in any treatment group. There were no noticeable changes in cell morphology at concentrations ≤40 mg active ingredient / L when compared to the control. However, enlarged cells were noted in the 78 mg active ingredient / L treatment groups at 72 hours.

The test was considered to be valid since the mean cell density in the control replicates increased by a factor greater than 16 within 3 days.

Results with reference substance (positive control):

No reference substance was used.

Reported statistics and error estimates:

The calculation of cell densities, areas under the growth curve, growth rates and percent inhibition values, as well as all statistical analyses, were conducted using “The SAS System for Windows”, Version 8.2 (4) or TOXSTAT version 3.5 (5).

EC50 values in this study represent the theoretical test concentration that would produce a 50% reduction in an effect relative to the control. Non-linear regression or linear interpolation was used to calculate EC50 values and their corresponding 95% confidence intervals for cell density (EC50), area under the growth curve (EbC50) and growth rate (ErC50) for each 24-hour exposure period, when possible (6,7). The 72-hour data were evaluated for normality and homogeneity of
variance (p=0.05) using the Shapiro-Wilk’s and Levene’s tests, respectively. The treatment groups then were compared to the negative control using Dunnett’s test (p=0.05). If the assumptions of normality and homogeneity of variances were not met an attempt was made to correct the condition by log transformation of the data. If the data failed the assumptions of normality and/or homogeneity of variances and transformations would not correct the problem, Dunnett’s test was still used to make the comparisons. The results of the statistical analyses, as well as an evaluation of the concentrationresponse pattern, were used to determine the NOEC relative to each parameter at 72 hours.

Category: Duration: 95% Confidence Interval (mg active ingredient/L)
Cell Density EC50
24 hr 2.0 to 58
48 hr 14 to 53
72 hr 18 to 26
Area Under the Growth Curve EC50
24 hr 0.041 to 27
48 hr 13 to 32
72 hr 18 to 25
Growth Rate EC50
24 hr 0.63 to 31
48 hr 31 to 60
72 hr Could not be calculated with the data obtained.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study calculates the EC50 for the test substance using various methods according to the criteria specified.