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

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 29 2005 to October 24 2005

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

GLP compliance:

yes

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/batch No.of test material: Sponsor amd R207
- Expiration date of the lot/batch: 2005-11-01

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Solubility and stability of the test substance in the solvent/vehicle: 3.5 mg/mL (unbuffered, final pH 3.9)

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 dilution water (well water) at a nominal concentration of 10 mg active ingredient / L
- Final dilution of a dissolved solid, stock liquid or gel: The stock solution was diluted to 0.63, 1.3, 2.5, and 5.0 mg active ingredient / L by proportional dilutation of the primary stock. The primary stock was also used as a final concentration of 10 mg active ingredient / L

FORM AS APPLIED IN THE TEST (if different from that of starting material)
-The test concentration were 0.63, 1.3, 2.5, 5.0, and 10.0 mg active ingredient / L of vehicle (filtered well water)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

-Concentrations: The test concentration were 0.63, 1.3, 2.5, 5.0, and 10.0 mg active ingredient / L of vehicle (filtered water)
- Sampling method: Samples were collected from each test chamber of each treatment and control group at test initiation, at the approximate mid-point of the test, and at test termination to measure concentrations of the test substance. All samples were collected from mid-depth, placed in glass vials, and analyzed as soon as possible without storage.
-Sample storage: No significant storage time existed and as such, the samples were never "stored".

Test solutions

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was mixed in dilution water (well water) for two hours using an electric top-down stainless steel mixer for the stock solution. The stock solution was diluted to make the remaining test concentrations with dilution water and then stirring with a stainless steel whisk.
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): water
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.63, 1.3, 2.5, 5.0, and 10.0 mg active ingredient / L of vehicle (filtered well water)

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: rainbrow trout
- Strain: Oncorhynchus mykiss
- Source: Thomas Fish Company, Anderson, California
- Age at study initiation (mean and range, SD):
- Length at study initiation (length definition, mean, range and SD): The average total length of 10 negative control fish measured at the end of the test was 5.3 cm, with a range of 4.8 to 6.1 cm
- Weight at study initiation (mean and range, SD): The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 1.2 grams, with a range of 0.82 to 1.9 grams

ACCLIMATION
- Acclimation period: 14 days
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food during acclimation: Commercially-prepared diet supplied by Zeigler Brothers, Inc., Gardners, Pennsylvania.
- Feeding frequency during acclimation: Daily, except for a two day fasting period prior to testing
- Health during acclimation (any mortality observed): None observed.

FEEDING DURING TEST
Not specified.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

26 h

Post exposure observation period:

None

Test conditions

Hardness:

Hardness 132 (mg/L as CaCO3)

Test temperature:

12 ± 1°C

pH:

8.1 to 8.5

Dissolved oxygen:

≥9.1 mg/L (≥84% of saturation) at test initiation, but decreased in some of the test solutions to a minimum of 62% saturation by 24 hours. Gentle aeration was added to each test chamber on Day 1 of the test and dissolved oxygen concentrations were ≥78% saturation throughout the remainder of the test.

Salinity:

Alkalinity 180 (mg/L as CaCO3)

Conductivity:

Specific Conductance 310 (µmhos/cm)

Nominal and measured concentrations:

Nominal Concentrations:
Negative Control Concentrations: 0 mg active ingredient/L
Test Concentrations: 0.63, 1.3, 2.5, 5.0, and 10.0 mg active ingredient / L of vehicle (filtered well water)

Measured Concentrations:
Negative Control Concentrations: < LOQ (The limit of quantitation (LOQ) was 0.400 mg a.i./L, calculated as the product of the concentration of the lowest calibration standard (0.400 mg a.i./L) and the dilution factor of the matrix blank samples (1.00).)
Test Concentrations: 0.651-0.683 mg active ingredient/L, 1.29-1.31 mg active ingredient/L, 2.57-2.60 mg active ingredient/L, 4.59-5.15 mg active ingredient/L , 8.98-9.79 mg active ingredient/L for the respectively closest in value nominal concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: 25L stainless steel aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Filled with 20L of test water to a depth of 23.3 cm
- Aeration: Yes, after Day 1 gentle aeration was used
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: 0.59 g fish / L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water was passed through a sand filter to remove particles greater than approximately 25 µm, and pumped into a 37,800-L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 µm to remove fine particles.
- Total organic carbon:
- Particulate matter:
- Metals:
- Pesticides:
- Chlorine: 6.9 mg/:
- Alkalinity: 178 – 184 (mg/L as CaCO3)
- Ca/mg ratio: 31.1 mg/L
- 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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Test temperature, culmulative mortality, measured concentrations of sildenfail citrate, dissolved oxygen, gerneral observations, and the pH of water in the test chambers was measured at 0, 24, 48, 72, and 96 hours

TEST CONCENTRATIONS
- Test concentrations:
Nominal Concentrations: 0.63, 1.3, 2.5, 5.0, and 10.0 mg active ingredient / L of vehicle (filtered well water)
Measured Concentrations: 0.651-0.683 mg active ingredient/L, 1.29-1.31 mg active ingredient/L, 2.57-2.60 mg active ingredient/L, 4.59-5.15 mg active ingredient/L , 8.98-9.79 mg active ingredient/L for the respectively closest in value nominal concentrations

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 9.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Effect concentrations 2

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

> 9.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Effect concentrations 3

Key result

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 9.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Effect concentrations 4

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 9.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 9.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: None
- Mortality of control: No mortalities were noted

Results with reference substance (positive control):

No reference substance was used.

Reported statistics and error estimates:

The absence of mortality in any sildenafil citrate treatment group precluded the statistical calculation of LC50 values. Therefore, the 24 and 48-hour LC50 values, as well as the no mortality concentration and the NOEC, were determined by visual interpretation of the mortality and observation data.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

This study determined that the LC50 and NOEC doses were >9.5 mg active ingredient / L according to the criteria specified in the respective guidelines.