Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-10-20 to 2007-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study performed according to OECD Guideline 203 (Fish, Acute Toxicity Test) and EU Method C.1 (Acute Toxicity for Fish). There were no deviations from the guideline(s) followed; however, the following deviation was noted from the protocol: Temperature in the control vessel was outside the range specified in the protocol, but this had no impact on the integrity of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
There were no deviations from the guideline(s) followed; however, the following deviation was noted from the protocol: Temperature in the control vessel was outside the range specified in the protocol, but this had no impact on the integrity of the study
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
There were no deviations from the guideline(s) followed; however, the following deviation was noted from the protocol: Temperature in the control vessel was outside the range specified in the protocol, but this had no impact on the integrity of the study
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate given by the Department of Health of the Government of the United Kingdom

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): VRT-753136 hcl
- Molecular formula: Not applicable
- Molecular weight: Not applicable
- Smiles notation: Not applicable
- InChl: Not applicable
- Structural formula attached as image file: Not applicable
- Substance type: No data
- Physical state: White powder
- Analytical purity: 100 area %
- Impurities: 0.03 area% of unknowns
- Composition of test material, percentage of components: No data
- Isomers composition: No data
- Purity test date: 2006-09-15
- Lot/batch No.: Batch number 25515/Lot No. AF6-001
- Expiration date of the lot/batch: 2008-05-01
- Stability under test conditions: No data
- Storage condition of test material: Room temperature
- Other: No data
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentrations
- Sampling method: Four mid-vessel samples (10 mL) of media, were taken from the control and test medium at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis.
- Sample storage conditions before analysis: On each occasion, two samples from each batch were analysed, the remaining samples were stored frozen in case further analysis was required.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: On each occasion, the test substance (2.5 g) was dissolved in diluent water in a volumetric flask (5 L). The contents of the flask were shaken before being poured into a test vessel (glass aquarium) containing diluent water (15 L). The volume in the test vessel was then adjusted to 25 litres.
- Controls: diluent water
- Eluate: Not applicable
- Differential loading: Not applicable
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Not applicable
- The test concentration of VRT-753136 hcl was measured using an LC-MS method of analysis. Four mid-vessel samples (10 mL) of media, were taken from the control and test medium at 0 and 72 hours (fresh media) and at 24 and 96 hours (expired media) for analysis.

Test Substance solutions:
- A weighed amount (corrected for purity) of VRT-753136 hcl was dissolved in acetonitrile: Water (50:50 v:v) to produce a stock standard solution.
- An aliquot of the stock standard solution was diluted appropriately in water to give fortification solutions.
- The appropriate fortification solution was further diluted with water to produce a series of calibration solutions in the range 25 to 200 ng/mL.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: No data
- Source: The fish were supplied by a commercial fish farm in the UK.
- Age at study initiation: approximately 5 months (juveniles)
- Length at study initiation: Their mean total length was 6.42 cm.
- Weight at study initiation: Their mean wet weight was 2.37 g.
- Method of breeding: Obtained from breeder
- Feeding during test: The fish were not fed during the 96 hour exposure period.
- Food type: Not Applicable
- Amount: Not applicable
- Frequency: Not applicable

ACCLIMATION
- Acclimation period: 14-day period immediately before the definitive test.
- Acclimation conditions: Same as definitive
- Type and amount of food: The fish were fed daily with commercial fish food (TROUW (UK) Ltd; Nutra Fry 02) an amount equivalent to between 0.8 and 1.3% of the total wet-weight of fish in the holding tank.
- Feeding frequency: No food was given during the 24-hour period immediately before exposure or during the exposure period itself.
- Health during acclimation (any mortality observed): Mortalities were recorded as <1% in the 14 days before the definitive test.

QUARANTINE (wild caught)
- Duration: Not applicable
- Health/mortality: Not applicable

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
None

Test conditions

Hardness:
172 (mg/L as CaCO3)
Test temperature:
The temperature ranged from 13.8-13.9 deg C at time 0 and 13.1-15.2 at time 96 h.
pH:
The pH ranged from 7.80-7.88 at time 0 and 7.36-8.16 at time 96 h
Dissolved oxygen:
The DO ranged from 92-98% of air saturation at time 0 and 88-104% at time 96 h
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal=100 mg/L
Mean measured=97 mg/L (The measured concentration of VRT-753136 hcl ranged between 94 and 104% of its nominal value in samples of freshly prepared and expired (24 hours old) medium, giving an overall mean measured level of 97 mg/L)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: Open
- Material, size, headspace, fill volume: Glass aquarium/25 litres of medium to a depth of 16.0 cm
- Aeration: Supplementary aeration was provided via narrow bore glass tubes.
- Type of flow-through (e.g. peristaltic or proportional diluter): Semi-static
- Renewal rate of test solution (frequency/flow rate): Daily renewal
- No. of organisms per vessel: Ten
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: < 1g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used to hold the fish and for the study was laboratory tap water, dechlorinated and softened by passage through a water purification system (Vivendi Water Systems; formerly Elga). It was passed through a high grade activated carbon filter to remove chlorine and any organic contaminants. A proportion of the supply then passed through a water softener before final reverse osmosis treatment to produce a highly purified water supply. The two grades of dechlorinated water were then remixed to give a supply with the desired water hardness. This water was then held in an intermediate tank where it was equilibrated to the test temperature and gently aerated before being supplied to the holding and test areas.
- Total organic carbon: 0.82 mg/L
- Particulate matter: <3 mg/L
- Metals: Trace
- Pesticides: Organochlorine pesticides (<0.020 g/L), Polychlorinated biphenyls (<0.02 g/L) and Organophosphorus pesticides (<0.0058 g/L)
- Chlorine: (total) 0.05; (free) 0.03 mg/L
- Alkalinity: No data
- Ca/mg ratio: Ca: 59 mg/L/ mg: 22.1 mg/L
- Conductivity: 330 tS/cm
- Culture medium different from test medium: No data
- Intervals of water quality measurement: Daily

OTHER TEST CONDITIONS
- Adjustment of pH: No data
- Photoperiod: A photoperiod of 16 hours light: 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase.
- Light intensity: No data

EFFECT PARAMETERS MEASURED
In addition to observations on mortality at approximately 2, 4, 24, 48, 72 and 96 hours, subjective assessments were also made on the incidence and type of any sub-lethal effects compared with control fish. The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence of response to physical stimulation of the caudal peduncle.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Single concentration limit test
- Justification for using less concentrations than requested by guideline: No data
- Range finding study
- Test concentrations: The range finding test employed nominal concentrations of 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 97 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
97 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- No mortality or treatment-related effects on the fish were exhibited during the study.
- Behavioural abnormalities: no data
- Observations on body length and weight: no data
- Other biological observations: no data
- Mortality of control: no data
- Other adverse effects control: no data
- Abnormal responses: no data
- Any observations that might cause a difference between measured and nominal values: no data
- Effect concentrations exceeding solubility of substance in test medium: no data


Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The “no observed effect concentration” (NOEC) was derived by direct inspection of the data for lethal and treatment-related-effects. An incidence rate of more than one affected fish per group is considered to be significant.

Any other information on results incl. tables

Not applicable

Applicant's summary and conclusion

Conclusions:
The 96-hour LC50 value for VRT-753136 hcl with rainbow trout could not be calculated but must be >97 mg/L.
The “no observed effect concentration” was 97 mg/L.
Executive summary:

Not applicable