Registration Dossier

Administrative data

bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 28 August 2008 and 6 January 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted to recognised testing methodology to GLP

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: Yakushokuhatsu No. 1121002 on November 21, 2003, Heisei 15.11.17 Seikyoku No. 2, Kanpokihatsu No. 031121002, final revision: November 20, 2006
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
- Name of test material (as cited in study report): JKY-214
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other tha n submission substance): Not applicable
- Substance type: Organic
- Physical state: White crystal
- Analytical purity: 99.5% w/w
- Impurities (identity and concentrations): Not specified
- Composition of test material, percentage of components:
- Isomers composition: Not applicable
- Purity test date: Not specified
- Lot/batch No.: RM05-58
- Expiration date of the lot/batch: Not specified
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Stable. After the completion of the study, infrared absorption spectrum of the test substance stored in a desiccator for test material storage. The spectrum obtained was consistent with that measured before the initiation of the study. Therefore it was determined that the test substance was stable during storage
- Storage condition of test material: Room temperature, dark, tightly sealed
- Other: Not specified

Sampling and analysis

Details on sampling:
- Sampling intervals/frequency for test organisms: On Days 4, 7, 14, 21, 42, and 61 after start of the exposure
- Sampling intervals/frequency for test medium samples: Before the exposure (Day 0) and Days 4, 7, 14, 21, 28, 35, 42, 48, and 61 after start of the exposure
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
- Two fish (One fish in the excretion test) Weight determination (about 12 g)
- Cut into pieces with scissors
- Homogenize (for about 1 min. at about 8000 rpm) × 4
- Collect 2.5 g of the pulverized sample extracted with Acetonitrile 20 mL
- Homogenize (for 5 min. at about 8000 rpm) and acetonitrile phase collected
Aspiration • filtration (Kiriyama funnel 40TYPE filter paper)
- Residue extracted with Acetonitrile 20 mL, Homogenized (for 3 min. at about 8000 rpm), Aspiration • filtration (Kiriyama funnel 40TYPE filter paper). Residue discarded. The acetonitrile added to the acetonitrile phase
- Combined acetonitrile phase made up to 50 ml
- 10 ml aliquot passed through Sep-Pak C8 (Conditioned with about 5 mL of acetonitrile) and added to To 0.5 mL of 0.1% (v/v) formic acid aqueous solution, the equivalent amount of passing solution and mixed well
- Analysis conducted by HPLC analysis.

Test solutions

Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 2g of test sample dissolved in vehicle and made up to 1000 ml to prepare 2000 mg/ml sample. Then further diluted in vehicle to prepare 1000 mg/ml sample
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): 2-methoxyethanol
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none reported

Test organisms

Test organisms (species):
Cyprinus carpio
Details on test organisms:
Species: Carp (Cyprinus carpio)
Source: Niikura Fish Farm (1217, Shimoya, Isehara-city, Kanagawa, Japan)

Date of purchase: January 15, 2008
Lot No.: 08-K-0115
Preacclimatization: At the start of acclimatization, macroscopic observation was made to remove the fish with any abnormalities. The remaining normal fish were reared in an aquarium with continuous flow water. No medical dipping was conducted during the acclimatization period.
Body length: 8 ± 4 cm
Body weight: about 6 g
Age: Within 1 year old after hatching

At the time of purchasing, the fish with any diseases, the debilitated fish or the fish with any other abnormalities were removed. The remaining normal fish were reared in an aquarium with continuous flow water. If necessary, medical dipping was made whereas such fish were not employed for the test for 2 weeks after medical dipping.

Aquarium number: A-13
Water temperature: 24 ± 2ºC
Period: July 9, 2008 - September 4, 2008
Feed: Babygold from Kyorin
Feeding: The food equivalent to about 2% of body weight was given once a day.
Mortality during 1 week before the test: < 5%

Study design

Route of exposure:
Test type:
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
61 d
Total depuration duration:
63 d

Test conditions

not reported
Test temperature:
24 ± 2ºC
6.0 - 8.5
Dissolved oxygen:
more than 60% of the saturated dissolved oxygen concentration (more than 5 mg/L in case of 24ºC)
not reported
not reported
Details on test conditions:
- Test vessel: glass vessel
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 800 L
- Aeration: yes
- Renewal rate of test solution (frequency/flow rate): 40 mL/day
-First concentration area: 32 fish/50 L of test water
(more than 1 L/day per 1.0 g)
-Second concentration area: 32 fish/50 L of test water
(more than 1 L/day per 1.0 g)
-Control area: 12 fish/50 L of test water
(more than 1 L/day per 1.0 g)

- Photoperiod: Approximately 16 hr/day, Hf fluorescent lamp (wave length 400 - 700 nm)
- Light intensity:

- Test concentrations:
- Results used to determine the conditions for the definitive study:
Nominal and measured concentrations:
2000 and 200 mg/l
Reference substance (positive control):

Results and discussion

Lipid contentopen allclose all
Lipid content:
6.2 %
Time point:
start of exposure
Lipid content:
6.4 %
Time point:
end of exposure
Bioaccumulation factor
<= 41 dimensionless
whole body w.w.
Time of plateau:
24.8 d
Calculation basis:
steady state
Details of metabolites are included in the attached sheet.
Results with reference substance (positive control):
Not applicable

Any other information on results incl. tables

96hr-LC50of the test substance inOryzias latipeswas >10 mg/L.

The test substance concentration mg/L

Accumulated mortality (%)

24 hr

48 hr

72 hr

96 hr













Based on the above findings, it was confirmed that the test concentration of the bioaccumulation tests was not more than 1/100 and 1/1000 of 96hr-LC50,respectively.

Applicant's summary and conclusion

Validity criteria fulfilled:
The test substance is considered to be not bioaccumulative in fish.
Executive summary:

The bioaccumulation of the substance has been assessed by continuous flow-through exposure of the substance to common carp over a period of 61 days with a 63 day depuration period. The BCF was calculated to be <=41 and is therefore considered to be not bioaccumulative.