Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 May to 25 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recent study conducted by a GLP certified laboratory in accordance with a suitable study guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
Not possible to verify the concentration was at least 80% of the nominal during the test as the saturated solution was below the level of analytical detection. This does not affect the validity of the study as there was no toxicity at a saturated solution
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
tris(propan-2-yl)silyl 2-methylprop-2-enoate
EC Number:
700-182-8
Cas Number:
134652-60-1
Molecular formula:
C13 H26 O2 Si
IUPAC Name:
tris(propan-2-yl)silyl 2-methylprop-2-enoate
Details on test material:
- Name of test material (as cited in study report): TIS-M
- Lot/batch No.: 9925
- Expiration date of the lot/batch: 2 December 2010
- Storage condition of test material: Under nitrogen at room temperature (15-25°C), continuously protected from all light.

Sampling and analysis

Analytical monitoring:
yes

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The method is based on the elution of the test substance with the test medium from a column, which is charged with an inert support material coated with an excess of the test substance. For this method glass beads serve as support material and aquarium water is used as eluent. The glass beads with a high surface area to volume ratio allows a relatively large amount of test substance to be coated on the surface and provides a large area over which dissolving of the test substance can take place.

An amount of 500 mg test substance was dissolved in 250 ml of acetone [supplier: REANAL; Lot no.: KBM60548; Exp. Date: January 2012 (the test substance information supplied by the Sponsor showed that acetone is a suitable solvent)]. This solution was added to about 500 g support material. The solvent was completely evaporated using a rotary evaporator. The dry support material was poured into the column then filled with aquarium water. The column was connected to a re-circulating pump within the aquarium. The flow through the column was started approximately 1 hour before the introduction of the fish (to achieve the constant saturated solution) and run over the whole test period. The flow rate of the water pumped through the column filled with test item allowed a theoretical recirculation of the whole tank volume approximately every 6 minutes, so 1 hour represented a recirculation of approximately 10 times.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Brachydanio rerio
- Source: “Black Molly” Fish Farm, Veszprém, Hungary
- Length at study initiation: 2.8 – 3.3 cm
- Feeding during test: The fish were not fed during the test

ACCLIMATION
- Acclimation period: >12 days

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
0 hours - all observations during exposure period.

Test conditions

Hardness:
Water hardness (CaCO3): 234 mg/L
Test temperature:
21.6 – 21.9°C
pH:
8.26 – 8.35
Dissolved oxygen:
91 – 93(%)
Salinity:
No data.
Nominal and measured concentrations:
Only one concentration used: 100 % v/v saturated solution.
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

OTHER TEST CONDITIONS
- Adjustment of pH: The pH was in the range of 6.0 to 8.5.
- Photoperiod: 16 hours light and 8 hours dark.

EFFECT PARAMETERS MEASURED
- Mortality and any sub-lethal effect were determined after 3, 6, 24, 48, 72 and 96 hours.
- The test conditions (pH, temperature, oxygen saturation) were examined daily during the test.
- The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (g fish/litre testing liquid).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Only one concentration used: 100 % v/v saturated solution.
- Test concentrations: One concentration: 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: yes - As no toxic effect was observed in the preliminary range-finding test at saturation, only this concentration (100 % v/v saturated solution) and one control were tested in a limit-test.
Reference substance (positive control):
no
Remarks:
Only an Untreated Control used: The dilution water (circulated and filtered water) was used without of addition of the test item.

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 100 other: % v/v saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Details on results:
- Behavioural abnormalities: no data
- Observations on body weight: There was no considerable difference observed concerning body weights between the groups.
- Other biological observations: none
- Mortality: There were no mortalities in 7 fish in the control group and in 7 fish exposed to a test concentration of 100 % v/v saturated solution.
- Sub-lethal effects: No sub-lethal effects were observed in the control and test group for a period of 96 hours.
- Mortality of control: none.
- Other adverse effects control: none
- Abnormal responses: none.



Reported statistics and error estimates:
No statistical analysis was performed because of the lack of toxic effects. The LC50, NOEC and LOEC were determined directly from the raw data.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Table 1: Cumulative mortality data in the definitive test:

Test Group

Mortality rate of treated fish (Dead fish/treated fish)

3h

6h

24h

48h

72h

96h

Control

0/7

0/7

0/7

0/7

0/7

0/7

100% v/v saturated solution

0/7

0/7

0/7

0/7

0/7

0/7

Table 2: Measured and calculated data of bodyweight:

Test Group

Measured weight of 7 fish (g)

Calculated mean weight of 1 fish (g)

Loading of testing aquarium (g fish/L testing liquid)

Control

2.96

0.42

0.30

100% v/v saturated solution

3.04

0.43

0.30

Table 3: Method validation results:

Selectivity

No interfering component was observed

Reinjection repeatability (7 injections)

Coefficient of Variation < 1.5 %

Linear range

0.2-10ng/ml

Limit of Detection

0.1 ng/ml

Limit of Quantification

0.2 ng/ml

Recovery from aqueous test media

51-62% (1 ng/ml)

Stability of the samples

At least 21 hours in the autosampler

Stock solution stability

At least seven days at 5 ± 3 °C

Stability of the test item in Water

Less than 30 minutes

Table 4: Data of the regression lines at the start and at the end of the study (Calibration)

Date of measurement

Analytical occasion

Constant

X Coefficient

R.Squared

21 May 2010

Start of the study

-4596

34875

0.999

25 May 2010

End of the study

-1332

35806

0.998

Table 5: Measured Concentrations from HPLC Analysis:

Sample Code

Measured concentrations

At the start

mg/L

At the end

mg/L

Control

Not detected.

Not detected.

Test Solution

<0.2

<0.2

<0.2

<0.2

<0.2

<0.2

<0.2

<0.2

<0.2

<0.2

Data of Test Conditions:

Table 6: Temperature (°C)

Test Group

Measuring (Day)

0

1

2

3

4

Control

21.7

21.6

21.8

21.8

21.9

100% v/v saturated solution

21.9

21.6

21.8

21.9

21.8

Table 7: Dissolved oxygen concentration (% of the air saturation value)

Test Group

Measuring (Day)

0

1

2

3

4

Control

91

92

91

93

92

100% v/v saturated solution

92

92

93

92

92

Table 8: pH

Test Group

Measuring (Day)

0

1

2

3

4

Control

8.33

8.30

8.34

8.32

8.35

100% v/v saturated solution

8.30

8.26

8.32

8.32

8.33

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
It was determined that for the registered substance, the 24h, 48h, 72h and 96h LC50 value was > 100% v/v saturated solution
The No-Observed Effect Concentration (NOEC) was determined to be 100% v/v saturated solution and the Lowest Observed Effect Concentration (LOEC) was determined to be > 100% v/v saturated solution. Based on the results of this study, the test item TIS-M had no toxic effect at saturation; the LC50 results and the LOEC are higher than the solubility level of the test item in the test medium.
Executive summary:

The short-term toxicity of the registered substance to Zebrafish (Brachydanio rerio) was assessed according to the EU Method C.1 (Acute Toxicity for Fish). An untreated Control of dilution water was also used. The 24, 48, 72 and 96-hour LC50 values were all > 100% v/v saturated solution and LOEC value was > 100% v/v saturated solution, so it was determined that the test item TIS-M had no toxic effect at saturation.