Registration Dossier

Administrative data

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-10-07 till 2006-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
This study was conducted in compliance with the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted November 26th, 1997 by decision of the OECD Council

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Unlabeled test item:
- Name of test material (as cited in study report): MeNigu
- Molecular formula (if other than submission substance): C2H6N4O2
- Molecular weight (if other than submission substance): 118.1 g/mol
- Substance type: intermediate
- Physical state: solid, white powder
- Analytical purity: 99.7%
- Impurities (identity and concentrations): water 0.2%, nitroguanidine 0.1%
- Purity test date: no data
- Lot/batch No.: 9077154
- Expiration date of the lot/batch: June 01, 2007
- Storage condition of test material: at room temperature in the dark

Labeled test item:
- Name: [14C]-MeNigu, N-Methyl-N’-nitroguanidine [14C]
- Lot/batch no.: 050923 (RCC substance No.: 170172/A)
- Radiochemical purity (if radiolabelling): [14C]-MeNigu, N-Methyl-N’-nitroguanidine [14C], radiochemical purity > 99 % as analysed on September
23, 2005 by American Radiolabelled Chemicals Inc.
- Specific activity (if radiolabelling): 55 mCi/mmol (466.1 µCi/mg or 17.25 MBq/mg)
- Locations of the label (if radiolabelling): at the C-atom of nitroguanidine component of the molecule
- Expiration date of radiochemical substance (if radiolabelling): The radiochemical purity was determined before use. Therefore no expiry date was
needed.
- Stability under test conditions: The radiochemical purity of [14C] MeNigu in the application solution at room temperature was determined by HPLC and TLC analyses. The initial radiochemical purity of the test item was 94.4% and after 7 days at room temperature 94.3% . The purities for the low
dose and the high dose administration solutions were determined and found to be 94.3% and 94.3% . An aliquot of the test item solutions used for
the initial purity test was analysed by TLC after 19 weeks storage at –20 °C. The purity was found to be 95.5% in SS1. These results proved that the
test item was sufficiently stable in the application solution (purified water) at test conditions.
- Storage condition of radioactive labelled test item: in the dark at -20°C.
- Other:

Test solutions

Vehicle:
yes
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method:
Stock solution 1
From the provided radiolabelled test item (solution in ethanol) 2.4 ml of the available material was transferred into a 50 mL volumetric flask. The
volume was made up to 50 mL with purified water. By liquid scintillation counting the amount was determined to be 104.06 MBq corresponding
6.03 mg (spec. activity: 17.25 MBq/mg).

Stock solution low dose:
Based on the needed amount of about 595 mg [14C] MeNigu at 75.48 kBq/mg and a target concentration of 595 mg/2000 mL purified water, to
592.4 mg unlabelled test item 21.2 mL of stock solution 1 (approximately 2.60 mg) were added. The volume was filled up to 2000 mL with purified
water. By liquid scintillation counting the amount was determined to be 44.91 MBq corresponding to 595 mg (specific activity 75.4769 kBq/mg). The
final concentration in this stock solution was 0.2975 mg/mL.

Stock solution high dose:
Based on the needed amount of about 5950 mg [14C] MeNigu at 7.53 kBq/mg and a target concentration of 5950 mg/2000 mL purified water, to
5947.5 mg unlabelled test item 21.2 mL of stock solution 1 (approximately 2.6 mg) were added. The volume was filled up to 2000 mL with purified
water. By liquid scintillation counting the amount was determined to be 44.82 MBq corresponding to 5950.1 mg (specific activity 7.5329 kBq/mg).
The final concentration in this stock solution was 2.975 mg/mL.

Preparation of Application Solutions
Aliquoted portions of the respective stock solution were diluted in purified water in order to reach test item concentrations of 0.25 and 2.5 mg/ml,
for high dose and low dose experiment, respectively. Of these application solutions a volume 0f 100 ml was to be dosed to 250 l of tank water per
day.


- Controls: fish tanks receiving an identical amount of solvent without test item
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not observations reported

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Strain: Oncorhynchus mykiss
- Source: Forellenzucht Hohler, Zeiningen, Switzerland
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (lenght definition, mean, range and SD): no data
- Weight at study initiation (mean and range, SD): 1-2 g (mean 1.3 g)
- Weight at termination (mean and range, SD): no data
- Method of breeding: no data
- Health status: After receipt, the health status of the fish was evaluated in an expert statement

- Description of housing/holding area: The flow-through system with tap water was used in the bioconcentration test and comprises three aerated
and temperature controlled tanks each containing 75 L.
- Feeding during test
- Food type: H.U. Hofmann AG, HOKOVIT, “Forellenfutter”, diet of known lipid and total protein content
- Amount: based on about 2% of the average fish body weight, taking into account increasing body weights and the decreasing number of fish per
sampling interval.
- Frequency: daily


ACCLIMATION
- Acclimation period: fish were allowed to acclimatize to the laboratory environment in tap water for at least two weeks
- Acclimation conditions (same as test or not): same as test
- Food type: H.U. Hofmann AG, HOKOVIT, “Forellenfutter”, diet of known lipid and total protein content
- Amount: based on about 2% of the average fish body weight, taking into account increasing body weights and the decreasing number of fish per
sampling interval.
- Frequency: daily
- Health during acclimation (any mortality observed): no observations reported

Study design

Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
ca. 14 d
Total depuration duration:
ca. 14 d

Test conditions

Hardness:
1.8 mmol/L
Test temperature:
13.2-14.5 °C
pH:
7.9-8.2
Dissolved oxygen:
9.1-10.6 mg/L
TOC:
The TOC value of untreated tap water was about 2.5 mg/l. TOC values during pre equilibrium and accumulation period ranged from 2.03-7.91 mg/L
for the two [14C]-tanks. The corresponding control tank values were 1.86-3.24 mg/L. During depuration, TOC values ranged from 1.56-4.69 mg/L
for the two [14C]-tanks.
Salinity:
Conductivity 735 µS/cm
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass tank, 75 l
- Aeration: stirring
- Type of flow-through: proportional diluter
- Renewal rate of test solution (frequency/flow rate): renewal rate of tank volume 3.3 times per day / flowrate 250 l per day
- No. of organisms per vessel: control 45, high dose and low dose experiment 80 fish each
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: control 0.2, high dose and low dose experiment 0.4


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Itingen tap water
- Particulate matter: not applicable
- Pesticides: contaminat assay of Itingen drinking water is supplied in the study report
- Chlorine: non chlorinated well water of drinking water quality
- Alkalinity: no data
- Ca/Mg ratio: 12:1 (w/w)
- Conductance: 735 µS/cm
- Holding medium different from test medium: no
- Intervals of water quality measurement:
- Intervals of test medium replacement: intervals of renewal of application solution every 4 to 5 days (flowrate 100 ml per day)


OTHER TEST CONDITIONS
- Adjustment of pH: not applicable
- Photoperiod: 16 hours light
- Light intensity: intensity at light period approximately 300-400 Lux


RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: no range finding study reported
- Results used to determine the conditions for the definitive study:
Nominal and measured concentrations:
Nominal test item concentrations 0.1 and 1.0 mg/l, respectively
Measured data:
0.1 mg/l, n=15, SD=0.01 (6.9 %)
1.0 mg/l, n=15, SD=0.03 (3.3 %)
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Measured/calculated logPow: The bioconcentration factors (BCF) of fish were calculated as average of total radioactivity in parent equivalents in the fish in µg eq/kg during exposure (Cf) related to the concentration of total radioactivity in the water (Cw, mg/L).
- Results from toxicokinetic study: no data
- Results from residue study: Average residual radioactivity in whole fish during exposure to [14C] labelled MeNigu at two dose levels during 14 days and during following depuration for 14 days are presented in the study report. The residues were expressed in µg parent equivalents/g according
to the total radioactivity found in whole fish and their fresh weights.
- Monitoring data: no data


BASIS FOR CALCULATION OF BCF

- Estimation software:
- Bioconcentration: Due to the low BCF values, no relevant accumulation kinetics were possible. The bioconcentration factors (BCF) of fish were
calculated as average of total radioactivity in parent equivalents in the fish in µg eq/kg during exposure (Cf) related to the concentration of total
radioactivity in the water (Cw, mg/L).
Levels of radioactivity in fish and in water were expressed as [14C]-MeNigu equivalents on a fresh weight basis based on the following equation:

ppb = µg eq/kg or l = (A x E) / (C x D x E) or ppm = µg eq/g or ml

where is:
A = dpm of the sample
B = Fresh weight of the sample in g or mL
C = Specific radioactivity in kBq/mg
D = Conversion factor: 60000 dpm/kBq
E = Conversion factor from mg into ng or µg: 106 (ppb) of to 103 (ppm)


- Depuration kinetics: Due to the negligible accumulation of the test item, the plateau level was determined as average parent equivalents concentration in fish during the accumulation period. Depuration curves of radioactivity in fish are described by first-order kinetics by the non-linear
parameter estimation program ORIGINTM (MicroCal Software Inc., Northampton, MA 01060, U.S.A.). R-square values were calculated to characterize the goodness of fit.
Cwf = C0 x e^(-kdep x t)
where is:
Cwf = Concentration of radioactivity in µg/g fish at depuration time point t
C0 = Concentration of radioactivity in µg/g fish at depuration time point t = 0
kdep = Depuration rate constant in day- based on depuration data
t = Depuration time points in days
The t0.5 (half-life) was calculated as follows:
t0.5 = -ln(0.5) / Kdep

- Result based on measured log Pow of: measured log Pow of test item -0.83
- Result based on calculated log Pow of:

Results and discussion

Lipid contentopen allclose all
Lipid content:
ca. 27 200 mg/kg bw w.w.
Time point:
other: day 4
Lipid content:
ca. 36 400 mg/kg bw w.w.
Time point:
other: day 14
Bioaccumulation factoropen allclose all
Type:
BCF
Value:
ca. 26
Basis:
total lipid content
Time of plateau:
4 d
Calculation basis:
steady state
Remarks on result:
other: Due to the low BCF values, no accumulation kinetics possible. BCF values based on lipid content (BCFlipid) were determined based on parent equivalents in lipid (µg eq/kg lipid; Cflipid) and the concentration in water (Cw, mg/L). BCF(lipid) = Cf(lipid)/Cw
Remarks:
Conc.in environment / dose:0.1 mg/l
Type:
BCF
Value:
ca. 28
Basis:
total lipid content
Time of plateau:
4 d
Calculation basis:
steady state
Remarks on result:
other: Due to the low BCF values, no accumulation kinetics possible. BCF values based on lipid content (BCFlipid) were determined based on parent equivalents in lipid (µg eq/kg lipid; Cflipid) and the concentration in water (Cw, mg/L). BCF(lipid) = Cf(lipid)/Cw
Remarks:
Conc.in environment / dose:1.0 mg/l
Type:
BCF
Value:
ca. 0.9
Basis:
whole body w.w.
Time of plateau:
4 d
Calculation basis:
steady state
Remarks on result:
other: The bioconcentration factors (BCF) of fish were calculated as average of total radioactivity in parent equivalents in the fish in µg eq/kg during exposure (Cf) related to the concentration of total radioactivity in the water (Cw, mg/L); BCF = Cf/Cw
Remarks:
Conc.in environment / dose:0.1 mg/l
Type:
BCF
Value:
ca. 0.9
Basis:
whole body w.w.
Time of plateau:
4 d
Calculation basis:
steady state
Remarks on result:
other: The bioconcentration factors (BCF) of fish were calculated as average of total radioactivity in parent equivalents in the fish in µg eq/kg during exposure (Cf) related to the concentration of total radioactivity in the water (Cw, mg/L); BCF = Cf/Cw
Remarks:
Conc.in environment / dose:1.0 mg/l
Depurationopen allclose all
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
12.3 d
Elimination:
yes
Parameter:
DT50
Depuration time (DT):
15.9 d
Details on kinetic parameters:
- Uptake rate constant (k1): Due to the rapid but negligible accumulation of the test item, the plateau level was determined as average parent
equivalents concentration in fish during the accumulation period.
- Depuration (loss) rate constant (k2): Depuration curves of radioactivity in fish are described by first-order kinetics
- Indication of bi- or multiphasic kinetics: none reported
- Computation / data analysis: Depuration curves of radioactivity in fish are described by first-order kinetics by the non-linear parameter estimation program ORIGINTM (MicroCal Software Inc., Northampton, MA 01060, U.S.A.). R-square values were calculated to characterize the goodness of fit.
Metabolites:
For both dose levels during the whole uptake period, exclusively parent item was detected, i.e. no metabolites occurred.
Results with reference substance (positive control):
not applicable
Details on results:
- Mortality of test organisms: no mortalities occurred
- Behavioural abnormalities: none
- Observations on body length and weight: none
- Other biological observations: none
- Organ specific bioaccumulation: not investigated
- Bound residues forming a plateau: The residues were expressed in µg parent equivalents/g according to the total radioactivity found in whole fish and their fresh weights.
- Mortality and/or behavioural abnormalities of control: none
- Loss of test substance during test period: no
- Results with vehicle control: valid
Reported statistics:
Depuration curves of radioactivity in fish are described by first-order kinetics by the non-linear parameter estimation program ORIGINTM
(MicroCal Software Inc., Northampton, MA 01060, U.S.A.). R-square values were calculated to characterize the goodness of fit.

Any other information on results incl. tables

Table 3:      Concentration of radioactivity in the whole fish during the test at the average dose levels of 0.1 mg/L and 1 mg/L.

Phase

Time interval

Concentration of radioactivity in fish (Cf)

Concentration of radioactivity in fish (Cf)

I

II

Low dose

High dose

µg eq/g

%*

µg eq/g

%*

A

4

---

0.0812

---

0.899

---

A

7

---

0.1016

---

0.996

---

A

10

---

0.0950

---

0.993

---

A

12

---

0.0869

---

0.866

---

A

14

---

0.0858

---

0.860

---

Mean

0.0901

---

0.923

---

± SD

% SD

0.0081

(9.0 %)

---

---

0.067

(7.3 %)

---

---

D

17

3

0.0696

77

0.835

91

D

20

6

0.0571

63

0.631

68

D

24

10

0.0469

52

0.526

57

D

28

14

0.0409

45

0.517

56

*:    Residual radioactivity in fish in percent of the average concentration in fish during exposure.

I:        Days after the onset of accumulation

II:       Days after the onset of depuration

A:      Accumulation

D:      Depuration

SD:    Standard deviation

Table 4:      Bioconcentration factors (BCF) in fish during the test at the dose levels of 0.1 mg/L and of 1 mg/L.

Phase

Time interval

BCF**

I

Low dose

High dose

A

4

0.8

0.9

A

7

1.0

1.0

A

10

0.9

1.0

A

12

0.9

0.9

A

14

0.9

0.9

Mean

0.9

0.9

 

± SD

% SD

0.1

(10.0 %)

0.1

(10.0 %)

 

**  Cf / Cw

I:        Days after the onset of accumulation

A:      Accumulation

SD:    Standard deviation


Table 5:      Levels in whole untreated fish during incubation (values given in parent equivalents on a fresh weight basis, i.e. µg eq/g).

Time

Whole fish

interval (days)

µg eq/g

Low dose

High dose

4

0.0364

0.3651

14

0.0293

0.2932

28

0.0317

0.3180

Mean

0.0325

0.3254

SD

0.0036

0.0365

SD:    Standard deviation


Table 6:      Bioconcentration factors calculated for the low dose level of 0.1 mg/L (A) and the high dose level of 1 mg/L (B) based on lipid content in control fish as determined on days 4 and 14.

A)

 Lipid/g fish

Cf

Cflipid

Cw

BCF*

(mg)

(µg eq/kg fish)

(µg eq/kg lipid)

(mg/L)

(lipid)

Day 4, whole fish

27.7

81.2

2'926

0.1

29

Day 14, whole fish

36.4

85.8

2'356

0.1

24

Mean BCF

 

 

 

 

26

B)

 Lipid/g fish

Cf

Cflipid

Cw

BCF*

(mg)

(µg eq/kg fish)

(µg eq/kg lipid)

(mg/L)

(lipid)

Day 4, whole fish

27.7

898.8

32'393

1

32

Day 14, whole fish

36.4

860.1

23'621

1

24

Mean BCF

 

 

 

 

28

          *  BCFlipid = Cflipid / Cw

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The present study in rainbow trout proved lack of bioconcentration of MeNigu at exposure concentrations of 0.1 mg eq/L (low dose) and
1 mg eq/L (high dose). BCF values of approximately 0.9 were calculated for the two dose levels.
Depuration half-life calculations were performed based on the Cf values from day 14 to day 28. For the low dose, a depuration half-life of 12.3 days was calculated. For the high dose a slightly longer half life of 15.9 days was obtained.
For both dose levels, the parent item (W1) was detected in all samples analysed. The average amounts of W1 were 96.3 ± 3.5% (low dose) and 98.4 ± 2.3% (high dose).
Executive summary:

The bioconcentration and depuration of MeNigu in rainbow trout was investigated by means of a dynamic flow through system according to OECD Guideline 305. The bioconcentration in whole fish was calculated.

The fish were continuously exposed to [14C]-MeNigu at an average low dose concentration of 0.1 mg eq/L and an average high dose concentration of 1 mg eq/L for 14 days. After the exposure, the fish were transferred to flowing untreated water and the depuration of radioactivity was followed for 14 days. The accumulation of MeNigu in fish at both dose levels was extremely low. The plateau levels could be determined as average concentration level in the fish, i.e. 0.0901 µg eq/g for the low dose and 0.923 µg eq/g for the high dose. Depuration half-life calculations were performed based on the Cf value on day 14 and the values measured during 14 days of depuration. For the low dose, a depuration half-life of 12.3 days was calculated (correlation r ² : 0. 9838). For the high dose a slightly longer half-life of 15.9 days was obtained (correlation r ²: 0.9094).

For the low dose, the residual radioactivity in fish during exposure amounted on average to 0.0901 ± 0.0081 µg eq/g. The residual radioactivity during depuration decreased from 0.0696 on day 17 to 0.0409 µg eq/g on day 28. For the high dose, the residual radioactivity in fish during exposure amounted on average to 0.923 ± 0.067 µg eq/g. The residual radioactivity during depuration decreased from 0.835 on day 17 to 0.517 µg eq/g on day 28.

For the low dose, based on the radioactivity levels in fish after exposure to [14C]-labelled MeNigu at an average dose level of 0.1 mg eq/L, the average bioconcentration factor (BCF) amounted to 0.9 ± 0.1.

For the high dose, based on the radioactivity levels in fish after exposure to [14C]-labelled MeNigu at an average dose level of 1 mg eq/L, the average bioconcentration factor (BCF) amounted to 0.9 ± 0.1.

BCF(lipid) based on the lipid content in representative fish amounted to 26 and 28 for the low dose and the high dose, resp.

For both dose levels, the parent item was detected in all samples analysed. The average amounts of W+ were 96.3±3.5% (low dose) and 98.4±2.3% 8high dose).

In conclusion, an extremely low accumulation of MeNigu in rainbow trout was detected, resulting in BCF values of approximately 0.9.