6-amino-1,3-dimethyluracil

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-04-24 - 2002-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

EU Commission Directive 92/69/EEC, Part C.1: Acute Toxicity for Fish, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature at about 20 °C, away from
direct sunlight
- Stability under test conditions: Stability in water: Max. 90 days

OTHER SPECIFICS:
Solubility in water: 5.5 g/L
pH in aqueous solution: 7

Analytical monitoring:

yes

Details on sampling:

For the analyses of the test item concentration in this static test, duplicate samples were taken from the freshly prepared test medium of the single test concentration and the control just before the start of the test.
To confirm the maintenance of the test item concentration throughout the test duration, duplicate samples from the test medium of the single test concentration and the control were taken after 48 hours and at the end of the test (after 96 hours).
All samples were taken from the approximate center of the aquaria without mixing of the test medium, and were deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (without GLP) the test item is stable in the test water under the storage conditions.
The concentration of the test item was analyzed in both test medium samples from the sampling times at 0 and 96 hours. From the control samples only one of the duplicate samples was analyzed from the corresponding sampling times.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium was prepared by dissolving 500 mg completely in 5 liter test water by ultrasonic treatment for 10 minutes and stirring for 1 hour at room temperature. The test medium was freshly prepared just before introduction of the fish (= start of the test).
- Controls: yes, negative control (test water only)
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): not stated

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebra fish
- Strain: Brachydanio rerio
- Source: Zoohaus Schaub, CH-4410 Liestal, Switzerland
- Length at study initiation (length definition, mean, range and SD): 2.9 ± 0.2 cm
- Weight at study initiation (mean and range, SD): 0.17 ±0.03 g

ACCLIMATION
- Acclimation period: In accordance with the test guidelines the fish were held in the laboratories for more than three weeks without any medication. Prior to test start, they were acclimated for one week to the test water and temperature.
- Type and amount of food during acclimation / Feeding frequency during acclimation: During holding and acclimatization until one day before the start of the test the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, D-49304 Melle, Germany).
- Health during acclimation (any mortality observed): During holding and acclimatization no fish died in the test fish batch and all fish were healthy.

FEEDING DURING TEST
The fish were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

none

Hardness:

2.5 mmol/l (=250 mg/l) as CaCO3

Test temperature:

21 - 22 °C during the test period

pH:

7.8 - 8.0

Dissolved oxygen:

8.2 - 8.7 mg/l
The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached.
The test medium and the control were slightly aerated during the test period.

Salinity:

Reconstituted water: analytical grade salts were dissolved in deionized water to obtain the following nominal concentrations:
CaCI2 x 2H2O: 2.0 mmol/L (= 294 mg/L)
MgSO4 x 7H2O: 0.5 mmol/L (= 123 mg/L)
NaHCO3: 0.75 mmol/L (= 65 mg/L)
KCI: 0.075 mmol/L (= 5.8 mg/L)

Nominal and measured concentrations:

0, 100 mg/l (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquarium with five liters test medium. At the start of the test 7 fish were introduced into each aquarium in a random order.
- Aeration: The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached. The test medium and the control were slightly aerated during the test period.
- Renewal rate of test solution (frequency/flow rate): None. A static test without test medium renewal was chosen since in a pre-experiment (without GLP) the test item concentration in the test medium was constant during the test period of 96 hours.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: The loading rate was 0.24 g fish wet weight per liter test medium.

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: The water temperature, pH-values and oxygen concentrations were measured in the single test concentration and the control at the start of the test and once every day during the test. At the same dates the appearance of the test medium was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: A 16-hour light to 8-hour darkness photoperiod, with a 30-minute transition period
- Light intensity: light intensity at light period approximately within the range of 50 to 500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed after approximately 3, 24, 48, 72 and 96 hours test duration for mortality and visible abnormalities.

TEST CONCENTRATIONS
A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test fish up to the test item concentration of 100 mg/l.Thus, the only test item concentration tested was nominal 100 mg/l.Additionally, a control was tested in parallel (test water without addition of the test item).

Reference substance (positive control):

not required

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortality and visible abnormalities

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

- Behavioural abnormalities: In the control and in the test medium of 100 mg/l all fish survived until the end of the test and no visible abnormalities were observed at the test fish.
- Mortality of control: none
- Other adverse effects control: none stated
- Abnormal responses: none stated
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test medium. The test medium was a clear solution throughout the entire test duration.
- Effect concentrations exceeding solubility of substance in test medium: none

Results with reference substance (positive control):

n/a

Reported statistics and error estimates:

could not be performed due to the absence of toxicity

Validity criteria fulfilled:

yes

Conclusions:

The test was performed scientifically reasonable according to OECD 203 under GLP, the report is well documented and the validity criteria are met. Hence, the results can be considered as sufficiently reliable to assess the toxicity of 1,3-Dimethyl-4-aminouracil to zebra fish.
In the control and in the test medium of 100 mg/l all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), and the 96-hour LC0 of 1,3-Dimethyl-4-aminouracil to zebra fish were determined to be at least 100 mg/L. The 96-hour NOEC and the 96-hour LC0 might even be higher than this concentration. The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 were clearly higher than 100 mg/l. These values could not be quantified due to the absence of toxicity of 1,3-DimethyI-4-aminouracil at the tested concentration.
With an LC50 > 100 mg/l, 1,3-Dimethyl-4-aminouracil does not need to be classified as hazardous to the environment acc. Regulation 1272/2008, neither acute nor chronic.

Executive summary:

The acute toxicity of the test item 1,3-Dimethyl-4-aminouracil to zebra fish (Brachydanio rerio) was determined in a 96-hour static test according to the EU Commission Directive 92/69/EEC, Part C.1 (1992), and the OECD Guideline for Testing of Chemicals No. 203, (1992), under GLP.

A limit test was performed in accordance with the guidelines to demonstrate that the test item has no toxic effect on the test organisms up to and including the concentration of nominal 100 mg/l.Thus the only concentration tested was nominal 100 mg/L and a control.

The analytically determined test item concentration in the test medium was 102 and 110% of the nominal value at the start and the end of the test. The test item 1,3-Dimethyl-4-aminouracil was stable under the test conditions during the test period of 96 hours. Therefore, all reported biological results are related to the nominal concentration of the test item.

In the control and in the test medium of 100 mg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours), and the 96-hour LC0 of 1,3-Dimethyl-4-aminouracil to zebra fish were determined to be at least 100 mg/L. The 96-hour NOEC and the 96-hour LC0 might even be higher than this concentration, but concentrations in excess of 100 mg/l have not been tested, according to the guidelines. The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 were clearly higher than 100 mg/l.These values could not be quantified due to the absence of toxicity of 1,3-Dimethyl-4-aminouracil at the tested concentration.

1,3-Dimethyl-4-aminouracil does not need to be classified as hazardous to the environment acc. Regulation 1272/2008, neither acute nor chronic.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Scientifically acceptable

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1.0009 g of the test substance was weighed into a beaker. The test vessel was then filled with 10 liters of test water. An aliquot was taken from the test water in the test vessel and mixed with the test substance in the beaker using an ultra turrax. Subsequently, the mixture was poured into the test vessel and the whole test batch was homogenized again with an ultra turrax.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Source: Zierfisch-Center Kloeckner, Ludwigshafen, Germany
- Age at study initiation (mean and range, SD): approx. 3 months
- Length at study initiation (length definition, mean, range and SD): 3.3 cm (range 3.0 - 3.7 cm)
- Weight at study initiation (mean and range, SD): 0.38 g (range 0.20 - 0.81 g)
- Duration of acclimation: about 14 days Mortality during the last week of acclimation: 0 % Medical treatment: none during acclimatization
- Feeding during test
- Food type:
- Amount:
- Frequency:


ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):


QUARANTINE (wild caught)
- Duration:
- Health/mortality:

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Hardness:

2.5 mmol/l

Test temperature:

22 - 23 °C

pH:

8.0 - 8.3

Dissolved oxygen:

8.0 - 8.5 mg/l

Nominal and measured concentrations:

Nominal test concentration: 100.0 mg/l, control
The analytically determined concentrations of the test substance in the test water were in a range of +/- 20 % of the nominal concentration in all tested concentrations, therefore all biological results refer to the nominal test concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass aquaria (30 x 22 x 24 cm)
- Type (delete if not applicable): open / closed
- Fill volume: 10 l
- Aeration: slight
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.4 g fish/l test water


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Non chlorinated charcoal filtered tap water (Frankenthal, Germany), aerated Dilution water chemistry:Ca content: Approx. 80 mg/l, Mg content: Approx. 10 mg/l, pH-value: 7.5 - 8.5
- Conductivity: Approx. 550 µS/cm (at 25 °C),
- Intervals of water quality measurement: temperature, pH and oxygen content after 0, 24, 48, 72 and 96 h


OTHER TEST CONDITIONS
- Photoperiod: 16 : 8 hours day-night regime


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality and symptoms of intoxication after 1, 4, 24, 48, 72 and 96 h

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Key result

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Reported statistics and error estimates:

The LC50 was determined directly from the raw data.

Sublethal observations / clinical signs:

During the 96 h exposure to 1,3-Dimethyl-4-aminouracil, mortalities were as follows:

Nominal test / Cumulative mortality (%) after x hours
concentration / 1 / 4 / 24 / 48 / 72 / 96 /
(mg/l)
Control / 0 / 0 / 0 / 0 / 0 / 0 /
100.0 / 0 / 0 / 0 / 0 / 0 / 0 /

Symptoms of intoxication:

Nominal test / Symptoms of intoxication after x hours
concentration / 1 / 4 / 24 / 48 / 72 / 96 /
(mg/l)
Control / - / - / - / - / - / - /
100.0 / - / - / - / - / - / - /

Definitions of Symptoms:
-: No symptoms

Description of key information

Short-term toxicity to fish:

- LC50 > 100 mg/l, LC0 ≥ 100 mg/l, NOEC ≥ 100 mg/l for Danio rerio over 96h (OECD 203, static, GLP)

- LC50 > 100 mg/l, LC0 ≥ 100 mg/l, NOEC ≥ 100 mg/l for Danio rerio over 96h (OECD 203, static, GLP)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

According to Regulation 1272/2008, Table 4.1.0, Classification categories for hazardous to the aquatic environment, a substance must be classified as Acute (short-term) aquatic hazard, Acute Category 1, if i.a. the 96 hr LC50 (for fish) is ≤ 1 mg/l. It must be classified as Chronic Category 3 if the 96 hr LC50 (for fish) is > 10 to ≤ 100 mg/l, and the substance is not rapidly degradable and/or the experimentally determined BCF ≥ 500 (or, if absent, the log Kow ≥ 4).In both available studies, the following values were determined: LC50 > 100 mg/l, LC0 ≥ 100 mg/l, NOEC ≥ 100 mg/l for Danio rerio. Hence, the substance does not need to be classified as hazardous to the aquatic environment, neither acute nor chronic.