N-(2-oxo-1,2-dihydropyrimidin-4-yl)benzamide

Short-term toxicity to fish

Administrative data

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 25 November 2012. Experimental Completion Date: 14 December 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

please see details on test solution section

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

yes

Remarks:

please see details on test solutions section

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N4-Benzoylcytosine

EC Number:

628-907-2

Cas Number:

26661-13-2

Molecular formula:

C11H9N3O2

IUPAC Name:

N4-Benzoylcytosine

Specific details on test material used for the study:

Identification: N-Benzoyl Cytosine
Description: off white powder
Batch: NBC-5-11001
Purity: 100%
Expiry Date: not supplied
Storage Conditions: room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and the 100% v/v saturated solution test vessel at
0 and 72 hours (fresh media) and at 24 and 96 hours (old media) for quantitative analysis. The 0,
24 and 72 hour samples were stored at approximately -20 °C prior to analysis with the 96 hour
samples.
Duplicate samples and samples at 24 (fresh media), 48 (old and fresh media) and 72 hours
(old media) were taken and stored at approximately -20 °C for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water
soluble test items, a modification of the standard method for the preparation of aqueous media
was performed. An approach endorsed by several important regulatory authorities in the EU and
elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a saturated solution of
the test item in cases where the test item is of high purity and is poorly soluble in water and in
the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was
prepared by stirring an excess (50 mg/L) of test item in dechlorinated tap water for a period of
24 hours prior to removing any undissolved test item present by filtration (0.2 pm Sartorius
Sartopore filter, first approximate 1 liter discarded in order to pre-condition the filter) to give a
saturated solution of the test item.
Pre-study solubility work conducted indicated that the test item was practically insoluble in
water using traditional methods of preparation e.g. ultrasonication and high shear mixing.
Based on this information the test item was categorized as being a ‘difficult substance’ as
defined by the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances
and Mixtures (OECD 2000). Therefore a media preparation trial was conducted in order to
determine the solubility of the test item under test conditions.

Saturated Solution Preparation
An amount of test item (550 mg) was dispersed, in duplicate, in 11 liters of deionized reverse
osmosis water with the aid of propeller stirring at approximately 1500 rpm for periods of either
24 or 48 hours. After stirring, samples were taken for chemical analysis after the following
pre-treatments:
Centrifugation at 10000 g for 30 minutes
Centrifugation at 40000 g for 30 minutes
Filtration through a 0.2 pm Gelman Acrocap filter (approximately 100 mL discarded in
order to pre-condition the filter)
Filtration through a 0.2 pm Gelman Acrocap filter (approximately 500 mL discarded in
order to pre-condition the filter)

Based the obtained information the test item was prepared using a saturated solution method of
preparation at an initial loading rate of 50 mg/L, stirred via propeller stirrer for 24 hours prior to
the removal of any undissolved test item by filtration through a 0.2 pm Gelman Acrocap filter
(first approximate 100 mL discarded in order to pre-condition the filter), or a 0.2 µm Sartorius
Sartopore filter (first approximate 1 liter discarded).
Although both 1 and 2 liter discard amounts were used in the test, this variation was not thought
to affect the validity of the results obtained as the results of the chemical analyses were
consistent between the different preparation methods.

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

The test was carried out using juvenile rainbow trout ( Oncorhynchus mykiss ). Fish were
obtained from Brow Well Fisheries Limited, Hebden, near Skipton, Yorkshire, UK and
maintained in-house since 17 October 2012. Fish were maintained in a glass fiber tank with a
"single pass" water renewal system .Fish were acclimatized to test conditions from
28 November 2012 to 10 December 2012. The lighting cycle was controlled to give a 16 hours
light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.

The water temperature was controlled at 13 °C to 15 °C with a dissolved oxygen content of
greater than or equal to 10.3 mg O2/L. These parameters were recorded daily. The stock fish
were fed commercial trout pellets which was discontinued approximately 24 hours prior to the
start of the definitive test. There was no mortality in the 7 days prior to the start of the test and
the fish had a mean standard length of 5.1 cm (sd = 0.5) and a mean - weight of 1.59 g (sd = 0.42)
at the end of the definitive test. Based on the mean weight value this gave a loading rate of
0.56 g bodyweight/liter (static volume).

The diet and diluent water are considered not to contain any contaminant that would affect the
integrity and outcome of the study.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

140 mg/L as CaC03

Test temperature:

Temperature was maintained at 13 °C to 15 °C throughout the test.
Measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

7.7 - 8.1
The pH was measured using a Hach HQ30d Flexi handheld meter.
There were no treatment related differences for pH

Dissolved oxygen:

9.7 - 10.0 mg O2/L
The oxygen was measured using a Hach HQ30d Flexi handheld meter.
There were no treatment related differences for oxygen.

Salinity:

not applicable as freshwater study

Nominal and measured concentrations:

Range-finding Test:100% v/v saturated solution
Definitive Test: 100% v/v saturated solution, mean measure test concentration 1.1 mg/L.

Details on test conditions:

Range-finding Test
The test concentration to be used in the definitive test was determined by a preliminary
range-finding test.
Based on the results of an Acute Toxicity to Daphnia magna test
the range-finding test was conducted using a single nominal concentration of
100% v/v saturated solution as no toxicity was expected at this concentration. In the range-finding
test fish were exposed to a single nominal test concentration of
100% v/v saturated solution.
An amount of test item (1125 mg) was dispersed in 22.5 liters of dechlorinated tap water with
the aid of a propeller stirrer at a rate of approximately 1500 rpm for a period of 24 hours. After
stirring, any undissolved test item was removed via filtration using a 0.2 pm Sartorius Sartopore
filter (initial approximate 2 liters discarded to pre-condition the filter) to give the
100% v/v saturated solution test concentration
In the range-finding test 3 fish were added to each 20 liter test and control vessel and maintained
at 14 °C to 15 °C in a temperature controlled room with a photoperiod of 16 hours light and
8 hours darkness with 20 minute dawn and dusk transition periods for a period of 96 hours under
static test conditions.
The control group was maintained under identical conditions but not exposed to the test item.
Data from the control group was shared with similar concurrent studies.
Each vessel was covered to reduce evaporation. After 3, 6, 24, 48, 72 and 96 hours any
mortalities or sub-lethal effects of exposure were determined by visual inspection of the test fish.
A sample of each test concentration was taken for chemical analysis at 0 and 24 hours in order to
determine the stability of the test item under test conditions. All samples were stored at
approximately -20 °C prior to analysis. Only concentrations within the range to be used for the
definitive test were analyzed.

Definitive Test
Based on the results of the range-finding test a "Limit test" was conducted at a nominal
concentration of 100% v/v saturated solution to confirm that at the highest attainable test
concentration, no mortalities or sub-lethal effects of exposure were observed.
Experimental Preparation
An amount of test item (1125 mg) was dispersed in 22.5 liters of dechlorinated tap water with
the aid of a propeller stirrer at a rate of approximately 1500 rpm for a period of 24 hours. After
stirring, any undissolved test item was removed via filtration using a 0.2 pm Sartorius Sartopore
filter (initial approximate 1 liter discarded to pre-condition the filter) to give the 100% v/v
saturated solution test concentration.
Whilst the discarded volume to pre-condition the filter differs from the range-finding test, this
was not thought to impact the validity of the results obtained as the results of the chemical
analyses were consistent between the range-finding and definitive tests.
The concentration and stability of the test item in the test preparations were verified by chemical
analysis at 0 and 72 hours (fresh media) and at 24 and 96 hours (old media).
Exposure Conditions
As in the range-finding test, 20 liter glass exposure vessels were used for each test concentration.
At the start of the test 7 fish were placed in each test vessel at random, in the test preparations.
The test vessels were then covered to reduce evaporation and maintained at 13 °C to 15 °C in a
temperature controlled room with a photoperiod of 16 hours light and 8 hours darkness with
20 minute dawn and dusk transition periods for a period of 96 hours. The test vessels were
aerated via narrow bore glass tubes. The fish were not individually identified and received no
food during exposure.
The control group was maintained under identical conditions but not exposed to the test item
Data from the control group was shared with similar concurrent studies.
A semi-static test regime was employed in the test involving a daily renewal of the test
preparations to ensure that the concentrations of the test item remained at the highest attainable
concentration and to prevent the build-up of nitrogenous waste products.
Any mortalities and sub-lethal effects of exposure were recorded at 3, 6, 24, 48, 72 and 96 hours
after the start of exposure. The criteria of death were taken to be the absence of both respiratory
movement and response to physical stimulation.
Physico-Chemical Measurements
The water temperature, pH and dissolved oxygen concentrations were recorded daily throughout
the test. The measurements at 0 hours, and after each test media renewal at 24, 48 and 72 hours,
represent those of the freshly prepared test preparations while the measurements taken prior to
each test media renewal, and on termination of the test after 96 hours, represent those of the used
or 24-Hour old test preparations. The pH and oxygen were measured using a Hach HQ30d Flexi
handheld meter and the temperature was measured using a Hanna Instruments HI 93510 digital
thermometer.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.1 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Range-finding Test
There were no sub-lethal effects of exposure during therange-finding test.
The results showed no mortalities at the test concentration of 100% v/v saturated solution.
Based on this information, a single test concentration of 100% v/v saturated solution was
selected for the definitive test. This experimental design conforms to a "Limit test" to confirm
that at the highest attainable test concentration, no mortalities or sub-lethal effects of exposure
were observed.
Chemical analysis of the 100% v/v saturated solution test preparation at 0 and 24 hours
showed measured concentrations of 1.18 and 1.17 mg/L respectively indicating
that the test item was stable under test conditions
The analytical methodology was not validated prior to the analysis of range-finding samples; full
validation was later conducted specifically for the concentration to be used in the definitive test.

Definitive Test
Verification of Test Concentrations
Analysis of the freshly prepared 100% v/v saturated solution at 0 and 72 hours (see Appendix 3)
showed measured concentrations of 1.14 and 1.05 mg/L respectively. Analysis of the old or
expired media at 24 and 96 hours showed measured concentrations of 1.16 and 1.22 mg/L
respectively.
Given the slight variation in measured test concentrations it was considered justifiable to base
the results on the mean measured test concentrations.

Mortality Data
There were no mortalities in 7 fish exposed to a test concentration of 1.1 mg/L for a period of
96 hours.
The results of the definitive test showed the highest test concentration resulting in 0% mortality
to be 1.1 mg/L. The No Observed Effect Concentration (NOEC) was 1.1 mg/L.

Sub- Lethal Effects
There were no sub-lethal effects of exposure observed in the test.

Observations on Test Item Solubility
The test preparation was observed to be a clear colorless solution for the duration of the test.

Physico-Chemical Measurement
Temperature was maintained at 13 °C to 15 °C throughout the test,
while there were no treatment related differences for oxygen concentration or pH.
The oxygen concentration in some of the test vessels was observed to have an air saturation
value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic
air bubbles in the media super -saturating the diluent and was considered not to have had an
impact on the outcome or integrity of the test as no adverse effects were observed.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the mean measured test concentrations of the test media the acute toxicity of the test
item to rainbow trout gave a 96-Hour LC50 value of greater than 1.1 mg/L. The No Observed
Effect Concentration was 1.1 mg/L.

Executive summary:

Introduction

A study was performed to assess the acute toxicity of the test item to rainbow trout

( Oncorhynchus mykiss ). The method followed was designed to be compatible with the OECD

Guidelines for Testing of Chemicals (1992) No 203, "Fish, Acute Toxicity Test" referenced as

Method C. l of Commission Regulation (EC) No. 440/2008.

Methods

Pre-study solubility work indicated that it was not possible to obtain a testable solution of the test

item using traditional methods of dispersal e.g., ultrasonication and high shear mixing.

A pre-study media preparation trial indicated that a dissolved test item concentration of

approximately 1.8 mg/L could be obtained from a saturated solution method of preparation

indicating this to be the limit of solubility for this test item under test conditions.

Following a preliminary range-finding test, seven fish were exposed to an aqueous solution of

the test item, at a single nominal concentration of 100% v/v saturated solution for a period of

96 hours at a temperature of 13 °C to 15 °C under semi-static test conditions. The test item

solution was prepared by stirring an excess (50 mg/ L) of test item in test medium using a

propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period any

undissolved test item was removed by filtration (0.2 pm Sartorius Sartopore filter, first

approximate 1 liter discarded in order to pre-condition the filter) to produce a 100 % v/v saturated

solution of the test item. The number of mortalities and any sub-lethal effects of exposure in

each test and control vessel were determined 3 and 6 hours after the start of exposure and then

daily throughout the test until termination after 96 hours.

Results

Analysis of the freshly prepared 100% v/v saturated solution at 0 and 72 hours showed measured

concentrations of 1.14 and 1.05 mg/L respectively. Analysis of the old or expired media at

24 and 96 hours showed measured concentrations of 1.16 and 1.22 mg/L respectively.

Given the slight variation in measured test concentrations it was considered justifiable to base

the results on the mean measured test concentrations.

The 96-Hour LC50 based on mean measured test concentrations was greater than 1.1 mg/L. The

No Observed Effect Concentration was 1.1 mg/L.

This study showed that there were no toxic effects at saturation.