1H-Pyrazolo[3,4-c]pyridine-3-carboxylic acid, 4,5,6,7-tetrahydro-1-(4-methoxyphenyl)-6-(4-nitrophenyl)-7-oxo-, ethyl ester

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 28th May 2014 and 18th June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

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. The test item was initially dissolved in dimethylformamide at a concentration of 100 mg/10 mL. An aliquot of this solvent stock solution was then dispersed in an appropriate volume of test media with the aid of magnetic stirring for approximately 10 minutes prior to removing any undissolved test item present by filtration (0.2 µm Gelman Acrocap, first approximate 500 mL discarded in order to pre-condition the filter) to give a nominal concentration of 1.0 mg/L.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

BMS-589152-01

IUPAC Name:

BMS-589152-01

Test material form:

solid: particulate/powder

Details on test material:

Identification: BMS-589152-01
Physical state/Appearance: tan-colored powder
Batch: 2G72631N
Purity: 99.5%
Expiry Date: 19 June 2014
Storage Conditions: room temperature in the dark
Intended use/Application: chemical intermediate

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
1.0 mg/L

- Sampling method:
The test samples were thawed with the aid of sonication. A volume (5 mL) of sample was pipetted into a volumetric flask (20 mL) with the addition of water (5 mL) then diluted to volume with 0.2% (v/v) acetic acid in acetonitrile.
Preparation of Spiked Recovery Samples:
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in 0.1% (v/v) acetic acid in acetonitrile to prepare a stock solution with a concentration of 1000 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 1.0 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

- Sample storage conditions before analysis:
Samples were taken from the solvent control and the 1.0 mg/L test group from the bulk test preparation at 0 and 24 hours (fresh media) and from the pooled replicates at 24 and 48 hours (old or expired media) for quantitative analysis. All samples were stored frozen prior to analysis.

Duplicate samples were taken and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances):
The results obtained from the preliminary media preparation trial conducted indicated that a dissolved test item concentration of approximately 0.75 mg/L could be obtained using a solvent spike solution method of preparation.

The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L.

Prior to use of the test item its appearance was checked against its documented description as a confirmation check used to verify its identity.

An amount of test item (100 mg) was dissolved in dimethylformamide and the volume adjusted to 10 mL to give the 100 mg/10 mL solvent stock solution. An aliquot (500 µL) of the solvent stock solution was added to 5 liters of test water and stirred using a magnetic stirrer for 10 minutes. After stirring, any undissolved test item was removed by filtration through a 0.2 µm Gelman Acrocap filter (first approximate 500 mL discarded in order to pre-condition the filter) to give a 1.0 mg/L test concentration. A series of dilutions was made from this test concentration to give further test concentrations of 0.0010, 0.010 and 0.10 mg/L.

All preparation was carried out under laboratory safety lighting due to the light sensitive nature of the test item. Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The control and solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (please see details below) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Reconstituted Water – Elendt M7 Medium
Ingredient Final Concentration (mg/L)
H3BO3 0.715
MnCl2.4H2O 0.090
LiCl 0.077
RbCl 0.018
SrCl2.6H2O 0.038
NaBr 0.004
Na2MoO4.2H2O - 0.016
CuCl2.2H2O 0.004
ZnCl2 0.013
CoCl2.6H2O 0.010
KI 0.0033
Na2SeO3 0.0022
NH4VO3 0.00058
Na2EDTA.2H2O - 0.625
FeSO4.7H2O 0.249
CaCl2.2H2O 293.8
NaHCO3 64.8
MgSO4.7H2O 123.3
Na2SiO3.9H2O - 10
KCl 5.8
NaNO3 0.274
K2HPO4 0.184
KH2PO4 0.143
Thiamine hydrochloride - 0.075
Cyanocobalamine (vitamin B12) - 0.0010
D(+) biotin (vitamin H) - 0.00075

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 ºC.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

The water temperature was recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at 24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-hour old test preparations. The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

The temperature was maintained at 20 °C throughout the test.

pH:

The pH of the prepared media was 7.8 ± 0.3 and stored at approximately 21 ºC.
The pH was measured using a WTW pH/Oxi 340I pH meter.
There were no treatment related differences for pH.

Dissolved oxygen:

The dissolved oxygen concentrations were recorded daily throughout the test. The measurements at 0 hours and after the test media renewal at
24 hours represent those of the freshly prepared test preparations while the measurements taken prior to the test media renewal, and on termination of the test after 48 hours, represent those of the used or 24-hour old test preparations. The dissolved oxygen concentration was measured using a Hach HQ30d Flexi handheld meter.

There was no treatment related differences for dissolved Oxygen concentration. The dissolved Oxygen conbcentration ranged between 9.0 and 9.3 mg O2/L

Salinity:

freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L.

Based on the results of the range-finding test, a limit test was conducted using 4 replicates at a concentration of 1.0 mg/L.

The time-weighted mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.53 mg/L.

Details on test conditions:

TEST SYSTEM: As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at 20 °C in darkness. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control and solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 100 µL/L of dimethylformamide.

Semi-static test conditions were employed in the test in an effort to maintain dissolved test item concentrations. For the test media renewal at 24 hours, the test concentrations were freshly prepared and the daphnids transferred by wide bore pipette from the 24-hour old test media into the fresh test media.

Samples were taken from the solvent control and the 1.0 mg/L test group from the bulk test preparation at 0 and 24 hours (fresh media) and from the pooled replicates at 24 and 48 hours (old or expired media) for quantitative analysis. All samples were stored frozen prior to analysis.

- Source/preparation of dilution water:
Reconstituted Water
Ingredient Final Concentration (mg/L)
CaCl2.2H2O 294
MgSO4.7H2O 123
NaHCO3 64.75
KCl 5.75

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.

- Culture medium different from test medium:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

- Intervals of water quality measurement:
Not recorded

OTHER TEST CONDITIONS

- Adjustment of pH:
The reconstituted water had a pH of 7.8 ± 0.3 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

- Photoperiod:
a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.

- Light intensity:
Not recorded

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

TEST CONCENTRATIONS

- Spacing factor for test concentrations:
Based on the results of the range-finding test a "limit test" was conducted at a nominal concentration of 1.0 mg/L to confirm that at the highest attainable test concentration, no immobilization or adverse reactions were observed.

- Justification for using less concentrations than requested by guideline:
Not applicable

- Range finding study:
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L.

- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test, a "limit test" was conducted at a nominal concentration of 1.0 mg/L to confirm that at the highest attainable test concentration, no immobilization or adverse reactions were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 0.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.53 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

Range finding test:

No immobilization was observed at the test concentrations of 0.0010, 0.010, 0.10 and 1.0 mg/L.

Based on this information, a single test concentration of four replicates, of 1.0 mg/L was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that at the highest attainable test concentration, no immobilization or adverse reactions to exposure were observed.

Chemical analysis of the 1.0 mg/L test preparation at 0 and 48 hours showed that measured concentrations of 71% and 53% of nominal were obtained respectively, indicating that the test item was possibly unstable under test conditions.

Observations made of the 1.0 mg/L test vessel at 24 and 48 hours showed a small amount of precipitate on the base of the vessel, however, this had no affect on the daphnia at this concentration.

Results for the Definitive test:
Verification of Test Concentrations
Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations of 0.62 and 0.64 mg/L, respectively. A decline in measured test concentration was observed in the old test media at 24 and 48 hours to 0.53 and 0.37 mg/L, respectively, and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentration only in order to give a “worst case” analysis of the data.

The time-weighted mean measured test concentrations were determined to be:

Nominal Test Concentration
(mg/L) Time-Weighted Mean Measured Test Concentration (mg/L) Expressed as a % of the 0-Hour Measured Test Concentration
1.0 0.53 86

Initial analysis of the 1.0 mg/L 24-hour old media showed that a measured concentration of 0.66 mg/L was obtained. Given that a decline in measured concentration was observed in both the range finding test and after the second 24–hour period in the definitive test at this concentration, it was considered appropriate to analyze the duplicate sample. Analysis of the duplicate sample showed that a measured concentration of 0.53 mg/L was obtained. Given that this result correlates with the expected profile of the test item, it was considered that the original result of 0.66 mg/L was anomalous and not considered to have had an impact on the outcome or integrity of the study.

There was no immobilization in 20 daphnids exposed to a time weighted mean measured test concentration of 0.53 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EC50 (mg/L)
24 >0.53
48 >0.53

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.53 mg/L.

Observations on Test Item Solubility:
The freshly prepared test preparation at 0 and 24 hours were observed to be clear colorless solutions, and the old test preparations at 24 and 48 hours were observed to be clear colorless solutions with very small amounts of precipitate on the base of the vessel.

Results with reference substance (positive control):

Analysis of the immobilization data by the maximum-likelihood probit method (Finney, 1971*) at 24 hours and by the trimmed Spearman-Karber method (Hamilton et al 1977 *) at 48 hours using the ToxCalc software package (ToxCalc 1999) based on the nominal test concentrations gave the following results:

Time Point(Hours) EC50 (mg/L) 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC (mg/L)
24 0.87 0.75 - 1.0 0.56 1.0
48 0.71 0.65 - 0.78 0.56 1.0

The No Observed Effect Concentration is based upon less than 10% immobilization at this concentration.

The results from the positive control with potassium dichromate were within the normal range for this reference item.

Any other information on results incl. tables

RESULTS

Results for Spiked Recovery Samples

Nominal Concentra­tion of Test Item cnom	Fortified Concentra­tion of Test Item in the Spiked Sample cfort	Measured Concentra­tion of Test Item in the Sample Vial x	Sample Preparation Factor F	Determined Concentra­tion of Test Item in the Spiked Sample c	Mean Analytical Recovery Rate R	Precision (Relative Standard Deviation of Recovery)
[mg/L]	[mg/L]	[mg/L]		[mg/L]	[%]	[%]
1.0	1.02	0.265	4	1.06	103	1.2
	1.02	0.265	4	1.06
	1.02	0.263	4	1.05
	1.02	0.263	4	1.05
	1.02	0.258	4	1.03
Acceptance Target					80-120	<10

Results for Test Samples:

Time Point	Nominal Concentration of Test Item in Test Sample cnom	Measured Concentration  of Test Item in Sample Vial x	Sample Preparation Factor F	Determined Concentration of Test Item in Test Sample   c	% of Nominal Concentration
[hours]	[mg/L]	[mg/L]		[mg/L]	[%]
0	Control	<LOQ	4	<LOQ	-
(Fresh)	1.0	0.155	4	0.619	62
24	Control	<LOQ	4	<LOQ	-
(Old)	1.0	0.165 / 0.132*	4	0.658 / 0.527*	66 / 53*
24	Control	<LOQ	4	<LOQ	-
(Fresh)	1.0	0.159	4	0.637	64
48	Control	<LOQ	4	<LOQ	-
(Old)	1.0	0.0915	4	0.366	37

*                             =             Duplicate sample, stored frozen prior to analysis

LOQ                      =             Limit of Quantification

-                              =             not applicable

For reporting purposes, x has been calculated from c retrospectively.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and based on the time-weighted mean measured test concentrations gave a 48-Hour EC50 value of greater than 0.53 mg/L. The No Observed Effect Concentration was 0.53 mg/L.

This study showed that there were no toxic effects at the limit of water solubility.

Executive summary:

INTRODUCTION:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

METHOD:

Information provided by the Sponsor indicated the test item was insoluble in water. Preliminary solubility work conducted indicated that it was not possible to obtain a testable solution of the test item using traditional methods of preparation e.g. ultrasonication and high shear mixing.

 

A preliminary media preparation trial indicated that a dissolved test item concentration of approximately 0.75 mg/L was obtained from a solvent spike solution method of preparation indicating this to be the limit of water solubility of this item in deionized reverse osmosis water.

 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at a nominal concentration of 1.0 mg/L for 48 hours at a temperature of 20°C under semi-static test conditions. The test item solution was prepared by dispersing 500µL of a 100 mg/10 mL solvent stock solution in 5 liters of test water with the aid of magnetic stirring for approximately 10 minutes. After stirring any undissolved test item was removed by filtration (0.2 µm Gelman Acrocap filter, first approximate 500 mL discarded in order to pre-condition the filter). Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

Due to the light sensitive nature of the test item, all test item preparation was performed under laboratory safety lighting.

RESULT:

Analysis of the freshly prepared test media at 0 and 24 hours showed measured test concentrations of 0.62 and 0.64 mg/L respectively. A decline in measured test concentration was observed in the old test media at 24 and 48 hours to 0.53 and 0.37 mg/L, respectively, and hence it was considered appropriate to calculate the results based on the time-weighted mean measured test concentration only in order to give a “worst case” analysis of the data.

.

Exposure of Daphnia magna to the test item gave EC₅₀ values based on the time-weighted mean measured test concentrations of greater than 0.53 mg/L. The No Observed Effect Concentration was 0.53 mg/L. This study showed that there were no toxic effects at the limit of solubility in test media.