3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

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 1 June 2009 and 18 June 2009.

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.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

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 materials, 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 material in cases where the test material 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 material in reconstituted water for a period of 24 hours prior to removing any undissolved test material present by centrifugation at 40000 g for 30 minutes to give a saturated solution of the test material.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 19/08/08 Date of Signature: 04/03/09

Test material

Test material information

Constituent 1

Reference substance name:

3-{[1,1'-biphenyl]-4-yl}-6-(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

EC Number:

700-341-1

Cas Number:

105194-34-1

Molecular formula:

C24H15ClN2O2

IUPAC Name:

3-{[1,1'-biphenyl]-4-yl}-6-(4-chlorophenyl)-1H,2H,4H,5H-pyrrolo[3,4-c]pyrrole-1,4-dione

Constituent 2

Reference substance name:

3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

IUPAC Name:

3-(biphenyl-4-yl)-6-(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione

Details on test material:

Sponsor's identification : PDE-28
Description : red powder
Purity : 97%
Batch number : 1RF-8017
Date received : 23 March 2009
Storage conditions : room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Standard solutions of test material were prepared in dimethylformamide at a nominal concentration of 1.0 mg/l.

- Sampling method:
A C18 solid phase extraction (SPE) cartridge was sequentially pre-conditioned with dimethylformamide and water*. A volume of test sample was acidified with phosphoric acid (with the exception of the pre-study media preparation trial), eluted through the cartridge and the cartridge dried. The test material was eluted from the cartridge with dimethylformamide and made to volume to give a final theoretical concentration of approximately 0.78 mg/l.

- Sample storage conditions before analysis:
A preliminary test sample was prepared, analysed initially and then after storage in sealed glass vessels at ambient temperature in light and dark conditions for approximately 48 hours

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
For the purpose of the definitive test the test material was prepared as a saturated solution in reconstituted water.
An amount of test material (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 21°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by centrifugation at 40000 g for 30 minutes to give the saturated solution.
The concentration and stability of the test material in the test preparations were verified by chemical analysis at 0 and 48 hours

- Eluate:
Not applicable

- Controls:
A positive control (Harlan Laboratories Ltd Project No: 0039/1069) conducted approximately every six months used potassium dichromate as the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

An amount of reference material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give a 100 mg/l stock solution. An aliquot (50 ml) of this stock solution was diluted in reconstituted water and the volume adjusted to 500 ml to give a 10 mg/l stock solution. Aliquots (16, 28, 50, 90 and 160 ml) of the 10 mg/l stock solution were each separately dispersed in a final volume of 500 ml of reconstituted water to give the test series of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l.

Each stock solution and prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
Exposure conditions for the positive control were similar to those used in the definitive test.
The temperature was maintained at approximately 20°C.


- Chemical name of vehicle :
Not applicable

- Concentration of vehicle in test medium:
Not applicable

- Evidence of undissolved material :
There was no evidence of any undissolved test material

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name:
Water Flea

- Source:
Derived from in-house laboratory cultures.

- Age at study initiation:
Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old.

- Feeding during test:
No food was provided during the test

ACCLIMATION
- Acclimation period:
Not stated


- Acclimation conditions:
Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 20 deg C.


- Type and amount of food:
Each culture was fed daily with a suspension of algae (Chlorella sp.).
The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

- Health during acclimation:
No mortality observed

Test Water:
The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.


Reconstituted Water:
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. 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.


QUARANTINE (wild caught)

- Duration:
Not applicable

- Health/mortality:
Not applicable

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

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

Test temperature:

Temperature was maintained at 21 to 22°C throughout the test,

pH:

The pH was calculated at the start and termination of the test. The pH was measured using a WTW pH/Oxi 340I pH meter

There were no treatment related differences for oxygen concentration or pH.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.

There were no treatment related differences for oxygen concentration or pH.

Salinity:

Freshwater used.

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.00010, 0.0010 and 0.010 mg/l.

Definitive Test:
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.167 mg/l to 0.197 mg/l. These measured concentrations were significantly higher than measured concentration of 0.010 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
In the definitive 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 21 to 22°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test material.

- Type (delete if not applicable):
closed

- Material, size, headspace, fill volume:
The test vessels were then sealed, with minimal headspace to prevent losses of the test material due to its volatile nature,

- Aeration:
None.

- Type of flow-through (e.g. peristaltic or proportional diluter):
None.

- Renewal rate of test solution (frequency/flow rate):
None.

- No. of organisms per vessel:
5 daphnids were placed in each test and control vessel at random, in the test preparations

- No. of vessels per concentration (replicates):
4 test vessels were used for each test and control group.

- No. of vessels per control (replicates):
4 test vessels were used for each test and control group.

- No. of vessels per vehicle control (replicates):
not stated

- Biomass loading rate:
Not recorded.




- Source/preparation of dilution water:

Appendix 1 Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l
ii) Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. 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.



- Total organic carbon:
Not recorded

- Particulate matter:
Not recorded

- Metals:
Not recorded

- Pesticides:
Not recorded

- Chlorine:
Not recorded

- Alkalinity:
Not recorded

- Ca/mg ratio:
Not recorded

- Conductivity:
Not recorded

- 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:
Not stated

- Photoperiod:
a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours 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

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

- Range finding study
An amount of test material (550 mg) was dispersed in 11 litres of reconstituted water with the aid of propeller stirring at approximately 1500 rpm at a temperature of approximately 20°C for 24 hours. After 24 hours the stirring was stopped and any undissolved test material was removed by centrifugation at 40000 g to give a saturated solution with a nominal test concentration of 0.010 mg/l from which serial dilutions were prepared in reconstituted water to give the remainder of the test series of 0.0010 and
0.00010 mg/l.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 to 22oC with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. The temperature at 24 and 48 hours were observed to be slightly in excess of the 20ºC ± 1ºC range given in the Protocol. This deviation was considered not to have any impact on the outcome or validity of the test as no adverse effects of exposure were observed throughout the test. Each 250 ml test and control vessel contained 200 ml of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.

- Test concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.00010, 0.0010 and 0.010 mg/l. The test material was prepared from a saturated solution.



- Results used to determine the conditions for the definitive study:

Based on the results of the preliminary study, a single test concentration of four replicates, prepared as a 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 significant immobilisation or adverse reactions to exposure were observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.12 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Details on results:

- Behavioural abnormalities: Not recorded

- Other biological observations: None recorded

- Immobilisation of control:

Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in Table 2 (see any other information on results section).
There was no immobilisation in 20 daphnids exposed to a geometric mean measured test concentration of 0.12 mg/l for a period of 48 hours. Inspection of the immobilisation data gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits
(mg/l)
24 > 0.12 not stated
48 > 0.12 not stated

The No Observed Effect Concentration after 24 and 48 hours exposure was 0.12 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

- Other adverse effects control: No other effects observed.

- Abnormal responses: None recorded

- Any other observations (e.g. precipitation) that might cause a difference between measured and nominal values:
Observations on test material solubility:
The test preparations were observed to be clear colourless solutions throughout the duration of the test.

Results with reference substance (positive control):

- Results with reference substance valid?
Yes

- Mortality:
No mortalities recorded.

- EC50/LC50:

For the results for the positive control, please see the any other information on results section.

Any other information on results incl. tables

Verification of test concentrations:

Analysis of the test preparations at 0 hours (see Appendix 3) showed measured test concentrations to range from 0.167 mg/l to 0.197 mg/l. These measured concentrations were significantly higher than measured concentration of 0.010 mg/l determined in the pre-study media preparation trial. The reason for the increased concentration in the definitive test was not apparent from a review of the data however this may have been due to differences in sampling techniques used to remove the supernatant after centrifugation between the media preparation trial and the definitive test.  This difference was considered not to have affected the results of the test which indicated no toxicity at the maximum attainable dissolved test material concentration.

Analysis of the old or expired test preparations at 48 hours (see Appendix 3) showed a slight decline in measured test concentrations of 0.0729 mg/l to 0.0757 mg/l.

Pre-study stability analyses indicated that the test material was stable in test medium over a 48-Hour period, however a Study to Determine the Partition Coefficient of the test material (Harlan Laboratories Ltd Project Number: 2811/0001) gave log₁₀ P_ow value of greater than 6.5.  Therefore it was considered that the decline in measured concentration over the 48-Hour test period was due to possible bioaccumulation.

Current regulatory advice is that in cases where a decline in measured concentrations is observed, geometric mean measured concentrations should be used for calculating EC₅₀ values. It was therefore considered justifiable to base the results on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were determined to be:

0-Hour Mean Measured Test Concentration (mg/l)	Geometric Mean Measured Test Concentration (mg/l)	Percentage of the 0-Hour Measured Test Concentration
0.18	0.12	67

 Positive Control

Cumulative immobilisation data from the exposure of Daphnia magna to the reference material (Harlan Laboratories Ltd Project No: 0039/1069) during the positive control are given in Table 3 (please see below). The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 1 and 2 and can be seen in attachment 1).

Inspection of the immobilisation data at 3 hours and analysis of the immobilisation data by the probit method (Finney 1971) at 48 hours based on the nominal test concentrations gave the following results:

Time (h)	EC50(mg/l)	95% Confidence limits (mg/l)
3	> 3.2		-
24	0.82	0.71	-	0.94
48	0.71	0.61	-	0.81

The No Observed Effect Concentration after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slopes and their standard errors of the response curves at 24 and 48 hours were 8.1 (SE = 1.7) and 8.6 (SE = 1.8) respectively.

The results from the positive control with potassium dichromate were within the normal range for this reference material. The mean 48-Hour EC₅₀ value calculated from all positive controls was 0.78 mg/l (sd = 0.21).

Table 1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Concentration[1] (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.00010	0	0
0.0010	0	0
0.010	0	0

 

[1]Concentration based on the results of the pre-study media preparation trial

Table 2              Cumulative Immobilisation Data in the Definitive Test

Geometric Mean Measured Test Concentration (mg/l)		Cumulative Immobilised Daphnia (Initial Population: 5 Per Replicate)
		24 Hours			48 Hours
		No. Per Replicate	Total	%	No. Per Replicate	Total	%
Control	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0
0.12	R1	0	0	0	0	0	0
	R2	0			0
	R3	0			0
	R4	0			0

R₁– R₄= Replicates 1 to 4

Table 3              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	3 Hours				24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0	0	0	0	0
0.56	0	0	0	0	1	1	2	10	2	2	4	20
1.0	0	0	0	0	7	8	15	75	9	9	18	90
1.8	0	0	0	0	10	10	20	100	10	10	20	100
3.2	0	0	0	0	10	10	20	100	10	10	20	100

 

R₁– R₂= Replicates 1 and 2

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and based on the geometric mean measured test concentration gave a 48-Hour EC50 of greater than 0.12 mg/l. Correspondingly the No Observed Effect Concentration was 0.12 mg/l.
This study showed that there were no toxic effects at saturation.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in 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.

Methods:

Information provided by the Sponsor indicated that the test material was insoluble in water.  Pre-study solubility work conducted indicated that it was not possible to obtain a testable solution of the test material using traditional methods of preparation e.g. ultrasonication and high shear mixing, and therefore a pre-study media preparation trial was conducted in order to determine a suitable method of preparation.  The results of this pre-study media preparation trial indicated that a saturated solution method of preparation maximised the dissolved test material concentration giving a result of approximately 0.010 mg/l.

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a saturated solution of the test material for 48 hours at a temperature of 21 to 22°C under static test conditions. The test material solutions were prepared by stirring an excess (50 mg/l) of test material in reconstituted water using a propeller stirrer at approximately 1500 rpm at a temperature of approximately 20°C for 24 hours. After the stirring period any undissolved test material was removed by centrifugation at 40000 gfor 30 minutes to produce the saturated solution. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C understatic test conditions. Immobilisation and any adverse reactions to exposure were recorded after 3, 24 and 48 hours.

Results:

Chemical analysis of the test preparations at 0 hours showed significantly higher measured concentrations (0.167 to 0.197 mg/l) than obtained in the pre-study media preparation trial. The reason for the increased concentration in the definitive test was not apparent from a review of the data however this may have been due to differences in sampling techniques used to remove the supernatant after centrifugation between the media preparation trial and the definitive test.  This difference was considered not to have affected the results of the test which indicated no toxicity at the maximum attainable dissolved test material concentration.

Analysis of the old or expired test preparations at 48 hours showed measured concentrations to range from 0.0729 to 0.0757 mg/l.

Pre-study stability analyses indicated that the test material was stable in test medium over a 48-Hour period, however a Study to Determine the Partition Coefficient of the test material (Harlan Laboratories Ltd Project Number: 2811/0001) gave log₁₀ P_ow value of greater than 6.5.  Therefore it was considered that the decline in measured concentration over the 48-Hour test period was due to possible bioaccumulation.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations of the test media in order to give a "worst case" analysis of the data. The 48-Hour EC₅₀ based on the geometric mean measured test concentration was greater than 0.12 mg/l and correspondingly the No Observed Effect Concentration was 0.12 mg/l.

The 48-Hour EC₅₀ for the reference material to Daphnia magna based on nominal concentrations was 0.71 mg/l with 95% confidence limits of 0.61 – 0.81 mg/l. The No Observed Effect Concentration was 0.32 mg/l.