(E)-N'-(2-cyano-4-(3-(1-hydroxy-2-methylpropan-2-yl)thioureido)phenyl)-N,N-dimethylformimidamide

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:

Experimental start date 06 January 2020 Experimental completion date 06 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

E-N'-{2-cyano-4-[3-(2-hydroxy-1,1-dimethylethyl)-thioureido]-phenyl}-N,N-dimethylformamidine

Cas Number:

1429755-57-6

Molecular formula:

C15H12N5OS

IUPAC Name:

E-N'-{2-cyano-4-[3-(2-hydroxy-1,1-dimethylethyl)-thioureido]-phenyl}-N,N-dimethylformamidine

Specific details on test material used for the study:

Identification: SPS 5290 (Stage 3)
Batch: 80034242B
Purity: 99.5%
Physical State/Appearance: Yellow crystalline powder
Expiry Date: 05 July 2020
Storage Conditions: Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All 0-Hour samples were stored frozen prior to analysis alongside the 48-Hour test samples. Duplicate sets of samples were taken at 0 and 48 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Preliminary Media Preparation Trial
Preliminary 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.
Saturated Solution Preparation
A nominal amount of test item (1100 mg) was dispersed, in duplicate, in 11 liters of Elendt M4 media 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 μm Whatman Polycap filter (approximately 1 liter discarded in order to pre-condition the filter)
• Filtration through a 0.2 μm Whatman Polycap filter (approximately 2 liters discarded in order to pre-condition the filter)
Discussion
It was evident from these results that the test item was soluble in Elendt M4 media with the aid of prolonged stirring for a period of 24 hours. As a precautionary measure, the preparation was filtered through a 0.2 μm Whatman Polycap filter (approximately 1 liter discarded in order to pre-condition the filter).

Range-finding Test
A nominal amount of test item (1100 mg) was dissolved in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded) to give the 100 mg/L test concentration. A series of dilutions was made from this test concentration to give further test concentrations of 10, 1.0 and 0.10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Initial Experiment
A nominal amount of test item (1100 mg) was dissolved in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded) to give the required test concentration of 100 mg/L.

Definitive Test
A nominal amount of test item (1100 mg) was dissolved in 11 liters of test water with the aid of propeller stirring at approximately 1500 rpm for 24 hours. After 24 hours the stirring was stopped and, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded) to give the required test concentration of 100 mg/L. A series of dilutions was made from this test concentration to give further test concentrations of 56, 32, 18 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in-house laboratory cultures.
Adult daphnids were maintained in 150 mL glass vesssels containing 100 mL Elendt M4 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °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 (Raphidocelis subcapitata) and GEMMA Micro 300 fish 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.

Study design

Test type:

static

Water media type:

other: Reconstituted water (Elendt M4 medium)

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20 °C to 21 °C

pH:

8.2 at 0 hours. 8.6 ot 8.7 at 48 hours
no treatment related differences for pH.

Dissolved oxygen:

8.7 to 8.8 mg/L 02 at 0 hours. 8.6 ot 8.8 mg/L 02 at 48 hours
no treatment related differences for oxygen concentration

Nominal and measured concentrations:

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Based on the results of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L (nominal)

Definitive Test
100, 56, 32, 18 and 10 mg/L (nominal)
106, 58.8, 32.7, 18.2, 8.99 mg/L measured at 0 Hours
95.5, 53.1, 29.6, 16.4, 8.52 mg/L at 48 hours

Details on test conditions:

Range-finding Test
In the range-finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 240 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
A sample of each test concentration was taken for immediate chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. Duplicate samples were taken on each occasion and stored frozen for further analysis if required.

Definitive Test
In the definitive test 120 mL glass vessels containing approximately 100 mL of test preparation were used. At the start of the test five 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C and a photoperiod of 16 hours light (between 200 and 1200 Lux) 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 item.
The test preparations were not renewed during the 48-Hour exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
No immobilization was observed at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.
No sub-lethal effects of exposure were observed throughout the test.
Based on this information, a single test concentration of four replicates, of 100 mg/L was selected for the initial experiment. This experimental design conforms to a "Limit test" to confirm that at highest test concentration given in the test guideline, no immobilization or adverse reactions to exposure were observed.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 82% to 103% of nominal concentrations. Analysis of the 0.10 and 1.0 mg/L test preparations at 48 hours showed a decline in measured test concentration occurred to less than the limit of detection (LOD) and 76% of nominal respectively. There was no significant decline in measured test concentration in the 10 and 100 mg/L test preparations.

Definitive Test
Verification of Test Concentrations
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 90% to 106% of nominal. There was no significant change in the measured concentrations at 48 hours (85% to 95% of nominal) and so the results are based on the nominal test concentrations only.

Immobilization Data
Visual inspection of the data at 24 hours and analysis of the immobilization data by Probit analysis using Linear Maximum-Likelihood regression at 48 hours gave the following results:
The No Observed Effect Concentration (NOEC) after 24 and 48 hours exposure was 100 mg/L.

Results with reference substance (positive control):

A positive control (Covance study number RQ16SK) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. The positive control was conducted between 20 August 2019 and 22 August 2019.
Analysis of the immobilization data was carried out using the Weibull analysis using linear maximum-likelihood regression at 24 and 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package. Accordingly the following results were obtained:
Time point (hours) EC50 mg/L 95% Confidence Limits (mg/L) NOEC (mg/L) LOEC(mg/L)
24 1.2 1.1 - 1.8 0.56 1.0
48 0.71 0.61 - 0.81 0.56 1.0
The NOEC is based upon equal to or 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

Validation Criteria

The test was considered to be valid given that none of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Observations on Test Item Solubility

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.

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 nominal test concentrations gave a 48 hour EC50 value of greater than 100 mg/L. The NOEC was 100 mg/L.

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.

Methods

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 the test item was however soluble with the aid of prolonged stirring.

Following a preliminary range-finding test and initial experiment, 20 daphnids (4 replicates of 5 animals) were exposed to aqueous solutions of the test item at nominal concentrations of 10, 18, 32, 56 and 100 mg/L for 48 hours at a temperature of 20 °C to 21 °C under static test conditions. The 100 mg/L test concentration was prepared by dissolving the test item in test water using a propeller stirrer at approximately 1500 rpm for 24 hours. After the stirring period, as a precautionary measure any undissolved test item was removed by filtration through a 0.2 μm Whatman Polycap filter (the first approximate 1 liter used to pre-condition the filter was discarded). This test concentration was then further diluted as necessary, to provide the remaining test concentrations.

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from 90% to 106% of nominal. There was no significant change in the measured concentrations at 48 hours (85% to 95% of nominal) and so the results are based on the nominal test concentrations only.

Exposure of Daphnia magna to the test item gave EC50 values based on the nominal test concentrations of greater than 100 mg/L. The No Observed Effect Concentration (NOEC) was 100 mg/L.