Registration Dossier

Ecotoxicological information

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:
2019-05-13 to 2019-11-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline; EU Method; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to guideline
Guideline:
other: OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures"
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidine-4,5,6-triamine
EC Number:
811-934-0
Cas Number:
1350653-30-3
Molecular formula:
C17 H14 F2 N8
IUPAC Name:
2-[5-fluoro-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidine-4,5,6-triamine
Test material form:
solid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test medium of the only test concentration and the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentration during the test period, duplicate samples from the test medium of the only test concentration and the control were collected at the end of the test (after 48 hours).
All samples were diluted by a factor of 2 with acetonitrile.
Additional samples of the untreated control and the dilution solvent acetonitrile were taken at test start and test end without any sample treatment.

Test solutions

Vehicle:
no
Details on test solutions:
Test Concentration: Filtrate of 100 mg test item/L and a control
corresponding to following mean measured concentration of the test item: 2.31 mg test item/L
Thus, a limit test in accordance with OECD Guideline 202 was performed to demonstrate that the test item has no toxic effect on daphnids up to the solubility limit in the used test water.
Control: In the control, test water was used without addition of the test item.
Dosage of Test Item: The test item was not well soluble in test water. To avoid physical effects of undissolved test item on the daphnids, no concentration above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 102 mg of test item in 1020 mL test water. The stock solution was stirred for 24.25 hours in the dark to dissolve as much test item as possible. Then, the undissolved test item was separated by filtration (0.45 µm cellulose acetate filter) and the filtrate was used as test medium.
The test media was prepared just before introduction of the daphnids (= start of the test).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Species: Daphnia magna (Straus), clone 5
Age at Test Start:
From 1 to 17.5 hours old
Sex: Female
Origin: The Daphnia introduced in the test were taken from ibacon's in house laboratory culture.
Breeding Conditions: The Daphnia were bred in the laboratories of ibacon under similar temperature and light conditions as used in the test. The cultivation of the parental Daphnia was performed in Elendt M4 medium. The test organisms were not first brood progeny. The Daphnia in the stock culture were fed at least on all working days with green algae (Desmodesmus subspicatus) freshly grown in the laboratories of ibacon.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h

Test conditions

Hardness:
Water Hardness: 2.5 mmol/L (= 250 mg/L) as Calcium Carbonate
Test temperature:
21 °C at test start;
20 °C at test end
pH:
8.0 - 8.1at test start;
7.9 - 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved oxygen:
5 .1to 8.9 mg/L at test start;
8.8 mg/L at test end
Nominal and measured concentrations:
Filtrate of 100 mg test item/L and a control
corresponding to following mean measured concentration of the test item:
2.31 mg test item/L
Thus, a limit test in accordance with OECD Guideline 202 was performed to demonstrate that the test item has no toxic effect on daphnids up to the solubility limit in the used test water.
Control: in the control, test water was used without addition of the test item.
Details on test conditions:
Test Environment: Controlled environment room
Measurement of pH, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and dissolved oxygen concentrations were determined at test start and test end in each treatment group.
Water Temperature: 20.6 to 21.5 °C at test start; 20.2 °C at test end
pH-Values: 8.0 to 8.1 at test start; 7.9 to 8.0 at test end; and thus the pH-value did not vary by more than 1.5 units
Dissolved Oxygen Concentration: 5.1 to 8.9 mg/L at test start; 8.8 mg/L at test end
Light Regime: 16 h light : 8 h dark
Light Intensity: The light intensity was 590 to 830 lux (measured once during the test).
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
Reference substance (positive control):
yes
Remarks:
For the evaluation of the quality of the Daphnia clone and the consistency of the experimental conditions, the reference item potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 2.31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 2.31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
> 2.31 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
After 48 hours of exposure no immobilisation of the test animals was observed in the control. At the concentration of 2.31 mg test item/L, the only concentration tested, one animal was immobile after 48h.
However this immobilisation rate was not estimated as a significant toxic effect, because according to the test guidelines this immobilisation rate is also tolerated in the control, and additionally all other animals did not show any signs of intoxication over the test period of 48h.
Results with reference substance (positive control):
In the most recent test with the reference item potassium dichromate the EC50 after 24 hours was determined to be 0.932 mg test item/L, indicating that the sensitivity of the Daphnia was consistent with the level proposed by the OECD 202 guideline (EC50-24 h between 0.6 and 2.1 mg potassium dichromate/L).
Reported statistics and error estimates:
No statistical analysis was performed.
The EC50 could not be quantified due to the absence of toxicity of the test item. The NOEC and the LOEC were determined directly from the raw data.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Control Immobilisation Rate was 0 % and furthermore no daphnid showed signs of disease or stress. Dissolved Oxygen Concentration was above or equal 8.8 mg O2/L in the control and test vessels at the end of the test.
Conclusions:
The toxic effect of the test item Fluorotrisamine to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L, corresponding to the mean measured concentration of 2.31 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than the mean measured concentration of 2.31 mg test item/L, the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of Fluorotrisamine up to 2.31 mg test item/L, the concentration representing the limit of solubility. The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to the arithmetic mean concentration, since the test item concentration was within ± 20 % of the measured initial concentration during the test.
Executive summary:

Title: Fluorotrisamine: Acute Toxicity to Daphnia magna in a Static 48-hour Immobilisation Limit Test

Purpose: The purpose of this study was to determine the influence of the test item Fluorotrisamine on the mobility of Daphnia magna in a static Limit test.

For this purpose, young daphnids (< 24 hours old) were exposed to the highest concentration which can be dissolved in test water (filtrate of nominal 100 mg test item/L) under defined conditions for 48 hours. The recorded effects were the mobility of the daphnids after 24 and 48 hours.

This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the daphnids up to this concentration.

The test method of application and the test system are recommended by the test guidelines and Daphnia magna is the recommended test species.

The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

Guidelines:

OECD Guideline for Testing of Chemicals No. 202: "Daphnia sp., Acute Immobilisation Test" adopted April 13, 2004

OECD Series on Testing and Assessment, No. 23, "Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals", 2nd Ed., February 08, 2019

SANCO/3029/99 rev.4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

 

Materials and Methods

Test Item: Fluorotrisamine; batch no.: FEY 7262-332-154; purity: 99 %, according to certificate of analysis

Test Species: Female Daphnia magna, clone 5;1 to 17.5 hours old

Source: The daphnids introduced in the test were taken from ibacon's in-house laboratory culture.

Test Design: This study encompassed two treatment groups (one test item concentration of a filtrate of nominal 100 mg/L, and one control) each containing 20 individuals. The mobility of the daphnids was determined in a static 48-hour test by visual observation after 24 and 48 hours.

Endpoints: Number of immobile organisms after 24 and 48 hours

Test Concentrations: Filtrate of 100 mg test item/L and a control

corresponding to following mean measured concentration of the test item:

2.31 mg test item/L.

Test Conditions: Water temperature: 20.2 to 21.5 °C; pH value: 7.9 to 8.1; dissolved oxygen concentration: 5.1 to 8.9 mg/L; photoperiod: 16 h light - 8 h dark; light intensity: 590 to 830 lux; and thus were within the ranges requested by guideline OECD 202

 

Results

Biological test results: After 48 hours of exposure no immobilisation of the test animals was observed in the control. At the concentration of 2.31 mg test item/L, the only concentration tested, one animal was immobile after 48h.

However this immobilisation rate was not estimated as a significant toxic effect, because according to the test guidelines this immobilisation rate is also tolerated in the control, and additionally all other animals did not show any signs of intoxication over the test period of 48h.

The 48-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L, corresponding to the mean measured concentration of 2.31 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than the mean measured concentration of 2.31 mg test item/L, the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of Fluorotrisamine up to 2.31 mg test item/L, the concentration representing the limit of solubility.

Analytical test results:

The quantification of the test item Fluorotrisamine in the test samples was performed using liquid chromatography with MS/MS detection.

The concentrations of the test item were determined in the duplicate test media samples from the only test concentration (filtrate of nominal 100 mg test item/L) and the duplicate control samples from all sampling times. The mean measured value is:

2.31 mg test item/L in the filtrate of nominal 100 mg test item/L.

 

Conclusion:

The toxic effect of the test item Fluorotrisamine to Daphnia magna was assessed in a static limit test. The 48-hour NOEC was determined to be at least the filtrate of nominal 100 mg test item/L, corresponding to the mean measured concentration of 2.31 mg test item/L. The NOEC might even be higher than this concentration, but concentrations in excess of nominal 100 mg test item/L or the limit of solubility have not been tested according to the recommendations of the test guidelines. The 48-hour LOEC and the 48-hour EC50 were clearly higher than the mean measured concentration of 2.31 mg test item/L, the filtrate of nominal 100 mg test item/L. These values could not be quantified due to the absence of toxicity of Fluorotrisamine up to 2.31 mg test item/L, the concentration representing the limit of solubility.

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to the arithmetic mean concentration, since the test item concentration was within ± 20 % of the measured initial concentration during the test.

This study is classified as acceptable and satisfies the guideline requirements for an acute toxicity study with freshwater invertebrates.