cis-N-methyl-N-(1H-pyrrolo[2,3-d]pyrimidin4-yl)cyclobutane-1,3-diamine phosphate (1:1)

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:

17 Dec 2018- 3 March 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Non-GLP. Marine copepod study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Guideline:

other: Guidance document on aqueous phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23, 2018.

Version / remarks:

If applicable

Test guideline 2

Guideline:

other: ISO International Standard 14669. "Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)", First edition 1999-04-01.

Deviations:

no

GLP compliance:

no

Remarks:

Study was conducted in accrdance to ISO International Standard 14669: "Water quality - Determination of acute lethal toxicity to marine copepods . This study is not a requirement for the submission of REACH registerationin accordarnce wth Artilce 17/18

Test material

Test material information

1

Reference substance name:

(1s,3s)-N1-methyl-N1-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutane-1,3-diamine; phosphoric acid

EC Number:

951-920-2

Molecular formula:

C11H15N5* H3PO4

IUPAC Name:

(1s,3s)-N1-methyl-N1-{7H-pyrrolo[2,3-d]pyrimidin-4-yl}cyclobutane-1,3-diamine; phosphoric acid

Specific details on test material used for the study:

Test item information

Identification: PF-03817968-09
Batch (Lot) Number: GR13472
Expiry date: 31 March 2020 (expiry date)
Physical Description: White to off-white solid
Purity/Composition: 100%
Storage Conditions: At room temperature
Test Facility test item number: 209806/A

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test samples were stored in the freezer (≤ -15°C). Storage stability of samples under these
conditions was demonstrated in Test Facility Study No. 20171437. On the day of analysis, the test samples were thawed at room temperature and analyzed. If necessary, the samples were further diluted with test medium to obtain concentrations within the calibration range.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of Test Solutions
The batch of PF-03817968-09 tested was a white to off-white solid with a purity of 100% and
was completely soluble in test medium at the concentrations tested. No correction was made
for the purity/composition of the test item. Preparation of test solutions started with the highest concentration of 100 mg

Stock and Spiking Solutions
Stock and spiking solutions of the test item were prepared in 25/75 (v/v) acetonitrile/water at
concentrations of 1000 and 2000 mg/L. The stock solutions were ultrasonicated for
5 minutes.

Calibration Solutions
Five calibration solutions in the concentration range of 0.07 – 12 mg/L were prepared from
two stock solutions. The end solution of the calibration solutions was test medium.

QC Samples
1 mL test medium was spiked with the test item at a target concentration of 0.1,
10 or 100 mg/L and analyzed. The QC samples were treated similarly as the test samples. Blank QC samples consisting of blank test medium were treated similar to the QC samples
spiked with test item and test samples.

Test organisms

Test organisms (species):

other aquatic crustacea: Marine Copepod: Tisbe Battagliai

Details on test organisms:

Species: Tisbe battagliai (Copepoda, Crustacea)(Volkmann-Rocco),
Source: Guernsey Sea Farms Ltd., Guernsey, United Kingdom.
Reason for selection: Species has been selected as an internationally accepted
invertebrate species.
Characteristics: 6 ± 2 days old copepodids
Breeding: Performed at Guernsey Sea Farms Ltd., Guernsey, United Kingdom. Less than 7
days old copepodids are transferred to Charles River Den Bosch. They were kept in the
transferral water until the start of the test.

Study design

Test type:

static

Water media type:

saltwater

Remarks:

5 µm filtered and UV treated natural marine water of the same origin as the water that was used for culturing the organisms. Water was obtained from Guernsey Sea Farms Ltd., Guernsey, United Kingdom and stored at 4ºC until test initiation.

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

24h, 48h

Test conditions

pH:

7.9

Dissolved oxygen:

>80% of the air saturation value

Salinity:

36%

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100 mg/L.
Measured concentrations: The measured concentrations varied between 85 and 100% relative to nominal throughout the test and thus remained within 80 – 120% of the nominal concentrations.

Details on test conditions:

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty copepodids per group (5 per replicate, quadruplicate) were exposed to a blank control, reference control (0.30 mg Zn/L), and to nominal concentrations of 10, 18, 32, 56, and 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

Reference substance (positive control):

yes

Remarks:

Zinc sulfate heptahydrate, CAS Number 7446-20-0, Identification number: RS454, Batch: AM1072083

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Results based on analytically confirmed nominal concentrations (95% confidence interval between 17 and 25 mg/L).

Details on results:

Mortality
No mortality was observed in the blank control and at the lowest concentration tested throughout the test. A concentration-related increase of mortality was found at 18 mg/L and
higher, resulting in total mortality at the two highest test concentrations at the end of the test.
The responses recorded in this test were in agreement with the responses found in the combined limit/range-finding test and allowed for reliable determination of a 24h and
48h-LC50.

Results with reference substance (positive control):

At the end of the test, 40% mortality was observed in the reference control. Since mortality in this treatment should be between 20 and 80%, it was concluded that the batch copepodids used for this test showed normal sensitivity to zinc sulphate heptahydrate.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Conclusions:

In conclusion, under the conditions of the present study with Tisbe battagliai, the 48h-LC50 was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence
interval between 17 and 25 mg/L).

Executive summary:

The objective of the study was to evaluate the test item for its ability to generate acute lethal toxic effects on marine copepod Tisbebattagliai during an exposure period of 48 hours and, if possible, to determine the LC50 at 24 and 48 hours of exposure (Static, Non-GLP). 

A final test was performed based on the results of a preceding combined limit/range-finding test. Twenty copepodids per group (5 per replicate, quadruplicate) were exposed to a blank control, reference control (0.30 mg Zn/L), and to nominal concentrations of 10, 18, 32, 56, and 100 mg/L. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test.

 The measured concentrations varied between 85 and 100% relative to nominal throughout the test and thus remained within 80 – 120% of the nominal concentrations. Based on these results, effect parameters were expressed in terms of analytically confirmed nominal concentrations.

A concentration-related increase of mortality was found at 18 mg/L and higher, resulting in total mortality at the two highest test concentrations at the end of the test.

In conclusion, under the conditions of the present study with Tisbebattagliai, the 48h-LC50 was 21 mg/L based on analytically confirmed nominal concentrations (95% confidence interval between 17 and 25 mg/L).