(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

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:

01 Feb 2019 - 06 Mar 2019

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study conducted in marine copepods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: ISO International Standard 14669. Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)

Version / remarks:

First edition 1999-04-01

Principles of method if other than guideline:

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.
The design of this study is based on the following study guideline:
• ISO International Standard 14669. "Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea)", First edition 1999-04-01.
In addition, the following guidance document was followed:
• Guidance document on aqueous-phase aquatic toxicity testing of difficult test chemicals, OECD series on testing and assessment number 23, 2018.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

EC Number:

950-860-4

Molecular formula:

C15H20N4O2

IUPAC Name:

(isopropyl (1s,3s)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)cyclobutane-1-carboxylate)

Test material form:

solid: bulk

Details on test material:

White Crystalline Solid

Specific details on test material used for the study:

Off-white crystalline powder
Batch AM1072083
Purity 100.4%
Storage conditions: At room temperature
Stable under storage conditions until 30 September 2021 (expiry date)
CAS Number 7446-20-0

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the controls.
Frequency at t=0h and t=48h
Volume 3.0 mL
Storage Samples were stored in a freezer (<-15°C) until analysis at the analytical laboratory of the Test Facility
At the end of the test, samples were taken from additional sample vessels containing
approximately 50 mL of test solution which were incubated under similar conditions as the
test vessels.
Additionally, reserve samples of 3.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (<-15°C) for a maximum of three months after delivery of the draft report,

Test solutions

Vehicle:

no

Details on test solutions:

The batch of PF-07094402 tested was a white solid with a purity of 100% which was not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Solutions containing 1.0, 2.2, 4.6, 10 and 22% of the a SS prepared at a loading rate of 100 mg/L Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring to ensure maximum dilution of the test item in test medium.
Thereafter, the aqueous Saturated Solution (SS) was filtered through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clearand colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):

other aquatic crustacea: Tisbe battagliai

Details on test organisms:

Breeding is 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.
Number of copepodids 20 per test group
Loading 5 per vessel containing 2.5 ml of test solution
Light A daily photoperiod of 16 hours
Temperature 18-22°C, constant within 2°C.
Oxygen concentration ≥ 4 mg/L at the end of the test
Feeding No feeding
Introduction of copepodids Within 1 hour after preparation of the test solutions.

Study design

Test type:

static

Water media type:

other: 5 µm filtered and UV treated natural marine water

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

The temperature continuously measured in a temperature control vessel remained constant at 19°C during the test,

pH:

7.8 - 8.1

Dissolved oxygen:

7.3 - 8.2

Salinity:

36 - 37%

Nominal and measured concentrations:

Samples taken from all test concentrations and the blank control were analysed. The
measured concentrations at the start of the test were 0.089, 0.20 , 0.43, 0.91, 2.0 and 8.6 mg/L
in solutions containing 1.0, 2.2, 4.6, 10 and 22% of the SS prepared at a loading rate of
100 mg/L, respectively. The measured concentrations were at the level of 59-86% relative to the initial concentrations at the end of the test.

Reference substance (positive control):

yes

Remarks:

0.30 mg Zn1 /L

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

0.17 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Results with reference substance (positive control):

At the end of the test 80% mortality was observed in the reference control.Since the effect 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

Any other information on results incl. tables

 

Average Exposure Concentration Versus Nominal Concentration
Nominal concentration PF-07094402 (mg/L)		Measured concentrations (mg/L)
				Average exposure con. (mg/L)
		t=0h	t=48h
	1.0	0.089	0.052	0.068
	2.2	0.20	0.14	0.17
	4.6	0.43	0.35	0.39
	10	0.91	0.74	0.82
	22	2.0	1.7	1.8

 

 

Number of IntroducedCopepodidsand Incidence of Mortality in the Final Test
								Reference
Time (h)	Replicate	Blank Control	Average exposure conc. (mg/L)					Control 0.30 mg Zn/L
			0.068	0.17	0.39	0.82	1.8
0	A	5	5	5	5	5	5	5
	B	5	5	5	5	5	5	5
	C	5	5	5	5	5	5	5
	D	5	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20	20
24	A	0	0 (1)	0 (2)	3 (1)	5	5	0
	B	0	0	2 (1)	5	5	5	4
	C	0	0	2*(1)	5	5	5	0
	D	0	0	0 (3)	4	5	5	2
	Total mortality	0	1	11	18	20	20	6
	Effect %	0	5	55	90	100	100	30
48	A	0	0 (1)	0 (3)	1 (3)	5	5	3
	B	0	0	0 (3)	5	5	5	5
	C	0	0	1 (2)	5	5	5	4
	D	0	0	0 (3)	5	5	5	4
	Total mortality	0	1	12	19	20	20	16
	Effect %	0	5	60	95	100	100	80
* A singlecopepodidwas scored dead after 24 hours of exposure and was observed to be alive at the end of the
test. Therefore, this specimen was not scored as dead after 24 hours of exposure.
( )– number ofcopepodidsthat were observed missing. Thesecopepodidswere considered to have died anddisintegrated during the exposure period.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

not specified

Conclusions:

In conclusion, under the conditions of the present study with Tisbe battagliai, the 48h-LC50 was 0.17 mg/L based on average exposure concentrations (95% confidence interval between 0.13 and 0.22 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 Tisbe battagliai during an exposure period of 48 hours and, if possible, to determine the LC50 at 24 and 48 hours of exposure (Static, Non-GLP). The study procedures described in this report were based on the ISO International Standard 14669: "Water quality - Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea).. The batch of PF-07094402 tested was a white solid with a purity of 100% which was not completely soluble in test medium at the loading rate initially prepared. A Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium. 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 solutions containing 1.0, 2.2, 4.6, 10 and 22% of a SS prepared at 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. Samples taken from all test concentrations and the blank control were analysed. The measured concentrations at the start of the test were 0.089, 0.20 , 0.43, 0.91, 2.0 and 8.6 mg/L in solutions containing 1.0, 2.2, 4.6, 10 and 22% of the SS prepared at a loading rate of 100 mg/L, respectively. The measured concentrations decreased to 59-86% relative to the initial concentrations at the end of the test. Based on these results, the average exposure concentrations were 0.068, 0.17, 0.39, 0.82 and 1.8 mg/L. No mortality was observed in the control throughout the test, while a concentration-related increase in mortality was observed at all concentrations tested. The study met the acceptability criteria prescribed by the study plan and was considered valid.