cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 Dec 2018 - 27 May 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:

cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

EC Number:

951-622-2

Molecular formula:

C12H15N5O2

IUPAC Name:

cis-N-hydroxy-3-[methyl(1H-pyrrolo[2,3-d]pyrimidin- 4-yl)amino]cyclobutanecarboxamide

Specific details on test material used for the study:

Off-white crystalline powder
Batch GR13449
Purity 100%
Storage conditions: At room temperature
Stable under storage conditions until 30 September 2021 (expiry date)
Test Facility test item number: 209804/A
Purity/Composition correction factor: No correction factor required

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-07085579 tested was a white solid with a purity of 100% which 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 a loading rate of 100 mg/L applying a two-day
period of magnetic stirring to accelerate dissolution of the test item in 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 clear and 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 - 8.4

Salinity:

36 - 37%

Nominal and measured concentrations:

Solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L

Details on test conditions:

The project started with a combined limit/range-finding test. Twenty copepodids per concentration (four replicates, 5 copepodids per vessel) were exposed to a blank control and a SS prepared at a loading rate of 100 mg/L. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Ten copepodids per concentration (in duplicate, 5 per vessel) were exposed to solutions containing 1.0 and 10% of the SS in the combined range-finding test.
• Dissolved oxygen concentrations, salinit and pH were only measured in the control and the highest test concentration.
• Samples for determination of actual exposure concentrations were taken from the control and the highest test concentration at the start and 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.:

11 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality

Details on results:

The 24h-LC50 could not be determined since the recorded effects were below 50% (i.e.LC50 > maximum concentration tested). The 48h-LC50 could not be determined using a regression method. Instead, the 48h-LC50 was
calculated by applying the Spearman-Karber procedure (non-linear; without trimming) on the percentages of mortality and the logarithms of the corresponding measured concentrations of the test item.

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-07085579(mg/L)		Measured concentrations (mg/L)
				Relative to initial [%]
		t=0h	t=48h
	0.58	0.582	0.599	103
	1.3	1.33	1.28	96
	2.7	2.7	2.65	98
	5.7	5.74	5.68	99
	13	12.7	12.8	101

 

 

Number of Introduced Copepodidsand 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 11 mg/L based on measured concentrations (95% confidence interval between 9.2 and 12 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); First edition 1999-04-01 and the OECD series on testing and

assessment number 23, July 06, 2018. The batch of  PF-07085579 tested was a white solid with a purity of 100% which was completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item. 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 4.6, 10, 22, 46 and 100% of the 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 0.58, 1.3, 2.7, 5.7 and 13 mg/L in solutions containing 4.6, 10, 22, 46 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The measured concentrations remained stable throughout the exposure period, i.e. were in the range of 96 – 103% relative to the initial concentrations at the end of the test. Based on this result, effect parameters were expressed in terms of the initially measured concentrations. No mortality was observed in the blank control and at concentrations up to 5.7 mg/L, while 70% mortality was observed at the highest test concentration of 13 mg/L at the end of the test. The study met the acceptability criteria prescribed by the study plan and was considered

valid. In conclusion, under the conditions of the present study with Tisbe  battagliai,  the 48h-LC50 was 11 mg/L based on measured concentrations (95% confidence interval between 9.2 and 12 mg/L).