tert-butyl {(1S)-2-[(1S,3S,5S)-3-cyano-2-azabicyclo[3.1.0]hex-2-yl]-1-[3-hydroxyadamantan-1-yl]-2-oxoethyl}carbamate

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:

21 February 2012 and 6 March 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Identification :BMS-528235-01
Description :off white powder
Batch :1L63487N
Purity :98.6%
Expiry / retest date :27 November 2014
Storage condition :room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-finding Test
The test concentrations to be used in the definitive test were determined by a preliminary range-finding test.

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. The test item was dissolved directly in water.

An amount of test item (50 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 500 mL to give the 100 mg/L test concentration. A series of dilutions was made from the 100 mg/L 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.

In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 20 °C to 22 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized Daphnia magna 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 chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored at approximately -20 °C prior to analysis.

Definitive Test
Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Test solutions

Vehicle:

no

Details on test solutions:

Experimental Preparation
For the purpose of the definitive test the test item was dissolved directly in water.

An amount of test item (250 mg) was dissolved in reconstituted water with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 2.5 liters to give the test concentration of 100 mg/L. A series of dilutions was made from the 100 mg/L test concentration to give further test concentrations of 56, 32, 18 and 10 mg/L.

Each stock prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours (see Appendix 5).

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.

Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 2) in a temperature controlled room at approximately 20 °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 (Desmodesmus subspicatus) and Tetramin® flake 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.

A positive control (Harlan Laboratories Ltd., Study Number: 41104946) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 3. The positive control was conducted between 29 November 2011 and 1 December 2011.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

EXAMPLE

Test conditions

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Temperature was maintained at 20 °C to 21 °C throughout the test, while there were no treatment related differences for oxygen concentration or pH.
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

pH:

The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
The pH was measured using a WTW pH/Oxi 340I pH meter.

Dissolved oxygen:

The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentration was measured using a dissolved oxygen meter.
See in appendix 6 for results.

Salinity:

freshwater used.

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. The test item was dissolved directly in water.

Details on test conditions:

Exposure Conditions
As in the range-finding test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test 5 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 at 20 °C to 21 °C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods with a light intensity of 867 to 891 lux. 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 exposure period. Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.


Physico-Chemical Measurements
Water temperature and light intensity were recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1.

No significant immobilization was observed at the test concentrations of 0.10, 1.0 and 10 mg/L. However, immobilization was observed at 100 mg/L.

Based on this information test concentrations of 10, 18, 32, 56 and 100 mg/L were selected for the definitive test.

Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 48 hours (see Appendix 5) showed near nominal measured test concentrations were obtained indicating that the test item was stable under test conditions.


Definitive Test
Immobilization Data
Cumulative immobilization data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 1.

There was no significant immobilization in 20 daphnids exposed to test concentrations 10, 18, 32, 56 and 100 mg/L for a period of 48 hours. Inspection of the immobilization data gave the following results:

Time (h) EC50 (mg/L) 95% Confidence limits
(mg/L)
24 >100 -
48 >100 -

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg/L. The No Observed Effect Concentration is based upon no significant immobilization at this concentration.

A single immobilized daphnid was observed in the 10, 18 and 56 mg/L test preparations. Given that no further immobilization occurred this was considered to be due to natural causes rather than a true toxic effect.


Observations on Test Item Solubility
All control and test item preparations were observed to clear colorless solutions throughout the duration of the test.

Physico-Chemical Measurements
The results of the physico-chemical measurements are given in Appendix 6. Temperature was maintained at 20°C to 21°C throughout the test, while there were no treatment related differences for oxygen concentration or pH.

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Verification of Test Concentrations
Analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed measured test concentrations to range from 94% to 101% of nominal value and so it was considered justifiable to estimate the EC50 values in terms of the nominal test concentrations only.

Results with reference substance (positive control):

A positive control (Harlan Laboratories Ltd., Study Number: 41104946) used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.

Analysis of the immobilization data by the geometric mean method at 24 and the trimmed Spearman-Karber method (Hamilton et al 1977 ) at 48 hours based on the nominal test concentrations gave the following results:

Time (h)
EC50 (mg/L) 95% Confidence limits
(mg/L)
24 1.3 1.0 - 1.8
48 1.1 1.0 - 1.3

The No Observed Effect Concentrations after 24 and 48 hours were 1.0 and 0.56 mg/L respectively. The No Observed Effect Concentration is based upon zero 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

Table1     Cumulative Immobilization Data in the Range-finding Test

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
0.10	0	0
1.0	1*	1*
10	1*	1*
100	2	2

*Single immobilized daphnid considered to be due to natural causes rather than a true toxic effect given that no further immobilization was observed to have occurred and that less than 10% of the daphnids were observed to be immobilized

Table 2     Cumulative Immobilization Data in the Definitive Test

Nominal Concentration (mg/L)	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)
	24 Hours						48 Hours
	R1	R2	R3	R4	Total	%	R1	R2	R3	R4	Total	%
Control	0	0	0	0	0	0	0	0	0	0	0	0
10	0	0	0	0	0	0	1*	0	0	0	1	5
18	0	0	0	0	0	0	0	0	0	1*	1	5
32	0	0	0	0	0	0	0	0	0	0	0	0
56	1*	0	0	0	1	5	1*	0	0	0	1	5
100	0	0	0	0	0	0	0	0	0	0	0	0

R₁– R₄= Replicates 1 to 4

*Single immobilized daphnid considered to be due to natural causes rather than a true toxic effect given that no further immobilization was observed to have occurred and that less than 10% of the daphnids were observed to be immobilized

Appendix 6   Physico-Chemical Measurements

Nominal Concentration (mg/L)		0 Hours				24 Hours	48 Hours
		pH	mg O2/L	%*	T°C	T ºC	pH	mg O2/L	%*	T°C
Control	R1	8.0	9.3	102	20	20	8.1	8.7	98	21
	R2	8.0	9.3	102	20	20	8.1	8.6	97	21
	R3	8.0	9.3	102	20	20	8.1	8.6	97	21
	R4	8.0	9.3	102	20	20	8.1	8.7	98	21
10	R1	8.0	9.1	100	20	20	8.1	8.6	97	21
	R2	8.0	9.1	102	21	20	8.1	8.6	97	21
	R3	8.0	9.1	100	20	20	8.1	8.6	97	21
	R4	8.0	9.1	100	20	20	8.1	8.6	97	21
18	R1	8.1	9.0	101	21	20	8.1	8.6	97	21
	R2	8.1	9.0	101	21	20	8.1	8.6	97	21
	R3	8.1	9.0	101	21	20	8.1	8.6	97	21
	R4	8.1	9.0	101	21	20	8.1	8.6	97	21
32	R1	8.1	8.9	100	21	20	8.1	8.5	96	21
	R2	8.1	8.9	100	21	20	8.1	8.6	97	21
	R3	8.1	8.9	100	21	20	8.1	8.5	96	21
	R4	8.1	9.0	101	21	20	8.1	8.6	97	21
56	R1	8.1	8.9	100	21	20	8.1	8.6	97	21
	R2	8.1	8.9	100	21	20	8.1	8.6	97	21
	R3	8.1	8.9	100	21	20	8.1	8.5	96	21
	R4	8.1	9.0	101	21	20	8.1	8.5	96	21
100	R1	8.1	8.9	100	21	20	8.1	8.6	97	21
	R2	8.1	9.0	101	21	20	8.1	8.6	97	21
	R3	8.1	9.0	101	21	20	8.1	8.6	97	21
	R4	8.1	8.9	100	21	20	8.1	8.5	96	21

*= Dissolved oxygen concentration expressed as a percentage of Air Saturation Value

R₁– R₄= Replicates 1 to 4

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 gave a 48-Hour EC50 of greater than 100 mg/L. The No Observed Effect Concentration 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

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to an aqueous solution of the test item at 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 number of immobilized Daphnia were recorded after 24 and 48 hours.

 

Results

The 48-Hour EC₅₀for the test item to Daphnia magna based on nominal test concentrations was greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

 

Analysis of the test preparations at 0 and 48 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.

 

Conclusion

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC₅₀of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.