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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 February 2012 and 8 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:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

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 :100%
Expiry / retest date :Not provided
Storage conditions :room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Range-Finding Test
The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was directly in culture medium.

An amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (10 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

The control group was maintained under identical conditions but not exposed to the test item.

At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron- Version 2 incubator) at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

A sample of each test concentration was taken for chemical analysis at 0 and 72 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 result of the range-finding test a "limit test" was conducted at a concentration of 100 mg/L to confirm that at the maximum concentration given in the OECD/EEC Test Guidelines no effect on algal growth was observed.

Test solutions

Vehicle:

no

Details on test solutions:

Experimental Preparation
For the purpose of the definitive test, the test item was dissolved directly in culture medium.

An amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 1 liter to give a100 mg/L stock solution.

The stock solution and the prepared concentration were inverted several times to ensure adequate mixing and homogeneity.

The concentration and stability of the test item in the test preparations verified by chemical analysis at 0 and 72 hours (see Appendix 3).

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and constant illumination at 21 ± 1 °C.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 103 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 105 cells/mL.

A positive control (Harlan Laboratories Ltd Project Number: 41104038) used potassium dichromate as the reference item. Details of the positive control are given in Appendix 1. The positive control was conducted between 10 October 2011 and 13 October 2011.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Test conditions

Hardness:

Not recorded.

Test temperature:

Temperature was maintained at 24 ± 1ºC throughout the test. The temperature within the incubator was recorded daily.

pH:

The pH of each control and test flask was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. Please see table 1.

Dissolved oxygen:

Not recorded.

Salinity:

freshwater used

Nominal and measured concentrations:

The test concentration to be used in the definitive test was determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

Details on test conditions:

Exposure Conditions
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and 100 mg/L treatment group.

The control group was maintained under identical conditions but not exposed to the test item.

Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 3.21 x 10E5 cells per mL. Inoculation of 1 liter of test medium with 15.6 mL of this algal suspension gave an initial nominal cell density of 5 x 103 cells per mL and had no significant dilution effect on the final test concentration.

The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron- Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.


Physico-Chemical Measurements
The pH of the control and 100 mg/L test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL not stated

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

cell number

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test
The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1.

The results showed no effect on growth at the test concentrations of 0.10, 1.0, 10 and 100 mg/L.

Based on this information a single test concentration of six replicates, of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC Test Guidelines no effect on growth was observed.

Chemical analysis of the 100 mg/L test preparation at 0 and 72 hours (See Appendix 3) showed measured test concentrations of 98% and 99% of nominal respectively were obtained indicating that the test item was stable under test conditions.

Definitive Test
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 2. Daily specific growth rates for the control cultures are given in Table 3. Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4.

The mean cell densities versus time for the definitive test are presented in Figure 1.


Validation Criteria
The following data show that the cell concentration of the control cultures increased by a factor of 185 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Mean cell density of control at 0 hours : 5.13 x 10E3 cells per mL
Mean cell density of control at 72 hours : 9.32 x 10E5 cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 7% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 – 72 h) was 1% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

Growth Data
From the data given in Table 2 and Table 4, it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of the test item at a nominal test concentration of 100 mg/L over the 72-Hour exposure period.

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Accordingly the following results were determined from the data:

Inhibition of Growth Rate
ErC10 (0 - 72 h) : >100 mg/L
ErC20 (0 - 72 h) : >100 mg/L
ErC50 (0 - 72 h) : >100 mg/L

where ErCx is the test concentration that reduced growth rate by x%.

Statistical analysis of the growth rate data was carried out for the control and 100 mg/L test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P0.05), between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on growth rate was 100 mg/L.

Inhibition of Yield
EyC10 (0 - 72 h) : >100 mg/L
EyC20 (0 - 72 h) : >100 mg/L
EyC50 (0 - 72 h) : >100 mg/L

Where:

EyCx is the test concentration that reduced yield by x%.

Statistical analysis of the yield data was carried out as in Section 4.2.2.1. There were no statistically significant differences (P0.05), between the control and 100 mg/L test group and therefore the "No Observed Effect Concentration" (NOEC) based on yield was 100 mg/L.


Observations on Cultures
All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.


Observations on Test Item Solubility
At the start of the test all control and 100 mg/L test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and 100 mg/L test cultures were observed to be green dispersions.


Physico-Chemical Measurements
The pH values of the control and 100 mg/l test preparation are given in Table 1. Temperature was maintained at 24 ± 1 ºC throughout the test.

The pH value of the control cultures (see Table 2) was determined to be pH 8.0 at 0 and 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.


Verification of Test Concentrations
Analysis of the test preparations at 0 and 72 hours (see Appendix 3) showed measured test concentrations to range from 99% to 100% of nominal 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):

Positive Control
A positive control (Harlan Laboratories Ltd Project No: 41104038) used potassium dichromate as the reference item at concentrations of 0.0625, 0.125, 0.25, 0.50 and 1.0 mg/L.
 
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
 
Exposure ofPseudokirchneriella subcapitata(CCAP 278/4) to the reference item gave the following results:
 
ErC50(0 – 72 h)          :          1.4 mg/L, 95% confidence limits 1.2 – 1.7 mg/L EyC50(0 – 72 h)          :          0.59 mg/L, 95% confidence limits 0.53 – 0.65 mg/L
 
No Observed Effect Concentration (NOEC) based on growth rate:          0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield:                     0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate:    0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield:              0.50 mg/L
 
The results from the positive control withpotassiumdichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical Analysis
A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L test concentration to determine any statistically significant differences between the test and control groups. All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Any other information on results incl. tables

Table1     Cell Densities and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/L)		Cell Densities*(cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	7.40E+03	1.43E+06	-	-
	R2	6.45E+03	1.51E+06
	Mean	6.92E+03	1.47E+06
0.10	R1	5.17E+03	1.44E+06	[7]	6
	R2	4.41E+03	1.32E+06
	Mean	4.79E+03	1.38E+06
1.0	R1	5.62E+03	1.43E+06	[3]	8
	R2	5.70E+03	1.28E+06
	Mean	5.66E+03	1.35E+06
10	R1	5.90E+03	1.51E+06	[1]	10
	R2	6.06E+03	1.13E+06
	Mean	5.98E+03	1.32E+06
100	R1	5.79E+03	1.10E+06	3	26
	R2	6.28E+03	1.07E+06
	Mean	6.04E+03	1.09E+06

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁and R₂= Replicates 1 and 2

[Increase in growth compared to controls]

Table2     Cell Densities and pH Values in the Definitive Test

Nominal Concentration (mg/L)		pH	Cell Densities* (cells per mL)				pH
		0 h	0 h	24 h	48 h	72 h	72 h
Control	R1	8.0	5.00E+03	2.85E+04	1.76E+05	8.86E+05	8.0
	R2		5.16E+03	3.23E+04	1.44E+05	9.88E+05
	R3		5.36E+03	2.80E+04	1.50E+05	7.93E+05
	R4		5.08E+03	3.12E+04	1.69E+05	9.72E+05
	R5		5.14E+03	3.24E+04	1.89E+05	9.99E+05
	R6		5.04E+03	2.75E+04	1.69E+05	9.53E+05
	Mean		5.13E+03	3.00E+04	1.66E+05	9.32E+05
100	R1	7.8	5.02E+03	2.37E+04	1.62E+05	9.57E+05	8.0
	R2		5.27E+03	3.22E+04	1.52E+05	9.68E+05
	R3		5.10E+03	2.26E+04	1.48E+05	9.55E+05
	R4		5.21E+03	3.22E+04	1.74E+05	9.66E+05
	R5		5.05E+03	2.49E+04	1.35E+05	9.63E+05
	R6		5.08E+03	2.87E+04	1.35E+05	9.64E+05
	Mean		5.12E+03	2.74E+04	1.51E+05	9.62E+05

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R₁- R₆= Replicates 1 to 6

Table3     Daily Specific Growth Rates for the Control Cultures in the Definitive Test

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 - 1	Day 1 - 2	Day 2 - 3
Control	R1	0.073	0.076	0.067
	R2	0.078	0.062	0.080
	R3	0.072	0.070	0.069
	R4	0.076	0.071	0.073
	R5	0.078	0.074	0.069
	R6	0.071	0.076	0.072
	Mean	0.075	0.072	0.072

 

R₁- R₆= Replicates 1 to 6

Table4     Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 – 72 h	% Inhibition	0 – 72 h	% Inhibition*
Control	R1	0.072		8.81E+05
	R2	0.073		9.83E+05
	R3	0.070		7.87E+05
	R4	0.073	-	9.67E+05	-
	R5	0.074		9.94E+05
	R6	0.073		9.48E+05
	Mean	0.073		9.26E+05
	SD	0.001		7.89E+04
100	R1	0.073	0	9.52E+05
	R2	0.073	0	9.63E+05
	R3	0.073	0	9.50E+05
	R4	0.073	0	9.61E+05
	R5	0.073	0	9.58E+05
	R6	0.073	0	9.59E+05
	Mean	0.073	0	9.57E+05	[3]
	SD	0.000		5.18E+03

 

*In accordance with the OECD test guideline only the mean value for yield is calculated

R₁-R₆= Replicates 1 to 6

SD= Standard Deviation

[Increase in growth as compared to controls]

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

Executive summary:

Introduction

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 440/2008.

 

Methods

Followinga preliminary range-finding test,Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at a concentration of 100 mg/L (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

 

Results

Exposure of Pseudokirchneriella subcapitata to the test item gave EC₅₀values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.

 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

 

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to range from 99% to 100% of nominal and so the results are based on nominal test concentrations only.

Conclusion

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EC50 values of greater than 100 mg/L. The No Observed Effect Concentration was 100 mg/L.