(1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-carboxamide

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:

29 December 2017 - 10 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-carboxamide

EC Number:

615-206-1

Cas Number:

709031-45-8

Molecular formula:

C6H10N2O.CH4O3S

IUPAC Name:

(1S,3S,5S)-2-azabicyclo[3.1.0]hexane-3-carboxamide

Test material form:

solid

Details on test material:

- Appearance: Light tan solid
- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

- Test item handling: use amber glassware or wrap container in aluminum-foil.
- The test item is soluble in water.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h, t=24 h and t=72 h
Volume: 1.0 and 2.0 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 exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: weighing of the test item and preparation of the test solution was performed under dimmed light.
The test solution was prepared at a loading rate of 100 mg test item/L applying a 37 minute (combined limit/range-finding test) and 16 minute (final test) period of magnetic stirring to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Evidence of undissolved material: no, all test solutions were clear and colorless at the end of the preparation procedure.
- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis)) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

PRE-CULTURE
- 3 (final test) or 4 (combined limit/range-finding test) days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium as used in the final test: M2 medium, according to OECD 201 Guideline.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21-23 °C

pH:

At the start of the test: 7.5 - 8.1
At the end of the test: 7.5 - 7.8

Nominal and measured concentrations:

Nominal concentrations: 1.0, 3.2, 10, 10 (without algae), 32 and 100 mg/L
TWA measured concentrations (cation): 0.86, 2.9, 8.7, 8.9, 22 and 54 mg/L, respectively to nominal concentrations stated above.
For cation and anion measured concentrations per time point, see table 1 and 2.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass vessels, containing 50 mL of test solution.
- Aeration: no
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 253 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6 for the highest concentration (100 mg/L), 3 for the other concentrations
- No. of vessels per control (replicates): 6
- In addition: 1 or 2 replicates of the highest concentration without algae; 1 or 2 replicates of each test concentration for sampling purposes.

GROWTH MEDIUM
- Medium used: M2 medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2; according to the OECD 201 Guideline, formulated using Milli-RO water.
- Intervals of water quality measurement: pH measured at beginning and end of test; temperature of medium continuously measured in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Continuously using TLD-lamps with a light intensity of 87-89 µE.m-2.s-1.

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm). Algal medium was used as blank (background for the treated solutions).
- Other: appearance of cells was observed with a microscope at the end of the final test on the highest test concentration to observe for any abnormal appearance of the algae.

LIMIT/RANGE-FINDING STUDY:
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes, at 10 and 100 mg/L growth was inhibited for 2.7 and 53%. Based on these results, the 72h-ErC50 was expected to be between a nominal concentration of 10 and 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate (test performed Jan 2018).

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 54 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

cation

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

20 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

cation

Basis for effect:

growth rate

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Remarks:

cation

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities: Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to a concentration of 54 mg/L (TWA) when compared to the control.
- Table 1 and 2 show the measured concentrations at different time points for the cation and the anion, respectively. The analysis showed that test concentrations varied in stability at different time points and concentrations. Due to unexpected results in the first analysis, reserve samples were analysed at the two highest concentrations for both the cation and the anion. As a worst case approach, the effect concentrations were based on the final exposure TWA, where the TWA concentrations of the two highest concentrations were calculated as the avaerage of the duplicate measurements.
- Only at the highest concentration a statistically significant effect of 32% growth inhibition was observed. Based on these findings, the 72h-ErC50 was determined to exceed the highest test concentration. Using linear regression, the EC50 was calculated to be 99 mg/L.
- No significant effect on growth rate was observed at a TWA concentration of 22 mg/L (NOEC), whereas the EC10 was calculated with the use of linear maximum likelihood regression to be 20 mg/L.
- Experimental conditions were within the range prescribed by the study plan throughout the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Tested concentrations: 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- 72-h ErC50: 0.86 mg/L, 95%-CI: 0.84-0.88 mg/L
- Other: the effect parameter was within the historical range of the test facility.

Reported statistics and error estimates:

- NOEC: Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller.
- ECx values: probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA concentrations of the test item. The two lowest concentrations were excluded as these values were not relevant and decreased the fit of the model by overestimating the effect parameters. The EC50 exceeded the highest tested concentration but was calculated using the linear regression model as well.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Table 1 - Measured concentrations in test samples - cation

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	0.00032^b	n.a.
	1	1.06	106
	3.2	3.37	105
	10	9.78	98
	10^a	9.64	96
	32	26.7	84
	100	53.1	53
24	0	0.00041^b	n.a.	n.a.
	1	0.904	90	85
	3.2	3.00	94	89
	10	9.11	91	93
	10^a	9.48	95	98
	32	24.1	75	90
	100	44.1	44	83
72	0	0.0016^b	n.a.	n.a.
	1	0.705	71	66
	3.2	2.43	76	72
	10	7.65	77	78
	10^a	7.83	78	81
	32	22.2	70	83
	100	64.6	65	121
0^c	32	26.0	81
	100	66.1	66
24^c	32	22.4	70	86
	100	50.3	50	76
72^c	32	14.5	45	56
	100	59.2	59	90

^a       Without algae.
^b       Estimated value, calculated by extrapolation of the calibration curve. A similar response was observed in the analytical blank. The response was lower than the lowest calibration standard. 

^c       Reserve samples.

n.a.   Not applicable.

Table 2 Measured concentrations in test samples - anion

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	1	0.757	76
	3.2	2.87	90
	10	10.2	102
	10^a	9.27	93
	32	31.7	99
	100	84.5	85
24	0	n.d.	n.a.	n.a.
	1	0.820	82	108
	3.2	2.76	86	96
	10	9.00	90	88
	10^a	9.73	97	105
	32	32.1	100	101
	100	76.0	76	90
72	0	n.d.	n.a.	n.a.
	1	1.13	113	149
	3.2	2.86	89	100
	10	10.2	102	100
	10^a	9.81	98	106
	32	28.3	89	89
	100	75.9	76	90
0^b	32	32.2	101
	100	96.2	96
24^b	32	33.3	104	103
	100	97.0	97	101
72^b	32	33.8	106	105
	100	95.8	96	100

^a       Without algae.

^b       Reserve samples.

n.d. Not detected.

n.a. Not applicable.

Table 3 Growth rate and percentage inhibition at different time intervals

Test item TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h		Total test period
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	2.408		1.658		1.463		1.843
0.86	3	2.343	2.7	1.653	0.3	1.498	-2.4	1.831	0.6
2.9	3	2.353	2.3	1.696	-2.3	1.539	-5.2	1.863	-1.1
8.7	3	2.355	2.2	1.626	1.9	1.487	-1.6	1.823	1.1
22	3	2.350	2.4	1.744	-5.2	1.513	-3.5	1.869	-1.4
54	3	2.130	12	0.967	42	0.673	54	1.257	32*

* - effect was statistically significant

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'Overall Remarks'

Conclusions:

Based on the results of a toxicity study towards algae, performed according to OECD guideline 201 and GLP principles, the 72h-ErC50 and the 72h-NOErC of BMS-482204-03 were determined to be >54 and 22 mg/L, respectively (based on TWA measured concentrations).