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

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:

29 December 2017 - 16 January 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

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 were taken from the highest test concentration (100 mg/L) and the control according to the schedule below.
Frequency: at t=0 and t=48 h
Volume: 1.0 and 2.0 mL
Storage: Samples were stored in a freezer (≤ -15°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: direct application to test medium; 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 30 minute 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg CaCO3/L

Test temperature:

19-20 °C (throughout the test)

pH:

7.8-8.1 (throughout the test, in the control vessel)
7.2-7.8 (throughout the test, in the test solution)

Dissolved oxygen:

At the start of the test: 9.5-9.7 mg O2/L (in all vessels)
At the end of the test: 8.6-8.7 mg O2/L (in all vessels)

Nominal and measured concentrations:

Nominal concentrations: 0.10, 1.0, 10 and 100 mg/L
Measured concentrations at 100 mg/L (cation): 109 mg/L at t=0; 109 mg/L at t=48
Measured concentrations at 100 mg/L (anion): 115 mg/L at t=0; 114 mg/L at t=48
The measured concentrations at the end of the study were 100% (cation) and 99% (anion) of initial measured concentrations at t=0.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass, fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4 for the highest concentration, 2 for the other concentrations
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, adjusted ISO medium
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours, daily
- Other: the test was performed in the dark

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, for the highest concentration and the control. Temperature of the medium: continuously in a temperature control vessel.

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: as only 1 daphnia was immobilised at the highest concentration after 48 hours, the combined limit/range-finding study was used as the final study and effect parameters were based on the results of this study.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (January 2018)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Analysis showed that test concentrations remained stable during the exposure concentrations: test concentrations after 48 hours were 99-100% of the initial measured concentrations at t=0 (see table 1). Since measured test concentrations were comparable to nominal concentrations (109-115% of nominal), the effect parameter was based on the nominal concentrations.
- At the end of the exposure concentration, only one daphnid was immobilised in the highest concentration and therefore the EC50 was determined to exceed the highest test concentration (i.e. >100 mg/L). No immobilisation was observed at any of the other test concentrations.
- The pH and temperature during the study remained within the limits prescribed by the study plan.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Tested concentrations: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- 48h-EC50: 0.54 mg/L, 95%-confidence interval: 0.49-0.61 mg/L
- Other: results fell within the historical range.

Reported statistics and error estimates:

No analysis was conducted as the test item proved to be non-toxic at the concentrations tested.

Any other information on results incl. tables

Table 1 Measured concentrations in the test samples - cation

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	100	109	109
48	0	n.d.	n.a.
	100	109	109	100

n.d. Not detected; n.a. Not applicable

Table 2 Measured concentrations in the test samples - anion

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	n.d.	n.a.
	100	115	115
48	0	n.d.	n.a.
	100	114	114	99

n.d. Not detected; n.a. Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'overall remarks'

Conclusions:

In an acute toxicity study, performed according to OECD 202 and GLP principles, the 48-h EC50 of BMS-482204-03 towards Daphnia magna exceeded the highest tested concentration (i.e. >100 mg/L).