ethyl (2S,3S)-3-[(2-chloro-5-fluoro-4-pyrimidinyl)amino]bicyclo[2.2.2]octane-2-carboxylate

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:

2018-02-16 to 2018-02-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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

Test material information

Constituent 1

Reference substance name:

ethyl (2S)-3-[(2-chloro-5-fluoropyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-carboxylate

EC Number:

821-183-0

Cas Number:

1777721-57-9

Molecular formula:

C15H19ClFN3O2

IUPAC Name:

ethyl (2S)-3-[(2-chloro-5-fluoropyrimidin-4-yl)amino]bicyclo[2.2.2]octane-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Physical state: solid
Appearance: White powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16DD2146
- Expiration date of the lot/batch: 2018-04-27 (retest date)
- Purity test date: 2017-11-14 (certificate of analysis release date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: 0.02 g/L in water

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for possible analysis were taken from all test concentrations and the control at t=0 and t=48h. 3.0 mL of volume was taken. At the end of the exposure period, the replicates were pooled at each concentration before sampling.
- Sample storage conditions before analysis: The samples were stored in a freezer. Additionally, reserve samples of 3.0 mL were taken for possible analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to an expected low water solubility of the test item, a saturated solution (SS) was prepared. The preparation of test solutions started with a loading rate of 100 mg/L applying three days of magnetic stirring at room temperature to ensure maximum dissolution of the test item in test medium. This resulted in a clear and colourless dispersion that contained undissolved floating material and precipitate. The obtained mixture was filtered through a 0.45 µm membrane filter (Whatman; RC55) to remove the undissolved fraction. The filter was pre-conditioned with a small volume of test solution that was discarded. The resulting SS was used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water-flea
- Source: in-house laboratory culture with a known history
- Age at study initiation (mean and range, SD): < 24 h (from parental daphnids of more than 2 weeks old, maximum age 4 weeks)
- Method of breeding: 250 newborn daphnids (i.e. less than 3 days old) are placed into 5 liters of medium in an all-glass culture vessel. Medium is M7. After 7 days of cultivation half of the medium is renewed twice a week.
- Feeding during cultivation: fresh water algae, daily
- Feeding during test: no

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L as CaCO3

Test temperature:

19-20°C

pH:

At t=0h: 8.1 - 8.2
At t=48h: 8.0 - 8.1

Dissolved oxygen:

At t=0h: 9.2 - 9.3
At t=48h: 9.1 - 9.4

Salinity:

not relevant

Nominal and measured concentrations:

Final test (final test):
Nominal concentrations: 0, 100 % SS prepared at a loading rate of 100 mg/L
Measured concentrations (mg/L) at t=0h: 0.0070*, 18.0
Measured concentrations (mg/L) at t=48h: n.d., 17.7
n.d. = not detected
* Estimated value, calculated by extrapolation of the calibration curve. Maximum contribution to the t=0 hours 100 % of SS sample is 0.034%, based on response and taking the dilution factor into account.

Details on test conditions:

TEST SYSTEM
- Test vessel: vessel
- Material, size, headspace, fill volume: 50 mL, all-glass filled with 50 mL
- Aeration: not during the test
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Test medium: adjusted ISO medium

OTHER TEST CONDITIONS
- Photoperiod: 16h light daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Immobility was defined as those organisms which are not able to swim within 15 seconds after gentle agitation of the test vessel and was monitored after 24 and 48 hours of continuous treatment with the test substance.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: combined limit/range-finding test
- Range finding study
- Test concentrations: 0, 10 and 100 % of a SS prepared at a loading rate of 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Mortality: no
- EC50/LC50: 48-h EC50 = 0.54 mg/L. The sensitivity of the daphnids was within the range determined with the historical data collected at Charles River Den Bosch.

Reported statistics and error estimates:

No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum concentration tested).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of T003688 to Daphnia magna was determined in a 48 hour static test according to OECD guideline 202. Based on initially measured concentrations, the 48-h EC50 was >18 mg/L (being considered the maximum solubility of test item in a filtrate prepared at a loading rate of 100 mg/L). The results of the test can be considered reliable without restriction.