Thieno[3,2-c]pyridine-5(4H)-acetic acid, .alpha.-(2-chlorophenyl)-6,7-dihydro-, methyl ester, (.alpha.S)-, (1R,4S)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-1-methanesulfonate (1:1)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study according to international accepted guidelines and GLP compliant.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[(1R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl]methanesulfonic acid; methyl (2S)-2-(2-chlorophenyl)-2-{4H,5H,6H,7H-thieno[3,2-c]pyridin-5-yl}acetate

EC Number:

601-680-7

Cas Number:

120202-68-8

Molecular formula:

C16H16ClNO2S.C10H16O4S

IUPAC Name:

[(1R)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptan-1-yl]methanesulfonic acid; methyl (2S)-2-(2-chlorophenyl)-2-{4H,5H,6H,7H-thieno[3,2-c]pyridin-5-yl}acetate

Test material form:

solid: crystalline

Details on test material:

Test item: Clopidogrel camphorsulphonic acid salt (CAS: 120202-68-8)
Batch No.: 648038N (0090D94)
Physical state: Solid, crystalline powder
Colour: Almost white
Storage: At a temperature of 15 to 30 °C, protected from light and moisture

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

In the test the following nominal concentrations were applied: 9.10; 4.55; 3.03; 2.28; 1.52; 0.91; 0.61 and 0.30 mg/L.
At the start of the test the concentration of the test item was determined in the remaining test solution of the concurrent test vessels at each dilution levels.
The test the concentration of the test item of pooled samples for each concentration was measured at the end.
Before the analysis the samples were filtered. The first 10 ml filtrates were disposed.

Test solutions

Vehicle:

yes

Details on test solutions:

OECD medium was used for inoculum and test cultures according to OECD 201 guideline. Deionised water was used for preparing the stock solutions and media. The stock solutions were sterilized by filtration. For the solid medium of seed stock cultures 5 % agar was added to the OECD medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

Test species: Pseudokirchneriella subcapitata (Korshikov) F.Hindák (formerly known as Selenastrum capricornutum)
Source: UTEX The Culture Collcetion of Algae (USA)
The seed stock culture is maintained on solid OECD medium. The alga cells are passed from old seed stock cultures to fresh solid medium in every two or three weeks. The new seed cultures are kept under continuous illumination for five or seven days. After this grow phase they are kept in refrigerator.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

21 ± 2 °C

pH:

At the beginning of the test the pH –values were measured in the remaining test solution of the concurrent test vessels at each dilution levels.
At the end of the test the pH-values were measured in each test vessels.
The pH of test solutions was not adjusted at the beginning of the test.

Nominal and measured concentrations:

In the test the following nominal concentrations were applied: 9.10; 4.55; 3.03; 2.28; 1.52; 0.91; 0.61 and 0.30 mg/L.
The measured concentraions at the start of the test: 8.60, 4.23, 2.80, 2.13, 1.43, 0.83, 0.56 and 0.25 mg/L.
The measured concentraions at the end of the test: 8.06, 4.04, 2.79, 2.09, 1.39, 0.81, 0.54 and 0.25 mg/l.

Details on test conditions:

Eight test solutions of different concentration were prepared by mixing appropriate volume of OECD medium and stock solution of the test item. These concentrations were arranged in a geometric series with a maximum separation factor 2.0.
Three replicates were prepared at every test and six at the control level. The initial alga concentration was 4 965 cells/ml. The vessels were closed with rubber plugs with a filter for ensuring the proper air exchange. The vessels were kept in incubator at 21 ± 2 °C with continuous
illumination. The vessels contained 75 ml test media. At 24, 48 and 72 hours after the beginning of the test the alga cell concentrations were determined by microscopically counting with a Bürker chamber. The initial alga cell density was calculated from the dilution ratio of inoculum culture.

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.44 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

4.86 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.43 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CL

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 1.41 mg/L

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The ECx for specific growth rate were determined by ToxRat® statistic software.
The No Observed Effect Concentration (NOEC) and Lowest Observed Effect Concentration (LOEC) of the test item were determined with Fisher’s exact binominal test with Bonferroni correction by ToxRat® software for specific growth rate.
The three lowest test concentrations were not involved in the statistical evaluation of the effective concentrations (ECx) of the test item as these concentrations were out of the validated concentration range.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

According to this Freshwater Alga and Cyanobacteria, Growth Inhibition Test for Clopidogrel camphorsulphonic acid salt the results indicated that the test item is hazardous to the aquatic environment [GHS Acute aquatic hazard Category Acute II. – 48 hr EC50 (for algae or other aquatic plants) > 1 mg/L but ≤ 10 mg/L].