METHYL (1R,2R,4S)-2-[HEX-5-EN-1-YL(METHYL)CARBAMOYL]-4-({7-METHOXY-8-METHYL-2-[4-(PROPAN-2-YL)-1,3-THIAZOL-2-YL]QUINOLIN-4-YL}OXY)CYCLOPENTANECARBOXYLATE

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:

2016-05-04 to 2016-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

methyl (1R,2R,4S)-2-[(hex-5-en-1-yl)(methyl)carbamoyl]-4-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)cyclopentane-1-carboxylate

EC Number:

922-147-8

Cas Number:

1042695-87-3

Molecular formula:

C32H41N3O5S

IUPAC Name:

methyl (1R,2R,4S)-2-[(hex-5-en-1-yl)(methyl)carbamoyl]-4-({7-methoxy-8-methyl-2-[4-(propan-2-yl)-1,3-thiazol-2-yl]quinolin-4-yl}oxy)cyclopentane-1-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-39125593-AAA (T003019)
- Physical state: solid
- Appearance: white powder

Specific details on test material used for the study:

- Batch n°: M15FB2925
- Analytical purity: 99.5%
- Expiration date: 2017-05-31 (re-test date)
- Storage condition: at room temperature protected from light
- Stability under storage conditions: stable until retest date. Analysis of stability, homogeneity and concentration of the test item under test conditions was not performed as part of this study.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples for possible analysis were taken form all test concentrations and the control at t=0h, t=24h and t=72h. 2.0 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 2.0 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: Preparation of test solutions started with a loading rate of 100 mg/L applying a two-day period of magnetic stirring to ensure maximum dissolution of the test item in test medium. The resulting aqueous mixture was filtered through a 0.45 μm membrane filter (Whatman; RC55) to remove the undissolved fraction of the test item. The obtained Saturated Solution (SS) was used as the highest test concentration. The lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All final test solutions were clear and colourless. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
- Controls: yes

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 (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algae stock cultures were started by inoculating growth medium 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.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as the test. 3 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.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21-22°C

pH:

7.7-8.3

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

Nominal test concentrations final test: 1.0, 10 and 100% of a SS prepared at 100 mg/L
Measured test concentration final test t= 0 h: <0.01, 0.462, 0.479 µg/L
Measured test concentration final test t= 24 h: <0.01, <0.01, <0.01 µg/L
Measured test concentration final test t= 72h: <0.01, <0.01, <0.01 µg/L

The concentration was not stable throughout the exposure duration, i.e. it decreased below the concentration of the lowest calibration solution within 24 hours of exposure.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass flasks
- Type (delete if not applicable): capped vessels
- Material, size, headspace, fill volume: 100 mL all-glass flasks filled with 50 mL test solution
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through system applied
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 10,000 cells/mL
- Control end cells density: 159.5 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 according to the OECD 201 guideline, formulated using Milli-RO water
- Culture medium different from test medium: no
- Intervals of water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: none
- Photoperiod: continuous illumination
- Light intensity and quality: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm, using TLD-lamps.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] At the beginning, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 10

- Range finding study
- Test concentrations: 1.00, 10 and 100 % of the SS
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: v/V % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 other: v/v % saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Additional peaks were observed in chromatograms at a different retention time than the test item. Intensity of the peaks increased with time. The appearance of the additional peaks occurred possibly due to degradation or reaction.
- EC50 (yield) = >0.019 µg T003019 /L
- EC10 (growth rate) = >0.019 µg T003019 /L
- EC10 (yield) = >0.019 µg T003019 /L
- NOEC (yield) = 0.019 µg T003019 /L

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.0 mg/L (95% C.L. 1.0-1.1 mg/L)

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the highest test concentration compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Two-sample t-test Procedure, α=0.05, onesided, smaller). No EC50-values could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested). The calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

A 72-h growth inhibition test with the unicellular green alga Pseudokirchneriella subcapitata was performed with the test substance T003019 according to the OECD guideline 201.
Under the test conditions, no significant inhibition of growth rate or inhibition of yield of this fresh water algae species was tested. The EC50 for growth rate inhibition (72h-ErC50) and the EC50 for yield inhibition (72h-EyC50) were beyond the concentration range tested, i.e. exceeded a TWA concentration of 0.019 μg/L (which is considered to be the maximum solubility of the test item in test medium prepared at a loading rate of 100 mg/L).
The results of the test can be considered reliable without restrictions.