N-phenyl-N-piperidin-4-ylpropionamide

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:

2016-05-09 to 2016-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to standard test guidelines (OECD, EC) and conform GLP requirements. The light intensity was not recorded but since all the validity criteria were met, it can be concluded that the study integrity was not adversely affected by this deviation. The results of the study can be considered reliable without restriction.

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

N-phenyl-N-piperidin-4-ylpropionamide

EC Number:

216-543-3

EC Name:

N-phenyl-N-piperidin-4-ylpropionamide

Cas Number:

1609-66-1

Molecular formula:

C14H20N2O

IUPAC Name:

N-phenyl-N-piperidin-4-ylpropionamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-1594255-AAA (T000425)
- Physical state: solid (powder)
- Appearance: white

Specific details on test material used for the study:

- Name of test material (as cited in study report): T000425
- Substance type: no data
- Physical state: white solid
- Analytical purity: 100%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: 00454795
- Expiration date of the lot/batch: 2005-12-31
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: solubility: 43 g/L in water, 500 g/L in ethano

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling, except for the 18 mg/L concentration where all three replicates were sampled individually.
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 and at the end of the test period.
Additionally, reserve samples of 1.4 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
- Sample storage conditions before analysis: stored in a freezer (< -15°C) until 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 the highest test concentration of 100 mg/L. No special treatment other than vigorous shaking was needed to fully dissolve the test item in the test medium. The pH was adjusted with HCl (1 mol/L, Merck, Darmstadt, Germany) from 9.6 to 7.1 and from 9.4 to 7.9 in the combined limit/range-finding and the final test, respectively. Lower test concentrations were prepared by subsequent dilutions of the highest concentration 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):

Pseudokirchneriella subcapitata (previous names: Raphidocelis 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): M1 according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

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:

21-23°C

pH:

t=0h: 7.9-8.2
t=72h: 7.8-7.9

Dissolved oxygen:

not reported

Salinity:

not relevant

Nominal and measured concentrations:

nominal test concentrations final test: 10, 18, 32, 56 and 100 mg/L
measured test concentration final test t= 0 h: 9.35, 17.3, 31.5, 32.1, 53.2, 96.8 mg/L
measured test concentration final test t= 72h: 9.58, 17.1, 17.4, 17.7, 32.0, 33.4, 54.1, 98.4 mg/L

Samples taken from all test concentrations and the control were analysed. The measured concentrations were in agreement with the nominal concentrations throughout the test period (93-104%).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all-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
- Initial cells density: 10,000 cells/mL
- Control end cells density: 211.579 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= 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: Continuously using TLD-lamps with a light intensity within the range of 84 to 90 μE/m2/s.

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 1.8
- Range finding study
- Test concentrations: 0.1, 1,0 and 10 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

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% C.L. 62-74 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

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: clear test solution
- EC50 (yield) = 22 mg T000425 /L
- EC10 (growth rate) = 22 mg T000425 / L (95% C.L. 18-26 mg/L).
- EC10 (yield) = 8.1 mg T000425 / L (95% C.L. 5.9-10 mg/L).
- NOEC (yield) = 10 mg T000425 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1 mg/L (95% C.L.: 1.0 to 1.1 mg/L)
- other: The historical ranges for growth rate inhibition lie between 0.82 and 2.6 mg/L. The observed 72h-ERC50 for the algal culture tested corresponds with this range

Reported statistics and error estimates:

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test item. 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 wit the test substance T000425 according to the OECD guideline 201 (GLP conditions). The measured concentrations were in agreement with the nominal concentrations throughout the test period (93-104%). Based on these results, effect concentrations were expressed in terms of
analytically confirmed nominal concentrations. The 72-h EC50 based on growth rate was determined to be 68 mg/L, the NOEC based on growth rate was 10 mg/L. The results of the test can be considered reliable without restrictions.