1-benzyl-N-phenylpiperidin-4-amine

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:

2016-08-26 to 2016-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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:

1-benzyl-N-phenylpiperidin-4-amine

EC Number:

214-583-6

EC Name:

1-benzyl-N-phenylpiperidin-4-amine

Cas Number:

1155-56-2

Molecular formula:

C18H22N2

IUPAC Name:

1-benzyl-N-phenylpiperidin-4-amine

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-1594008-AAA (T000293)
- Physical state: solid (powder)
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15JB3618
- Expiration date of the lot/batch: 2019-10-09 (retest date)
- Purity: 99.8% (based on base titration assay)
- Purity test date: 2016-02-12 (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: 0.04 g/L in water

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=0, t=24h and t=48h. 1.8 mL of volume was taken from the approximate centre of the test vessels. In addition, the filter containing undissolved residue was kept for possible analysis. 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 (< -15°C). Additionally, reserve samples of 1.8 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 three-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.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): final test solutions were clear and colourless

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. M1 medium was used.
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in M2 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.

ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before the start of the test)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21-23°C

pH:

7.8-8.4

Dissolved oxygen:

not reported

Salinity:

not applicable

Conductivity:

not relevant

Nominal and measured concentrations:

nominal test concentrations combined limit/range-finding test: 1.0 and 100% of a SS at 100 mg/L
measured test concentration combined limit/range-finding test t= 0 h: 0.414, 43.8 mg/L
measured test concentration combined limit/range-finding test t= 24h: 0.393, 44.5 mg/L
measured test concentration combined limit/range-finding test t= 72h: 0.243, 44.2 mg/L

nominal test concentrations final test: 0.32, 1.0, 3.2, 10, 32 and 100% of a SS at 100 mg/L
measured test concentration final test t= 0 h: 0.114, 0.371, 1.24, 3.66, 11.7, 37.6 mg/L
measured test concentration final test t= 24h: 0.107, 0.324, 1.19, 3.79, 12.0, 40.4 mg/L
measured test concentration final test t= 72h: 0.0476, 0.254, 0.997, 3.79, 12.1, 40.5 mg/L

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: yes continuously
- 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: 150 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Standard medium used: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis
- Detailed composition if non-standard medium was used:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

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: Yes (M1 versus M2). Three days before the start of the test the algal stock culture (culture in M1) was inoculated in the same culture medium (M2) used in the test. The culture was maintained under the same conditions as used in the test.
- Intervals of measurements: pH was measured at the beginning and at the end of the test. Temperature was continuously measured in a control vessel. At the end of the final test microscopic observations were performed on all test concentrations to observe for any abnormal appearance of the algae.

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 680 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: x 3.2
- 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.:

5.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CL 95%: 5.2-6.3 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.084 mg/L

Nominal / measured:

meas. (TWA)

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:
- EC50 (yield) = 2.1 mg T000293 /L
- EC10 (growth rate) = 2.6 mg T000293 / L (95% C.L. 2.2-2.9 mg/L).
- EC10 (yield) = 0.27 mg T000293 / L (95% C.L. 0.096-0.48 mg/L).
- NOEC (yield) = 0.084 mg T000293 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50:
72-h EC50 (growth rate) = 1.0 mg/L (95% CI 0.97 - 1.0 mg/L)
72-h EC50 (yield) = 0.43 mg/L (95% CI 0.42 - 0.44 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 test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, alpha=0.05, one-sided, smaller).
Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.
Calculation of ECx values was based on weibit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding TWA 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 with the test substance T000293 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, T000293 iinhibited growth rate of this fresh water algae species significantly at TWA concentrations of 3.8 mg/L and higher. The EC50 for growth rate inhibition (72h-ERC50) was 5.7 mg/L with a 95% confidence interval ranging from 5.2 to 6.3 mg/L The results of the test can be considered reliable without restrictions.