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Diss Factsheets

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
FROM 28 OCT 2022 TO 17 NOV 2022
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
according to guideline
other: Guidance Document on Aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD series on testing and assessment number 23 (2nd edition)
Version / remarks:
Adopted February 08, 2019
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide
Cas Number:
Molecular formula:
1-amino-N-[6-cyano-5-(trifluoromethyl)-3- pyridyl]cyclobutanecarboxamide
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: JNJ-63632283-AAA (T003665)
- Physical state: solid (powder)
- Appearance: white powder
Specific details on test material used for the study:
- Source and lot/batch number of test material: 22BN0551
- Purity: 98.3%
- Physical appearance: off-white powder
- Expiration date of the lot/batch: 2023-04-28 (retest date)
- Purity test date: 2022-05-13 (certificate of analysis release date)
- Storage condition of test material: at room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not indicated
- Treatment of test material prior to testing: no treatment

Sampling and analysis

Analytical monitoring:
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t=24 h and t=72 h.
Volume 1.8 mL from the approximate centre of the test solutions.
Storage Samples were stored in a freezer (set to maintain -20°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at 10% of the SS prepared at a loading rate of 100 mg/L but without algae (abiotic control) and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.
Additionally, reserve samples of 1.8 mL were taken from all test solutions for possible analysis.

Test solutions

Details on test solutions:
- 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 material in the test medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1.0 mL of an algal suspension was added to each replicate providing a cell density of 1 x 10⁴ cells/mL.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final test solutions were clear and colourless

Test organisms

Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Method of cultivation: Algal 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 10⁴ cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.

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

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
72 h

Test conditions

24 mg CaCO3/L
Test temperature:
Between 21-24 °C
At t=0h 7.9-8.0
At t=72h 8.1-9.0
Dissolved oxygen:
Not reported
Not relevant
Not relevant
Nominal and measured concentrations:
nominal test concentrations final test: 0 (blank), 0.32, 1.0, 3.2, 10, 32 and 100% of a S.S. prepared at 100 mg/L with algae and 10% of a S.S. prepared at 100 mg/L without algae
measured test concentration final test t= 0 h: measured test concentration final test t= 24 h: measured test concentration final test t= 72 h:
Time-weighted average (TWA; mg/L) determined with the measured concentrations reported above, were: 0.12, 0.54, 2.0, 7.1, 7.2, 23 and 76 mg/L, for the test concentrations prepared at 0.32, 1.0, 3.2, 10 (with algae), 10 (without algae), 32 and 100% SS, respectively.

LOQ = 0.1 mg/L
*result of repeated analysis after re-dilution since the response of initial analysis was outside the calibrated range
** sample without algae
Details on test conditions:
- Test vessel: glass flasks with aluminium caps, perforated for ventilation
- 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
- 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 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 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

- 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 were 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 water quality measurement: temperature measured continuously, pH at the beginning and the end of the test.
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 84 to 91 μE/m²/s.
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: At the beginning of the test, 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 (path length = 20 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.

Range finding study
- Test concentrations: 0.32, 1.0, 3.2, 10, 32 and 100% of the SS
- Results used to determine the conditions for the definitive study: yes.
Reference substance (positive control):
Potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
72 h
Dose descriptor:
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (TWA)
Basis for effect:
Key result
72 h
Dose descriptor:
Effect conc.:
0.12 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
72 h
Dose descriptor:
Effect conc.:
24 mg/L
95% CI:
>= 23 - <= 25
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
72 h
Dose descriptor:
Effect conc.:
5 mg/L
95% CI:
>= 4.2 - <= 5.9
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
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: no
Other effect parameters:
- 72-h EC10 (yield) 1.1 (0.70 - 1.4) mg/L
- 72-h EC10 (growth rate) 5.8 (5.4 - 6.2) mg/L
- 72-h EC20 (yield) 1.9 (1.4 - 2.3) mg/L
- 72-h EC20 (growth rate) 9.8 (9.1 - 10) mg/L
Results with reference substance (positive control):
Experiment performed: August 2022.
The batch of Raphidocelis subcapitata tested, showed higher sensitivity to Potassium dichromate (EC50: 1.6 mg/L with a 95% confidence interval ranging from 1.5 to 1.8 mg/L) based on the range specified in ISO International Standard 8692, February 2012. Sensitivity of the batch of algae was however within the historical range of reference tests performed by the Test Facility in the last ten years. This has no impact on the results of the toxicity studies as increased sensitivity provides the worst-case.
The raw data from this study are kept in the Charles River Den Bosch archives. The test described above was performed non-GLP.
Reported statistics and error estimates:
For determination of the NOEC and the ECx 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, α=0.05, one-sided, smaller). This statistical comparison was preceded by a check on normal distribution of the data (Shapiro-Wilk’s Test) and a test for homogeneity of the variances (Levene’s test).
For both yield and growth rate, both the normality (p > 0.01) and variance homogeneity (p > 0.01) checks were passed. The Trend analysis by Contrasts to justify the use of the Williams Multiple Sequential t-test Procedure showed a significant linear trend (p < 0.001) and a significant quadratic trend (p < 0.001).

Calculation of ECx-values was based on a 3-parameter logistic cumulative distribution function (CDF) using non-linear regression analysis with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test material.

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata, JNJ 63632283-AAA (T003665) inhibited growth rate and yield of this freshwater algae species significantly at TWA concentrations of 0.54 mg/L higher.
The 72h-EC50 for growth rate inhibition (ErC50) was 24 mg/L with a 95% confidence interval ranging from 23 to 25 mg/L.
The 72h-EC50 for yield inhibition (EyC50) was 5.0 mg/L with a 95% confidence interval ranging from 4.2 to 5.9 mg/L.
The 72h-NOEC for both growth rate and yield inhibition was 0.12 mg/L based on statistical significance.