[No public or meaningful name is available]

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:

FROM 21 OCT 2022 TO 28 OCT 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23, 2006; Annex 5 corrected 28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide

Cas Number:

1950587-20-8

Molecular formula:

C20H17F4N5O2

IUPAC Name:

4-[(1-[[6-cyano-5-(trifluoromethyl)pyridin-3-yl]carbamoyl]cyclobutyl)amino]-2-fluoro-N-methylbenzamide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 22CN0611
- Expiration date of the lot/batch: 2023-05-11 (retest date)
- Purity: 99.9%
- Purity test date: 2022-05-31 (certificate of analysis release date)
- Physical appearance: brown powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under storage conditions: not indicated
- 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:

yes

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 2.0 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 22% of the SS 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 2.0 mL were taken from all test solutions 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 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 10,000 cells/mL.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All test solutions were clear and colorless at the end of the preparation procedure

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- 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 1E+04 cells/mL. The pre-culture was maintained under the same conditions as used in the test. Cell density was measured before use.
ACCLIMATION
- Acclimation period: not relevant

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:

21-24°C

pH:

At t=0h: 8.1
At t=72h: 8.7-8.9

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Conductivity:

Not relevant

Nominal and measured concentrations:

nominal test concentrations final test: 0 (blank), 4.6, 10, 22, 46, 100% of a S.S. prepared at 100 mg/L with algae and 22% of a S.S. prepared at 100 mg/L without algae (abiotic control)
measured test concentration final test t= 0 h: measured test concentration final test t= 24 h: measured test concentration final test t= 72 h:
LOQ is 0.07 mg/L
*sample without daphnids and algae (abiotic control at 22% of 100 mg/L S.S.)

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
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- 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

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 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.
OTHER TEST CONDITIONS
- Photoperiod: continuous illumination
- illumination: TLD-lamps with a light intensity within the range of 80 to 85 μE/m²/s.
- Adjustment of pH: none

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter;
spectrophotometer; colorimeter] 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.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Test concentrations: 4.6, 10, 22, 46 and 100 % of the SS (range finding study)
- 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:

NOEC

Effect conc.:

0.28 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.28 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: C.L. 95% not determined

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
- 72-h EC50 (yield) >0.28 mg/L
- 72-h EC10 (growth rate) >0.28 mg/L
- 72-h EC10 (yield) >0.28 mg/L
- 72-h NOEC (yield) 0.28 mg/L

Results with reference substance (positive control):

Experiment performed: August 2022
The batch of Raphidocelis subcapitata tested showed expected sensitivity to Potassium dichromate, based on the historical range of reference tests performed by the Test Facility in the last ten years.
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:

ECx VALUES
The ECx-values (based on both growth rate and yield) could not be determined because the observed effects were below 10%.

NOEC value (growth rate)
For determination of the NOEC value, a Shapiro-Wilk's test indicated that the treatment data did not significantly deviated from a normal distribution (p = 0.560), passing the normality check. A subsequent Levene's test on Variance Homogeneity (with residuals) indicated that the homogeneity of variance check was also passed (p = 0.320).
To justify the use of Williams test, a trend analysis by contrasts was performed, indicating the absence of both a linear trend (p = 0.416) and quadratic trend (p = 0.176). Therefore, the selected Williams test was replaced by a Dunnett's multiple t-test procedure, using a significance level Alpha of 0.050 (one-sided smaller; multiple level). The growth rate at any of the test concentrations did not significantly differ from the growth rate of the control. Hence, the NOEC for growth rate appeared was ≥ 100 %ss 100 mg/L.

NOEC value (yield)
For determination of the NOEC value, a Shapiro-Wilk's test indicated that the treatment data did not significantly deviated from a normal distribution (p = 0.963), passing the normality check. A subsequent Levene's test on Variance Homogeneity (with residuals) indicated that the homogeneity of variance check was also passed (p = 0.300).
To justify the use of Williams test, a trend analysis by contrasts was performed, indicating the absence of both a linear trend (p = 0.412) and quadratic trend (p = 0.167). Therefore, the selected Williams test was replaced by a Dunnett's multiple t-test procedure, using a significance level Alpha of 0.050 (one-sided smaller; multiple level). The growth rate at any of the test concentrations did not significantly differ from the growth rate of the control. Hence, the NOEC (yield) appeared was ≥ 100 %ss 100 mg/L.

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

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of JNJ-63632335-AAA (T003671) tested.
The 72h-NOEC for growth rate and yield inhibition was 0.28 mg/L based on statistical significance.
Due to the very low solubility of JNJ-63632335-AAA (T003671) in test medium, concentration levels that might be toxic for algae could not be reached.