2-Pyridinecarbonitrile, 5-amino-3-(trifluoromethyl)-

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:

08 Jan 2021 to 26 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile

EC Number:

815-137-9

Cas Number:

573762-62-6

Molecular formula:

C7H4F3N3

IUPAC Name:

5-amino-3-(trifluoromethyl)pyridine-2-carbonitrile

Details on test material:

- Appearence : Slightly yellowish brown fine powder
- CAS Number: 573762-62-6
- Molecular formula: C7H4F3N3
- Molecular weight: 187.12

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- lot/batch number of test material: BI03K2005005
- Expiry date: 02 June 2021 (retest date)
- Physical Description: Almost white to yellow powder (determined by Charles River Den Bosch)
- Purity: 99.7%
- Purity correction factor: no correction factor required

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

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 vessels.
- Storage Samples were stored in a freezer (≤-15°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 32 mg/L but without algae (abiotic control) and samples for analysis were taken at the start and at the end of the test period. Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional 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 concentration of 100 mg/L applying one hour of ultrasonic treatment to accelerate dissolution of the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): All final 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 (according to the OECD 201 Guideline) 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 immediately before use.
ACCLIMATION
- Acclimation period: not relevant (except pre-culture 3 days before 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:

Between 21-22 °C

pH:

7.9-8.2

Dissolved oxygen:

Not reported

Salinity:

Not relevant

Conductivity:

Not relevant

Nominal and measured concentrations:

final test:
- nominal concentrations: 0 (control), 10, 18, 32, 56 and 100 mg/L
- measured concentrations (0 h): nd, 9.40, 17.3, 32.0, 65.1, 95.2 mg/L
- measured concentration (72 h): nd 10.1, 18.3, 32.2, 66.9, 101 mg/L

Samples taken from all test concentrations and the control were analysed. The measured concentrations were in agreement with the nominal concentrations, i.e. varied between 94 and 119% relative to the nominal values at the start and the end of the 72-hour exposure period. No test item was detected in the samples taken from the control solution. Based on these results, effect parameters were expressed as analytically confirmed nominal concentrations. The concentrations measured in the solution without algae (abiotic control) were comparable with the concentrations measured in the samples with algae. This indicates that the presence of algae does not affect the test item concentrations in test medium.

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: 132.8 x 10,000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 5

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 2O 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 60 to 120 μE/m²/s.
- 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 (range-finding test)
- Spacing factor for test concentrations: 10 (range finding study); 1.8 (final test)
- Range finding study
- Test concentrations: 0.10 to 100 mg/L (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:

EC50

Effect conc.:

89 mg/L

95% CI:

>= 85 - <= 93

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.:

18 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: no
The 72h-NOEC for yield was 10 mg/L based on statistical significance.
The 72h-EC50 for yield was 41 mg/L based on statistical significance.

Results with reference substance (positive control):

Experimental Start Date: 15 Jan 2021
Experimental Completion Date: 21 Jan 2021
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:

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, 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). 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. ToxRat Professional v 3.2.1 (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, JNJ-61232574-AAA (T003630) inhibited growth rate of this freshwater algae species significantly at analytically confirmed nominal concentrations of 32 mg/L and higher.
The 72h-EC50 for growth rate inhibition (ERC50) was 89 mg/L with a 95% confidence interval ranging from 85 to 93 mg/L.
The 72h-EC50 for yield inhibition (EYC50) was 41 mg/L with a 95% confidence interval ranging from 37 to 46 mg/L.
The 72h-NOEC for growth rate inhibition was 18 mg/L based on statistical significance.
The 72h-NOEC for yield inhibition was 10 mg/L based on statistical significance.
The results of the test can be considered reliable without restriction.