4H-Pyrido[1,2-a]pyrimidin-4-one, 9-hydroxy-3-(2-hydroxyethyl)-2-methyl-

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:

2015-09-14 to 2015-10-08

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:

9-hydroxy-3-(2-hydroxyethyl)-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one

EC Number:

605-935-3

Cas Number:

181525-38-2

Molecular formula:

C11H12N2O3

IUPAC Name:

9-hydroxy-3-(2-hydroxyethyl)-2-methyl-4H-pyrido[1,2-a]pyrimidin-4-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Name of test material (as cited in study report): JNJ-200395-AAA (T002416)
- Physical state: solid
- Appearance: light yellow
- Analytical purity: >97.4%
- Batch n°: I14FB2740
- Expiration date: 26 June 2016
- Storage condition: at room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test and after 24 and 72 hours from all test concentrations and from the control. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling. Reserve samples were taken from all test solutions for possible 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: The highest test concentration was prepared at a loading rate of 100 mg/L applying a magnetic stirring (combined limit/range-finder: 43 minutes; final test: 73 minutes). Adequate volumes of the highest test concentration were diluted with test medium to prepare the test media with the lower test item concentrations.
- Controls: yes

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: 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): same as 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:

22-22.5°C

pH:

7.6 - 8.4

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

nominal test concentrations final test: 4.6, 10, 22, 46 and 100 mg/L
measured test concentration final test t= 0 h: 4.06, 8.74, 17.3, 31.2 and 63.8 mg/L
measured test concentration final test t= 24h: 0.410, 0.0819, 0.011, n.d. and 0.0082 mg/L (n.d.: not detected)
measured test concentration final test t= 72h: 0.0118, 0.029, n.d., n.d. and 0.0063 mg/L (n.d.: not detected)

At the start of the test, the actual test concentrations were close to or just below nominal. The concentrations all decreased rapidly, i.e. by 90% or more already during the first 24 hours of exposure. It can be seen from the analysis that the decrease of the test item is related to degradation processes due to the formation of degradation products. In general Time Weight Average (TWA) exposure concentrations are calculated in similar cases with decreasing concentrations. However, this calculation resulted in TWA concentrations that showed no dose related increase and as such could not be used to explain the observed dose related increase in biological effects. As a consequence, the only alternative was to base the biological results on the initial measured exposure concentrations.
The range tested based on initial measured concentrations was 4.1, 8.7, 17, 31 and 64 mg/L.

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: 153.7 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: 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 720 nm using a spectrophotometer with immersion probe.
- effect calculated parameters: specific growth rate and yield

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2

- Range finding study
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L (nominal)
- 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.:

45 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%CL: 45-46 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

31 mg/L

Nominal / measured:

meas. (initial)

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: All concentrations decreased rapidly, i.e. by 90% or more within the first 24 hours of exposure. Additional peaks were observed, increasing in time. The rate of decrease differed between test concentrations, i.e. the % decrease in the lowest test concentration was much less than the % decrease in the highest test concentration1. Due to the varying rates of concentration decreases among the test concentrations, it was assumed that factors other than degradation had a significant influence. Decreases in the solution incubated at 22 mg/l with and without algae showed a similar decrease indicating that adsorption to algae did not add to the loss of test substance. A possible explanation for the different decrease rates could be degradation due to photolysis. In that case light shielding in the lower test concentrations (from the increasing algal biomass) might have reduced the rate of degradation. Photolysis as a possible cause was supported by the fact that the decreases in test concentrations observed in an acute daphnia study (WIL Research project 509058) performed at lower light intensity were much less significant.
- EC50 (yield) = 35 mg T002416 /L (95% cl 34-37 mg/L).
- EC10 (growth rate) = 32 mg T002416 / L (95% cl 31-33 mg/L).
- EC10 (yield) = 22 mg T002416 / L (95% cl 20-23 mg/L).
- NOEC (yield) = 8.7 mg T002416 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L (95% CL: 1.1 to 1.2 mg/L)

Reported statistics and error estimates:

- NOEC and EC values were determined using a Williams Multiple Sequential t-test Procedure (alpha=0.05, one-sided, smaller).
- Calculation of ECx values was based on probit analysis using linear max. likelihood regression.
- Time Weight Average (TWA) exposure concentrations are calculated in similar cases with decreasing concentrations. However, this calculation resulted in TWA concentrations that showed no dose related increase and as such could not be used to explain the dose related increase in biological
effects. As a consequence, the results were based on the initial measured exposure concentrations.

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 T002416 according to the OECD guideline 201. The concentrations of the test item decreased rapidly (i.e. by 90% or more already during the first 24 hours of exposure likely due to photolysis).
Under the conditions of the present study, T002416 reduced growth rate of this fresh water algae species significantly at an initial measured concentration of 64 mg/L. The 72-h ECr50 was 45 mg T002416/L based on initial measured test concentrations.
The results of the test can be considered reliable without restrictions.