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

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:

2015-10-12 to 2015-11-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Principles of method if other than guideline:

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 pH at the end of the test was below 6.0 at a nominal concentration of 100 mg/L. The effect at 100 mg/L fits well into the dose-response curve, therefore it was considered that the pH did not significantly influence the effect. The results of the study can be considered reliable without restriction.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-(2-CHLOROETHYL)-9-HYDROXY-2-METHYL-4H-PYRIDO[1,2-A]PYRIMIDIN-4-ONE HYDROCHLORIDE (1:1)

EC Number:

617-648-0

Cas Number:

849727-62-4

Molecular formula:

C11H11ClN2O2.HCl

IUPAC Name:

3-(2-CHLOROETHYL)-9-HYDROXY-2-METHYL-4H-PYRIDO[1,2-A]PYRIMIDIN-4-ONE HYDROCHLORIDE (1:1)

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification JNJ-1692405-AAC (T002006)
Appearance Almost white to ochre powder (determined by WIL Research Europe at receipt of the test item)
Batch I15BB0722
Test item storage conditions At room temperature
Stable under storage conditions until 21 February 2017 (retest date)
- Analytical purity: 97.5% (calculated on the anhydrous basis)
- Impurities (identity and concentrations): R117091 <0.10%, unspecified <0.10%, water content <0.2%
- Purity test date: 2015-02-22
- Stability under storage conditions: stable
- Other: solubility in water: 60g/L

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples were taken before the start of the test and after 24h 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 test medium of the highest nominal test item concentration of 100 mg/L was prepared applying a vigorous shaking. 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:

21-24°C

pH:

4.6 - 8.0

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

nominal test concentrations : 1.0, 3.2, 10, 32 and 100 mg/L
measured test concentration t= 0 h: 0.853, 2.85, 9.28, 31.1 and 98.8 mg/L
measured test concentration t= 24h: 0.113, 0.370, 1.62, 5.55 and 45.1 mg/L
measured test concentration t= 72h:

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: 194.8 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: no.
- Photoperiod: continuous illumination
- Light intensity and quality: 92 to 94 µE.m-2.s-1 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: x 3.2
- Range finding study
- Test concentrations: 0.10, 1.0 and 10 mg/L
- 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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 4.6 - 6.2 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.41 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: clear test solution
- EC50 (yield) = 1.4 mg T002006 /L (95% cl 1.4-1.5 mg/L)
- EC10 (growth rate) = 0.72 mg T002006 / L (95% cl 0.53-0.92 mg/L).
- EC10 (yield) = 0.55 mg T002006 / L (95% cl 0.47-0.62 mg/L).
- NOEC (yield) = 0.41 mg T002006 / L.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: 72-h EC50 (growth rate) = 1.2 mg/L (95% CI 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) (yield) or Welch-t-test for inhomogeneous Variance with Bonferroni-Holm Adjustement (alpha=0.05, one-sided, smaller)(growth rate).

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 T002006 according to the OECD guideline 201 (GLP conditions).
Under the test conditions with Pseudokirchneriella subcapitata, the 72-h ECr50 was 5.3 mg/L (95% cl 4.6-6.2 mg/L), and the 72h-NOEC was 0.41 mg/L.
The results of the test can be considered reliable without restrictions.