4-amino-2-methylpyrimidine-5-methylamine

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:

7 July 2010 to 29 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-2-methylpyrimidine-5-methylamine

EC Number:

202-384-7

EC Name:

4-amino-2-methylpyrimidine-5-methylamine

Cas Number:

95-02-3

Molecular formula:

C6H10N4

IUPAC Name:

5-(aminomethyl)-2-methylpyrimidin-4-amine

Details on test material:

- Name of test material (as cited in study report): "GREWE-DIAMIN"
- Chemical name: 4-Amino-2-methyl-5-pyrimidinmethanamine
- Trade name: Grewediamin
- Supplier: Sponsor
- Molecular formula: C6H10N4
- CAS Nr.: 95-02-3
- Charge Nr.: M-622-035-200
- Appearance: White to yellowish crystals
- Melting point: 128-132 °C
- Solubility in water: Ca. 430 g/L
- Density: 1.083 g/cm3
- pH: 10.3 (430 g/L)
- Conditions of storage: Room temperature, in the dark, may be used under light
- Stability at room temperature: Stable
- Stability at conditions of storage: Stable
- Expiry date: February 2011

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: nominal 0.15, 0.46, 1.37, 4.11, 12.30, 37.00, 111.10, 333.30 and 1000.00 mg/L
- Sampling method: At the start and at the end of the incubation period, samples of the test media were drawn and the concentrations of the test substance were determined in aliquots of the blank containing test substance, but no algae and of one replicate of each of the test and control cultures (the 72 hours samples were filtered through a 0.20 µm filter).
One sample was taken of each of the above listed aliquots.
- Sample storage conditions before analysis: The samples drawn at the start of the incubation were deep frozen. The samples drawn at the end of the test were analysed on the day of sampling together with the thawn samples taken at the start of the test.

Test solutions

Vehicle:

no

Details on test solutions:

- Method: A stock solution with a nominal test substance concentration of 1111.1 mg/L was prepared. The test substance was first grinded with a morter, afterwards 1121.8 mg test substance were dissolved in 1 L of nutrient medium by manual homogenisation. Aliquots of this stock solution were diluted with nutrient medium to obtain the test solutions, nominally spaced by a factor of 3.
- Controls: For the negative control group only nutrient medium was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle was used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: ATCC (American Type Culture Collection) 22662.
- Source (laboratory, culture collection): LGC Promochem GmbH, Germany
- Method of cultivation: Stock cultures, initially cultivated from deep frozen algae, are continuously maintained in 250 mL conical flasks (Erlenmeyer), each containing 100 mL nutrient medium, at a temperature in the range of 21 to 24 (± 2) °C under permanent light with an intensity between 4400 and 8800 lux. In about weekly intervals 1 mL of the stock culture is diluted 100-fold with nutrient medium for precultivation and incubation is continued.

ACCLIMATION
- Acclimation period: Precultures were prepared by incubating a new stock culture for four days.
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: no.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None.

Test conditions

Test temperature:

Between 22 and 23°C.

pH:

Due to the basic nature of the test substance, the pH was between 7.6 and 9.5 at the start of the incubation in the test cultures and it was 7.4 in the control cultures.
After 72 hours of incubation the pH was between 7.9 and 9.7 in the test cultures and it was 9.5 in the control cultures. The pH in the control cultures changed by 2.1 units during the 72 hours of incubation, i.e. by more than 1.5 units recommended by the guideline. As this did not adversely affect algal growth or the outcome of the experiment the change in pH was tolerated.

Nominal and measured concentrations:

Nominal: 0.15, 0.46, 1.37, 4.11, 12.30, 37.00, 111.10, 333.30 and 1000.00 mg/L
Measured: 0h: 0.12, 0.44, 1.45, 3.98, 12.79, 35.50, 120.96, 336.53 and 1018.50 mg/L
72h: 0.11, 0.43, 1.39, 3.73, 12.92, 35.35, 117.34, 346.29 and 1020.01 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL closed Erlenmeyer flasks filled with 100 mL medium .
- Initial cells density: 10 000 cells/mL
- Control end cells density: the cell density in the control cultures increased by a factor of about 140.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes.
- Sterile deionised water and sterile concentrated nutrient medium 9 + 1 was used.
- Conductivity of the deionised water: <5 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: The test cultures are prepared under steril conditions.
- Adjustment of pH: no.
- Photoperiod: 24 h.
- Light intensity and quality: between 5540 and 6350 lux. Wavelength of 400 to 700 nm.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic cell counter with Casy Cell Counter after 24, 48, and 72 hours of incubation
- Chlorophyll measurement: no.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.0
- Justification for using less concentrations than requested by guideline: not applicable.
- Range finding study: no.

Reference substance (positive control):

yes

Remarks:

Reference test 72h EC50 (K2Cr2O7): for growth rate 1.32 mg/L and yield 0.62 mg/L

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

37 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

35.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

35.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

81.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

85.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): During the 72 hours incubation period the cell density in the control cultures increased by a factor of about 140, corresponding to about 7.1 generations.
- Observation of abnormalities (for algal test): None
- Colour differences: The test vessels containing the highest test substance concentration were coloured slightly yellow during the first 48 hours of incubation. No other change in the colour of the media and no precipitation of test substance was noted during the incubation period.
- Any stimulation of growth found in any treatment: Yes. A maximum of 1.8 % growth stimulation was observed.
- Effect concentrations exceeding solubility of substance in test medium: No.

Growth Inhibition:
- Based on the yield and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 8.1 % enhancement to 99.8% inhibition.
- Based on the average growth rates and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 1.8 % enhancement to 94.8 % inhibition.

Results with reference substance (positive control):

The last reference test with K2Cr2O7 was conducted from the 21st to the 24th of June 2010 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg K2Cr2O7/L, respectively. These results establish the reliability of the test procedures for this kind of study type.

Reported statistics and error estimates:

Based on the yield as well as on the average growth rates two "lowest observed effective
concentrations" (LOECs) are calculated by comparison of the data of the three replicates of
each test substance culture with the negative control (analysis of variance, followed by the
Scheffé-test, P = 0.05). The "no observed effect concentrations" (NOECs) are then derived
from these results (highest concentration with no statistically significant difference to the
control).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

As the test substance concentrations have been satisfactorily maintained to within 80 % of the nominal concentrations throughout the duration of the test the effective concentrations may be based on the nominal concentrations:
EC50 (0-72h) based on the average growth rates = 81.3 mg/L
NOEC (0-72h) based on the average growth rates = 37.0 mg/L

Executive summary:

A Pseudokirchneriella subcapitata growth inhibition test according to the EC regulation 761/2009 Part C.3 and the OECD-Guideline 201 (adopted by the Council on 23rd March 2006)was performed to determine the possible effects of"GREWE-DIAMIN"on the growth of a unicellular green algal species.

Nine different concentrations of"GREWE-DIAMIN"between nominal 0.15 and 1000.00 mg per litre nutrient medium, spaced by a factor of about 3.0, were tested against one concurrent negative control (nutrient medium only).

Three replicates were used for each test culture and six replicates for the control culture. The cell count of the algae was about 10 000 cells/mL at the start of the exposure in each vessel.

Possible test substance effects were determined by comparison of the yield and of the growth rates.

At the start and at the end of the experiment, samples of the cultures exposed to the test substance, of the control cultures and also of the blank with the highest concentration of the test substance, but no algae, were taken and analysed in duplicate by HPLC.

As the test substance concentrations have been satisfactorily maintained to within 80 % of the nominal concentrations throughout the duration of the test the effective concentrations may be based on the nominal concentrations:

EC50 (0-72h) based on the average growth rates = 81.3 mg/L

NOEC (0-72h) based on the average growth rates = 37.0 mg/L