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EC number: 280-622-9 | CAS number: 83732-72-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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:
- 2016-11-21 to 2016-11-24
- Reliability:
- 1 (reliable without restriction)
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- 6-methoxy-N2-methylpyridine-2,3-diamine dihydrochloride
- EC Number:
- 280-622-9
- EC Name:
- 6-methoxy-N2-methylpyridine-2,3-diamine dihydrochloride
- Cas Number:
- 83732-72-3
- Molecular formula:
- C7H11N3O.2ClH
- IUPAC Name:
- 6-methoxy-N~2~-methylpyridine-2,3-diamine dihydrochloride
Constituent 1
- Specific details on test material used for the study:
- Test item: A130 6-Methoxy-2-methylamino-3-Aminopyridin HCl
Trade name: 3-Amino-2-methylamino-6-methoxypyridine HCl
Batch number: 20151207
CAS number: 83732-72-3
Purity (certified):
93.36/ 93.69 % (Photometric)
99.63% (HPLC area-%)
Sum formula: C7H13Cl2N3O
Appearance: Grey, red, brown powder
Water solubility: >100 g/L
Vapour Pressure: ≤ 1.4*101 Pa (20 °C)
Expiry date : 2017-12-07
Recommended storage : Keep containers tightly closed in a cool and well-ventilated place
Sampling and analysis
- Analytical monitoring:
- yes
- Details on sampling:
- Determination of the test item
The test concentrations of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl were analytically verified via LC/MS-MS at the start (0 hours), after 12, 18 and at the end of the exposure (72 hours) with algae.
Test solutions
- Vehicle:
- no
- Details on test solutions:
- Stock solution, media preparation
10 mg test item/L was freshly prepared with dilution water. The dispersion was agitated until the solution was homogeneous.
Test concentrations (nominal)
5 concentrations were tested in a geometrical series with a dilution factor of 2.5: 20.5 - 51.2 - 128 - 320 - 800 µg/L, corresponding to the time weighted average test item concentration of 3.02 – 3.26 – 3.50 – 4.56 – 5.00 µg/L.
The concentrations are based on the results of two preliminary range finding tests.
Control
Six replicates (without test item) were tested under the same test conditions as the test item replicates. Additionally six replicates were tested in dilution water without ascorbic acid.
Test organisms
- Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.
Study design
- Test type:
- static
- Water media type:
- freshwater
- Limit test:
- no
- Total exposure duration:
- 72 h
- Remarks on exposure duration:
- The method was modified according to OECD 23. The test item solutions are orange. Since this may affect the light intensities for algae cultures the volumes of test media in test vessels were lowered in order to minimize the water layer and path length.
- Post exposure observation period:
- No
Test conditions
- Hardness:
- Not specified
- Test temperature:
- min.: 22.0 max.: 23.0 mean value: 22.5
- pH:
- Nominal test item concentration pH-values
[µg/L] Start; 0 hours End; 72 hours
800 7.40 7.26
320 7.40 7.41
128 7.43 7.47
51.2 7.41 7.46
20.5 7.57 7.59
Control without ascorbic acid 7.51 7.58
Control 7.41 7.47 - Dissolved oxygen:
- Not measured
- Salinity:
- Not measured, freshwater conditions
- Nominal and measured concentrations:
- Nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100 mg/L
- Details on test conditions:
- TEST SYSTEM
- Test vessel: 25 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 25 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6305 cells/mL
- Control end cells density: Mean 2712769 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:
Three-fold concentrated AAP medium according to the guidelines
Composition of Dilution water
Component Concentration [mg/L]
NaNO3 76.5
MgCl2 6 H2O 36.492
CaCl2 2 H2O 13.23
MgSO4 7 H2O 44.1
K2HPO4 3.132
NaHCO3 45
H3BO3 0.5565
MnCl2 4 H2O 1.246
ZnCl2 9.81 x 10-3
CoCl2 6 H2O 4.29 x 10-3
CuCl2 2 H2O 0.036 x 10-3
NaMoO4 2 H2O 21.78 x 10-3
FeCl3 6 H2O 0.4794
Na2-EDTA 0.9
Ascorbic acid 1
pH 7.5 0.1
The pH of the test medium had to be 7.5 0.1 and was adjusted prior to testing with addition of 1 M NaOH or HCl.
OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5370 lux,
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the range finding test. A separate replicate per test item concentration was prepared without algae and treated the same as the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation. - Reference substance (positive control):
- yes
- Remarks:
- (Potassium dichromate)
Results and discussion
Effect concentrationsopen allclose all
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 128 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 380 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: CI: 329 - 669
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 128 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- 213 µg/L
- Nominal / measured:
- nominal
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: CI: 132 - 289
- Details on results:
- - Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Effect concentrations exceeding solubility of substance in test medium: No - Results with reference substance (positive control):
- The toxicity of potassium dichromate (SIGMA ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2016-10-11 to 2016-10-14 (with headspace). The reference item toxicity is in the valid range following test facility SOPS.
EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.436 0.764 ± 0.513
95% confidence interval 0.414 – 0.458
Yield inhibition
EyC50 0.230 0.402 ± 0.293
95% confidence interval 0.179 – 0.322
SD = Standard deviation - Reported statistics and error estimates:
- EC-values and statistical analyses
EC10-, EC20- and EC,50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.
NOEC, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield. As a standard, One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.
The difference between control and control without ascorbic acid was determined by calculation of statistically significant differences of growth rates and yield. A t-test was used for calculations. When running a t-test, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.
Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
•Excel, MICROSOFT CORPORATION
•SigmaPlot, SPSS INC.
•GraphPad Prism, GRAPHPAD SOFTWARE, INC.
Any other information on results incl. tables
Cell Densities
Nominal test item concentration |
Replicate |
Cell density [cells/mL] |
|||||||||
[µg/L] |
No. |
0 hours |
24 hours |
48 hours |
72 hours |
||||||
800 |
1 |
6305 |
20893 |
14717 |
13911 |
||||||
2 |
6305 |
29057 |
25683 |
25426 |
|||||||
3 |
6305 |
18483 |
16262 |
13550 |
|||||||
Mean |
6305 |
22811 |
18887 |
17629 |
|||||||
320 |
1 |
6305 |
18771 |
76884 |
216509 |
||||||
2 |
6305 |
25198 |
106422 |
389235 |
|||||||
3 |
6305 |
35191 |
192204 |
598179 |
|||||||
Mean |
6305 |
26387 |
125170 |
401308 |
|||||||
128 |
1 |
6305 |
34357 |
232634 |
1701426 |
||||||
2 |
6305 |
31267 |
171699 |
1562844 |
|||||||
3 |
6305 |
39916 |
309514 |
2381200 |
|||||||
Mean |
6305 |
35180 |
237949 |
1881823 |
|||||||
51.2 |
1 |
6305 |
24643 |
161350 |
1347797 |
||||||
2 |
6305 |
28699 |
193842 |
1843659 |
|||||||
3 |
6305 |
39180 |
332136 |
2420764 |
|||||||
Mean |
6305 |
30841 |
229109 |
1870740 |
|||||||
20.5 |
1 |
6305 |
19165 |
220966 |
2221548 |
||||||
2 |
6305 |
32541 |
219252 |
1523367 |
|||||||
3 |
6305 |
22520 |
203438 |
2212415 |
|||||||
Mean |
6305 |
24742 |
214552 |
1985777 |
|||||||
Control without ascorbic acid |
1 |
6305 |
40686 |
347472 |
3123741 |
||||||
2 |
6305 |
45052 |
339742 |
2298564 |
|||||||
3 |
6305 |
40178 |
246185 |
2711603 |
|||||||
4 |
6305 |
30061 |
323972 |
2927088 |
|||||||
5 |
6305 |
33525 |
316959 |
3037480 |
|||||||
6 |
6305 |
38473 |
347122 |
2502321 |
|||||||
Mean |
6305 |
37996 |
320242 |
2766800 |
|||||||
Control |
1 |
6305 |
34766 |
372284 |
2113806 |
||||||
2 |
6305 |
45080 |
412224 |
3020058 |
|||||||
3 |
6305 |
46349 |
441801 |
3190439 |
|||||||
4 |
6305 |
31476 |
301325 |
1868328 |
|||||||
5 |
6305 |
41480 |
418065 |
2946708 |
|||||||
6 |
6305 |
40291 |
405819 |
3137272 |
|||||||
Mean |
6305 |
39907 |
391920 |
2712769 |
Evaluation after 72 hours
Statistically significant differences of growth rates and yield compared to control values (with ascorbic acid) are marked (+), not significant differences are marked (-).
Nominal test item concentration |
Replicate |
Growth rate |
Inhibition of growth rate |
Yield |
Inhibition of yield |
||
[µg/L] |
No. |
[d-1] |
[%] |
[cells/mL] |
[%] |
||
800 |
1 |
|
0.264 |
87 |
|
7606 |
100 |
2 |
|
0.465 |
77 |
|
19121 |
99 |
|
3 |
|
0.255 |
87 |
|
7245 |
100 |
|
Mean |
(+) |
0.328 |
84 |
(+) |
11324 |
100 |
|
320 |
1 |
|
1.18 |
41 |
|
210204 |
92 |
2 |
|
1.37 |
32 |
|
382930 |
86 |
|
3 |
|
1.52 |
25 |
|
591874 |
78 |
|
Mean |
(+) |
1.36 |
33 |
(+) |
395003 |
85 |
|
128 |
1 |
|
1.87 |
7 |
|
1695121 |
37 |
2 |
|
1.84 |
9 |
|
1556539 |
42 |
|
3 |
|
1.98 |
2 |
|
2374895 |
12 |
|
Mean |
(-) |
1.89 |
6 |
(-) |
1875518 |
31 |
|
51.2 |
1 |
|
1.79 |
11 |
|
1341492 |
50 |
2 |
|
1.89 |
6 |
|
1837354 |
32 |
|
3 |
|
1.98 |
2 |
|
2414459 |
11 |
|
Mean |
(-) |
1.89 |
6 |
(-) |
1864435 |
31 |
|
20.5 |
1 |
|
1.95 |
3 |
|
2215243 |
18 |
2 |
|
1.83 |
9 |
|
1517062 |
44 |
|
3 |
|
1.95 |
3 |
|
2206110 |
18 |
|
Mean |
(-) |
1.91 |
5 |
(-) |
1979472 |
27 |
|
Control without ascorbic acid |
1 |
|
2.07 |
|
|
3117436 |
|
2 |
|
1.97 |
|
|
2292259 |
|
|
3 |
|
2.02 |
|
|
2705298 |
|
|
4 |
|
2.05 |
|
|
2920783 |
|
|
5 |
|
2.06 |
|
|
3031175 |
|
|
6 |
|
1.99 |
|
|
2496016 |
|
|
Mean |
(-) |
2.03 |
|
(-) |
2760495 |
|
|
Control |
1 |
|
1.94 |
|
|
2107501 |
|
2 |
|
2.06 |
|
|
3013753 |
|
|
3 |
|
2.08 |
|
|
3184134 |
|
|
4 |
|
1.90 |
|
|
1862023 |
|
|
5 |
|
2.05 |
|
|
2940403 |
|
|
6 |
|
2.07 |
|
|
3130967 |
|
|
Mean |
|
2.01 |
|
|
2706464 |
|
Section-by-Section and Average Specific Growth Ratesof the Control Group (0 - 72 hours)
|
Replicate No. |
Specific growth rate [d-1] |
Mean (0 - 72 hours) |
SD ± |
CV |
Mean CV [%] |
||
section-by-section |
||||||||
0 - 24 hours |
24 - 48 hours |
48 - 72 hours |
||||||
Control |
1 |
1.71 |
2.37 |
1.74 |
1.94 |
0.375 |
19.4 |
12.2 |
2 |
1.97 |
2.21 |
1.99 |
2.06 |
0.136 |
6.59 |
||
3 |
1.99 |
2.25 |
1.98 |
2.08 |
0.155 |
7.49 |
||
4 |
1.61 |
2.26 |
1.82 |
1.90 |
0.332 |
17.5 |
||
5 |
1.88 |
2.31 |
1.95 |
2.05 |
0.229 |
11.2 |
||
6 |
1.85 |
2.31 |
2.05 |
2.07 |
0.229 |
11.0 |
||
|
|
|
Mean |
1.92 |
|
|
||
|
|
|
SD ± |
0.02 |
|
|||
|
|
|
CV [%] |
0.86 |
|
SD = Standard deviation CV = Coefficient of variation
Applicant's summary and conclusion
- Validity criteria fulfilled:
- yes
- Conclusions:
- In this study, A130 6-Methoxy-2-methylamino-3-aminopyridine HCl was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on nominal test item concentrations): The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 380 (329 – 669) µg/L and 213 (132 – 289) µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 128 µg/L.
The EC50-values based on time weighted average test item concentrations with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 4.73 (4.59 – 4.97) µg/L and 4.02 (3.53 – 4.39) µg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 3.50 µg/L.
Since no data on volatility was provided by the sponsor, the loss of test item over time is considered to be an effect of abiotic or biotic degradation. Therefore, toxicity effects are considered to be related to the test item and, most likely, a formed mixture of degradation products. - Executive summary:
The toxicity of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl (Batch no.:20151207) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 761/2009 C.3 from2016-11-21 to2016-11-24 at the test facility. The aim of the study was the determination of NOEC, LOEC, EC10, EC20- and EC50-values of growth rate and yield over a period of 72 hours.
The study was conducted under static conditions with an initial cell density of 6305 cells/mL. A stock solution of 10.0 mg test item/L was freshly prepared with dilution water. The definitive test was carried out with five concentrations in a geometrical series with a dilution factor of 2.5:20.5 - 51.2 - 128 - 320 - 800 µg/L, corresponding to the time weighted average test item concentration of 3.02 – 3.26 – 3.50 – 4.56 – 5.00 µg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.
The test media were clear and light yellow at the start of the exposure. A concentration related color-change was observed throughout the test period from light yellow to yellow. The concentrations of thetest itemA1306-Methoxy-2-methylamino-3-aminopyridine HClin all test concentrations and the control were analytically verified by LC-MS/MS at the start, after 12, 18 and 72 hours (end of exposure). Details of the analytical method are presented in part14. The analytical results are presented inTable 11.
The measured concentrations ofA1306-Methoxy-2-methylamino-3-aminopyridineHClin the fresh media (0 h) were between 97 and 116% of the nominal values. The measured concentrations in the media after 12 hours were between < LOQ and 4% of the nominal values. After 18 hours and 72 hours (end of exposure) all concentrations in the test media were < LOQ.Since no information about volatility was provided by the sponsor, loss of test item is considered to be an abiotic or biotic degradation effect. The observed effects can therefore not be related to the test item A130 6-Methoxy-2-methylamino-3-aminopyridine HCl itself. Concentration control analysis showed complete degradation of the test item after 18 hours at the latest under test conditions, while inhibitory effects are observed over the whole 72 hours. No specific degradation product could be detected in the LC-MS/MS analysis. Therefore, toxicity effects are considered to be related to the test item and, most likely, a formed mixture of degradation products.
Based on this, all effect values given are based on the nominal test item concentrations of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl.
NOEC, LOEC, ECx-values and 95% Confidence Intervals of A130 6-Methoxy-2-methylamino-3-aminopyridine HCl (0 - 72 hours)
based on nominal test item concentrations [µg/L]; the test item was found to be completely degraded after 18 hours
Growth Rate Inhibition
Nominal test item concentration [µg/L]
NOEC
128
LOEC
320
ErC10
211 (109 – 307)
ErC20
272 (150 – 314)
ErC50
380 (329 – 669)
Yield Inhibition
NOEC
128
LOEC
320
EyC10
< 20.5
EyC20
< 20.5
EyC50
213 (132 – 289)
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Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.