6-methoxy-N2-methylpyridine-2,3-diamine dihydrochloride

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:

2017

Report date:

2017

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

Test material

Test material information

Constituent 1

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

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 concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

128 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

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

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

128 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

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, EC_x-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)