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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-06-28 to 2017-06-30

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

83732-72-3

Details on test material:

Name of the test material (as cited in the report): A130 6-Methoxy-2-methylamino-3-Aminopyridin HCl

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All tested concentration levels and the control will be analytically
verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the exposure (24 and 48 hours).
The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000). Details of the analytical method are presented in section 14. Analytical results are presented in section 7.2.3.

Sampling for the analytical monitoring
At the start of the exposure and at the renewal (0 and 24 hours),
samples were taken after preparation of the test item concentrations and analyzed.
At the renewal and at the end of the exposure (24 and 48 hours), samples were taken directly from the test vessels for analyses.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal concentrations.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution
A stock solution (10.0 mg/L of the test item were weighed out) was freshly prepared with dilution water (see Table 2) prior to the start of the exposure (0 hours) and prior to the renewal of the test solutions (24 hours). The stock solution was mixed thoroughly by manual agitation.

Test concentrations
5 test item concentrations in a geometric series with a separation factor of 2, prepared by dilution of the stock solution 10 mg/L with dilution water (see Table 2), was tested as follows:
0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L.
This corresponds to geometric mean measured test item concentrations of: 0.0570 – 0.0787 – 0.211 – 0.472 – 1.157 mg/L
The test concentrations were selected based on the results of a non-GLP preliminary range finding test, which are presented in section 7.1.

Control
Dilution water without test item incubated under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system Daphnia magna STRAUS (Clone 5).

Reason for the selection of the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.

Origin
Institut für Wasser-, Boden- und Lufthygiene (WaBoLu),
14195 Berlin, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004), modified to a total hardness of 160 to 180 mg CaCO3/L, is used. The composition of the culture medium is presented in Table 2.

Feeding of the culture stocks
The daphnids were fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with a cell density of > 106 cells/mL. The algae were cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG),
Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany
 

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)
Component Concentration [mg/L]
CaCl2 x 2 H2O 176*
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8
* = original recipe: 293.8 mg/L, modified to achieve a total water hardness of
160 to 180 mg CaCO3/L

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Dilution water
0 hours:Total hardness [mg CaCO3/L]: 165
24 hours:Total hardness [mg CaCO3/L]: 168

Test temperature:

18 - 22°C, constant within ± 1°C
19 °C

pH:

Water Quality Parameters in the fresh Media at the Start of the Exposure and at the Renewal (0 and 24 hours)
(measured in one additional replicate (without daphnids) per concentration level and control)
Geometric mean
measured concentration
of the test item
[mg/L] 0 hours 24 hours
pH-value Dissolved
O2 concentration [mg/L] pH-value Dissolved
O2 concentration [mg/L]
1.157 7.60 8.98 7.71 8.87
0.472 7.62 8.95 7.73 8.96
0.211 7.67 8.99 7.78 8.96
0.0787 7.65 8.92 7.76 8.79
0.0570 7.39 8.81 7.70 8.47
Control 7.46 9.17 7.47 9.08

Water Quality Parameters in the old Media at the Renewal (24 hours)
(measured in all replicates (containing daphnids) per concentration level and control)
Geometric mean
measured concentration
of the test item
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
1.157 7.18 7.92 7.90 7.89 8.50 8.52 8.46 8.55
0.472 7.16 7.89 7.86 7.86 8.57 8.63 8.67 8.67
0.211 7.10 7.84 7.84 7.83 8.43 8.56 8.63 8.65
0.0787 7.66 7.82 7.79 7.81 8.52 8.60 8.55 8.59
0.0570 7.61 7.77 7.79 7.79 8.56 8.54 8.55 8.60
Control 7.54 7.75 7.74 7.74 8.35 8.42 8.51 8.56

Water Quality Parameters in the old Media at the End of the Exposure (48 hours)
(measured in all replicates (containing daphnids) per concentration level and control)
Geometric mean
measured concentration
of the test item
[mg/L] pH-values Dissolved O2 concentration [mg/L]
Replicates Replicates
1 2 3 4 1 2 3 4
1.157 7.67 7.67 7.68 7.69 8.45 8.54 8.63 8.55
0.472 7.63 7.64 7.65 7.67 8.66 8.81 8.77 8.68
0.211 7.56 7.58 7.59 7.62 8.74 8.86 8.76 8.81
0.0787 7.52 7.54 7.54 7.57 8.63 8.77 8.84 8.79
0.0570 7.43 7.48 7.45 7.49 8.77 8.71 8.78 8.84
Control 7.56 7.48 7.44 7.42 8.68 8.67 8.73 8.75

Dissolved oxygen:

see above

Conductivity:

Dilution water day 0: 423 [µS/cm]
Dilution water day 1: 429 [µS/cm]

Details on test conditions:

Test method
The study was performed under semi-static conditions (with a renewal of the test solutions after 24 hours), since the test item concentrations are not stable during an exposure period of 48 hours (Table 7).

Test duration
48 hours

Test vessels
Glass beakers (4 (ID) x 7 (H) cm), 50 mL capacity, loosely covered with watch glasses

Test volume
20 mL

Dilution water
Same as culture medium (see Table 2)

Number of daphnids and replicates
20 daphnids, divided into 4 replicates, each with 5 daphnids, were
used for each concentration level and the control.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for
the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles bornwithin the following period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatisation
Acclimatization of the daphnids was not necessary, because the dilution water was equivalent to the culture medium.
 
Application
20 g test solution per replicate was weighed out into each test vessel. This corresponds to 20 mL per test vessel. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (renewal of the test solutions) by pipette.

Renewal of the test solutions
The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).


Test temperature (target)
18 - 22 °C, constant within ± 1 °C

Illumination (target)
Diffuse light, light intensity of max. 1500 lx

Photoperiod (target)
16/8 hours light/dark cycle

Feeding
The daphnids were not fed during the study.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

other: EC10

Effect conc.:

1.068 other: mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

other: EC10

Effect conc.:

0.569 other: mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 1.157 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 1.157 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 1.157 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

A reference test was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium (Table 2) under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility. The results of the most recent test are presented in section 7.3.

Reference item Potassium dichromate p.a. (SIGMA ALDRICH)

Purity 99.0%

Batch No. MKBV0900V

Expiry date 2021-11-25

Test concentrations 1.00 – 2.00 – 4.00 mg/L

Ranges of validity EC50 (24 hours): 0.6 - 2.4 mg/L, according to AQS P 9/2 (clone 5)
EC50 (24 hours): 0.6 - 2.1 mg/L, according to OECD 202 (clone A)

Test duration 2017-06-01 to 2017-06-02

Reported statistics and error estimates:

Methods of evaluation
The EC50-value after 24 hours and the EC100-values (after 24 and 48 hours) were empirically derived from the observation data.
All effect levels (EC10 / 50 / 100) given are based on the geometric mean measured test item concentrations, since the measured test item concentrations were not within ± 20% of the nominal concentrations. The concentration-effect relationships of the test item after 24 and 48 hours is shown graphically.

EC-values and statistical analyses
The EC10-values (after 24 and 48 hours) and the EC50-value after
48 hours were calculated by sigmoidal dose-response regression. Wherever appropriate, the respective confidence limits were calculated from the standard error and the t distribution. Otherwise, the confidence limits were empirically derived from the observation data. All calculations were carried out from the best-fit values with the software GraphPad Prism.
The EC50-value for the reference item and its 95% confidence limits were calculated accordingly.

Software
All data were computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software:
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- Excel, MICROSOFT CORPORATION

Any other information on results incl. tables

A non-GLP preliminary range finding test with a semi-static test design over a period of 48 hours was conducted at the test facility. Three nominal concentrations of the test item of 1, 10 and 100 mg/L prepared with dilution water (seeTable2) were tested.

A stock solution 100 mg/L was prepared with dilution water, mixed thoroughly by manual agitation and used as the highest concentration level and for the preparation of the concentration levels 1 and 10 mg/L by dilution with dilution water. All tested concentration levels were visually clear throughout the exposure. During the exposure intervals, the color of the solutions changed from light yellow to orange in the concentration level 100 mg/L, from colorless to yellow in the concentration level of 10 mg/L and from colorless to light yellow in the concentration level 1 mg/L.

In the range finding test, two replicates per treatment group and control, each with ten daphnids, were tested. The results are presented inTable 3.

Since the analytical system has to be changed from HPLC-DAD to LC/MS/MS, an additional stability test was performed with the concentration level 10 mg/L, which is chosen as the highest concentration level in the definitive test. For results, seeTable4.

 

Table3:      Immobilization Rates in the non-GLP Preliminary Range Finding Test

                (n = 20, divided into 2 replicates with 10 daphnids each)

Nominal test item concentration [mg/L]	IMMOBILIZATION [%]
	24 hours			48 hours
	Replicates			Replicates
	1	2	MV	1		2	MV
100	100	100	100	100% mortality after 24 hours
10	30	20	25	90	100		95
1	0	0	0	10		0	5
Control	0	0	0	0		0	0

 

Table4:      Measured Concentrations of the Test ItemA1306-Methoxy-2-methylamino-3-aminopyridineHClduring thenon-GLP Stability Test

                Analytical system: LC-MS/MS

Sampling date	2016-09-05 0 hours Start of the exposure		2016-09-07 48 hours End of the exposure
Nominal test item concentration [mg/L]	A1306-Methoxy-2-methylamino-3-aminopyridineHCl
	Meas. conc. [mg/L]	%	Meas. conc. [mg/L]	%
10	10.6	106	0.0234	< 1

Meas. conc.    = measured concentration of the test item, dilution factors taken into account

%                     = percent of the nominal test item concentration

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the geometric mean measured concentrations of the test item A130 6-Methoxy-2-methylamino-3-aminopyridine HCl, the 48 hour-EC50 for Daphnia magna was 0.990 (confidence limits: 0.750 – 1.122).

Executive summary:

In the acute immobilization test withDaphnia magna(STRAUS), the effects of the test itemA1306‑Methoxy-2-methylamino-3-aminopyridineHCl(batch number:20151207) were determined at the test facility according to OECD 202 (2004) from2017-06-28 to 2017-06-30.

The study was conducted under semi-static conditions over a period of 48 hours with5concentrations of the test itemA1306-Methoxy-2-methylamino-3-aminopyridineHClin the range of0.625 - 10.0 mg/L, prepared in a geometric series with a separation factor of2.

Twenty daphnids (divided into 4 replicates with 5 daphnids each) were exposed to each concentration leveland the control.

The concentrations of the test itemA1306-Methoxy-2-methylamino-3-aminopyridineHClwere analytically verified via LC-MS/MS in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) and in the old media at the renewal and at the end of the exposure (24 and 48 hours) in all concentration levels and in the control. Details of the analytical method are presented in section14.

The measured concentrations of the test item were in the range of 89 to 107% of the nominal values in the fresh media at the start of the exposure and at the renewal (0 and 24 hours) and < LOQ to 2% in the old media at the renewal and at the end of the exposure (24 and 48 hours). The analytical results are presented in part7.2.3.

The geometric mean measured concentrations of the test itemA1306-Methoxy-2-methylamino-3-aminopyridineHClare: 0.0570 – 0.0787 – 0.211 – 0.472 – 1.157 mg/L.

Since the measured test item concentrations were not within the range of±20% of the nominal concentrations, the effect levels given are based on the geometric mean measured concentrations of the test itemA1306-Methoxy-2-methylamino-3-aminopyridineHCl(Table1).

The validity criteria of the test guidelines were fulfilled.

 

Table1:   EC₁₀-, EC₅₀-(with Confidence Limits), EC₁₀₀-Values

(based on the geometric mean measured concentrations of the test item)

A132 2-Amino-3-hydroxypyridine
Effect levels		Test duration [h]		Geometric mean measured test item concentrations [mg/L]
EC10 (with confidence limits)		24		1.068		(Cl: 0.215 - > 1.157)
		48		0.569		(Cl: 0.390 – 0.834)
EC50 (with confidence limits)		24		> 1.157		(Cl: Not applicable)
		48		0.990		(Cl: 0.750 – 1.122)
EC100		24		> 1.157
		48		> 1.157