3-amino-p-toluamide

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:

2014-03-19 to 2014-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: performed in accordance with OECD and GLP guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3-amino-p-toluamide

EC Number:

243-039-0

EC Name:

3-amino-p-toluamide

Cas Number:

19406-86-1

Molecular formula:

C8H10N2O

IUPAC Name:

3-amino-p-toluamide

Details on test material:

- Name of test material (as cited in study report): 3-Amino-4-methylbenzamid
- Physical state: Light brown powder
- Analytical purity: 99.4 % (w/w)
- Sum formula: C8H10N2O
- Molecular weight: 150.18 g/mol
- Purity test date: 2013-02-21
- Lot/batch No.:120808
- Expiration date of the lot/batch: 2014-10-17
- Storage condition of test material: Room temperature, protected from light, in original container under nitrogen and in evacuated desiccator.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
Six test item concentrations in a geometric series with a separation factor of 2.2, prepared by diluting the stock solution of 100 mg/L with dilution water, were tested as follows: 1.94 - 4.27 - 9.39 - 20.7 - 45.5 - 100 mg/L.

- Sampling method:
All tested concentration levels and the control were analytically verified at the start of the exposure (0 h) and at the end of the exposure (48 h) as specified below. At the start of the exposure, sampling was carried out immediately after preparation of the test concentrations.
At the end of the exposure (48 h), samples were taken directly from the test vessels.
The method was validated according to SANCO 3029/99 rev.4 (2000-07-11).

- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Before weighing out, an appropriate amount of the test item was crushed with a mortar.
The stock solution (100 mg/L of the test item were weighed out) was freshly prepared with dilution water (0 h). The stock solution was treated with ultrasound for 5 minutes at room temperature. Six test item concentrations in a geometric series with a separation factor of 2.2, prepared by diluting the stock solution of 100 mg/L with dilution water, were tested as follows: 1.94 - 4.27 - 9.39 - 20.7 - 45.5 - 100 mg/L.


- Dilution water : ISO Test water, according to OECD 202, Annex 3, composition see below

Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123



- Controls: Dilution water without test item tested under the same conditions as the test goups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation: 2 to 24 h
- Method of breeding:
In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2°C, in an incubator, 16 h illumination, light intensity of max. 20 µExm-2 x s-1;
Culture feeding: at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus with an algae cell density of > 10^6 cells/mL.
Culture medium: Elendt M4, according to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L

- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: at least 2 h in dilution water
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

DILUTION WATER:

Component Concentration [mg/L]
KCl 5.76
NaHCO3 64.8
CaCl2  2 H2O 294
MgSO4  7 H2O 123

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 265 (mg CaCO3/L)

Test temperature:

At test start: 21.5°C (Water)

pH:

Please refer to "Any other information on materials and methods"

Dissolved oxygen:

Please refer to "Any other information on materials and methods"

Salinity:

Not measured, freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, closed with parafilm
- Material, size, headspace, fill volume:
Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): Static test (no water renewal)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see above
- Conductivity: Dilution water at test start: 658 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and at test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Immobilisation was determined in all groups after 24 and 48 h. An animal was considered to be immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel.

TEST CONCENTRATIONS
- Range finding study:
Immobilisation 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] IMMOBILISATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 80 80 80 100 100 100
10.0 10 0 5 30 20 25
1.00 0 0 0 0 0 0
Control 0 0 0 0 0 0


MV = mean value

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

6.9 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Remarks on result:

other: 9.56 - 10.5

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

1.94 mg/L

Nominal / measured:

nominal

Basis for effect:

mobility

Details on results:

- Mortality of control: 0 %
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.96 (CI: 1.68 - 2.28) mg/L

Reported statistics and error estimates:

The EC100-values after 24 and 48 h, the NOEC and LOEC after 48 h were deduced directly from the observation data, without mathematical calculation.

The EC10- and the EC50-values were calculated by sigmoidal dose-response regression. The 95 % confidence limits were calculated from the best-fit values, the standard error and the t-distribution with the software GraphPad prism5. The EC50-value for the reference item was calculated accordingly.

Any other information on results incl. tables

Immobilisation Rates after 24 and 48 h of Exposure in the Definitive Test

(n = 20, divided into 4 replicates with 5 daphnids each)

3-Amino-4-methylbenzamid	IMMOBILISATION [%]
Nominal test item concentration   [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
100	80	80	60	60	70	100	100	100	100	100
45.5	60	80	80	60	70	100	100	100	100	100
20.7	80	60	100	60	75	100	100	100	100	100
9.39	0	60	20	40	30	40	80	80	100	75
4.27	0	0	0	0	0	40	0	0	20	15
1.94	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

MV  = mean value

All test item concentrations and the control were visually clear throughout the exposure period.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the nominal concentrations of the test item 3-Amino-4-methylbenzamid, the 48 h-EC50 for Daphnia magna was 6.90 mg/L (95 % confidence limits: 5.71 – 8.07 mg/L). The NOEC after 48 h was 1.94 mg/L. The LOEC after 48 h was 4.27 mg/L.

Executive summary:

In the acute immobilisation test with Daphnia magna (STRAUS), the effects of the test item 3-Amino-4-methylbenzamid (batch number:120808) were determined according to OECD 202 (2004) from 2014-03-19 to 2014-03-21 at Dr.U.Noack-Laboratorien, Käthe-Paulus-Str.1, 31157 Sarstedt, Germany.

The study was conducted under static conditions over a period of 48 h with 6 concentrations of the test item 3-Amino-4-methylbenzamid in the range of 1.94 to 100 mg/L, prepared with dilution water in a geometric series with a separation factor of 2.2. Twenty daphnids were exposed to each concentration level and the control.

The test item 3-Amino-4-methylbenzamid was analytically verified by HPLC-DAD in samples of all test concentrationsand the control at the start of the exposure (0 h) and at the end of the exposure (48 h).
The measured concentrations of the test item were in the range of 98 to 101 % of the nominal values at the start of the exposure (0 h) and also at the end of the exposure (48 h).

The recoveries of the test item were within ±20 % of the nominal values. This indicates that the test item was successfully maintained for the duration of the test. The effect values given are based on the nominal concentrations of the test item3-Amino-4-methylbenzamid.

The water quality parameters (i.e. pH-value and dissolved oxygen concentration), measured at the start (0 h) and at the end of the exposure (48 h), were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC₁₀-, EC₅₀- (with 95 % Confidence Limits), EC₁₀₀-Values, NOEC and LOEC

(based on the nominal concentrations of the test item)

Endpoints	Test duration [h]	3-Amino-4-methylbenzamid
		Nominal test item concentration [mg/L]	Confidence limits p = 95% [mg/L]
EC10	24	8.61	< 1.94 – 9.16
	48	3.80	< 1.94 – 5.15
EC50	24	10.1	9.56 – 10.5
	48	6.90	5.71 to 8.07
EC100	24	> 100	Not applicable
	48	20.7	Not applicable
NOEC	48	1.94	Not applicable
LOEC	48	4.27	Not applicable