4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

Sediment toxicity

Administrative data

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-01-06 to 2010-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-study compliant to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Draft ISO/DIS 10872 (2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

according to German Chemikaliengesetz and Directive 88/320/EEC

Test material

Test material information

Constituent 1

Reference substance name:

4-[[5-[[[4-(aminocarbonyl)phenyl]amino]carbonyl]-2-methoxyphenyl]azo]-N-(5-chloro-2,4-dimethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

EC Number:

261-785-5

EC Name:

4-[[5-[[[4-(aminocarbonyl)phenyl]amino]carbonyl]-2-methoxyphenyl]azo]-N-(5-chloro-2,4-dimethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide

Cas Number:

59487-23-9

Molecular formula:

C34H28ClN5O7

IUPAC Name:

4-({5-[(4-carbamoylphenyl)carbamoyl]-2-methoxyphenyl}diazenyl)-N-(5-chloro-2,4-dimethoxyphenyl)-3-hydroxy-2-naphthamide

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Name of test material (as cited in study report): Prüfung von Farbstoff-Pigmenten: Haut- und Schleimhautverträglichkeit: Permanentcarmin FBB extra
Analytical purity: Not specified

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

yes

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Pooling or mixing of different substrates:
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.3 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 44 %.
- Details of spiking: The respective test item amount for the limit concentration was weighed out and dissolved in acetone. Subsequently, the solution was thoroughly mixed with quartz sand (20 % of the entire sediment amount) in a glass vessel. After complete evaporation of the solvent, the dry quartz sand was given to the sediment and thoroughly mixed with the sediment.
Additional M9 medium* was added to the sediment to obtain a water content of 44 %. Subsequently, 0.5 g sediment wet weight was filled into each test well.
- Equilibration time: 1 day
- Equilibration conditions: 8 +/- 2 °C, dark
- Controls: Moistened test sediment was tested under the same conditions as the test replicates.

*) M9 medium: 13.95 g Na2HPO4+ 12 H2O corresponding to 6 g Na2HPO4, 3 g KH2PO4, 5 g NaCI and 0.25 g MgSO4 x 7H2O dissolved in 1000 mL demineralised water and autoclaved.

Test organisms

Test organisms (species):

Caenorhabditis elegans

Details on test organisms:

TEST ORGANISM
- Common name: Nematode
- Strain/clone: Wilde type strain N2 var Bristol
- Source: Caenorhabditis Genetics Center, Minneapolis
- Breeding conditions:Breeding is performed at the test facility at 20 +/- 2 °C in the dark on agar plates containing NGM (nematode growth medium) agar inoculated with Escherichia coli OP 50.
- Age of animals at beginning of exposure: First stage juveniles were obtained by filtering nematode solutions from culture plates through nylon nets (10 µm and subsequently 5 µm mesh size). The size of 30 representative juvenile nematodes, which were not used in the test, was determined prior to nematode insertion.
- Feeding during test
LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 21.5 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.



ACCLIMATION
- Acclimation period: None, test conditions = breeding conditions

Study design

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

yes

Exposure duration

Duration:

96 h

Exposure phase:

total exposure duration

Test conditions

Nominal and measured concentrations:

Nominal: 1000 mg test item/kg sediment dry weight

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Nunc Polystyrol Multiwells (12 well microtiter plates)
- Sediment volume: 0.5 g sediment wet weight per replicate
- Overlying water volume: 0.5 mL food medium (see above)
- Aeration: no
- Replacement of evaporated test water, if any: No

Controls:
Control: Moistened test sediment with the addition of quartz sand (20% of entire sediment amount) was tested under the same conditions as the test replicates.
Solvent control: Moistened test sediment with the addition of quartz sand with acetone was tested under the same conditions as the test replicates.
Liquid Control: Tap water (0.55 ml) without sediment was tested under the same test conditions as the test replicates.



EXPOSURE REGIME
- No. of organisms per container (treatment): 40
- No. of replicates per treatment group: 6
- No. of replicates per control / vehicle control: 6
- No. of replicates for the liquid control: 4
- Type and preparation of food: LB-medium* was inoculated with E. coli OP50 one day prior to the insertion of the test organisms. About 21.5 hours after inoculation the food bacteria were washed with M9 medium and concentrated in M9 medium by centrifugation. 0.5 mL of the food medium was provided once prior to the addition of the nematodes to each test replicate.
*) LB-medium: 10 g casein peptone, 5 g yeast extract, 10 g NaCl dissolved in 1000 mL demineralised water and autoclaved.


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- Composition (if artificial substrate):
Modified artificial sediment:
- 70 % quartz sand (with > 50 % particles sized between 50 and 200 µm)
- 20 % Al2O3
- 4.5 % Fe2O3
- 4 % peat, air dried and finely ground
- 0.3 % CaCO3
- 0.5 % dolomite (clay)
The pH-value and the moisture of the artificial sediment were determined. One day prior to introduction of the nematodes the sediment was moistened with M9 medium to a moisture of 44 %.
- Maturation of artificial substrate (if any): no

OTHER TEST CONDITIONS
- Continuous dark
The initial pH of the artificial sediment at test start was 6.7. The initial sediment moisture was
5.5 %. The room temperature ranged from 19.0 to 21.0 °C.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The recovery / mortality of the inserted nematodes was analysed by counting the adult nematodes under a microscope. The body length of each female adult nematode was determined with a microscale.
The reproduction was analysed by inserting the extracted nematodes in a counting chamber. The produced number of offspring was determined in the specified volume of the counting chamber under a microscope. The total number of offspring per replicate was calculated by using the entire volume of extract.


VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: none
- Range finding study
Range Finding Test (96 h): Recovery, Mortality and Reproduction
Test Item Concentration Recovery of adult nematodes Fertility of adult nematodes Reproduction
[mg test item/kg soil dry weight] [%] [%] [Offspring per inserted test organism]
Control 83.3 100 42.0
Solv. control 90.0 100 44.8
10 90.0 100 46.0
100 96.7 100 51.7
1000 93.3 100 42.6

Reference substance (positive control):

yes

Remarks:

Benzyldimethylhexadecylammonium chloride

Results and discussion

Effect concentrations

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: mortaliy, fertility, reproduction, growth

Details on results:

- Mortality of test animals at end of exposure period: In the artificial sediment a recovery of 90 % was observed in the pooled control after 4 days of exposure. At the limit test item concentration of 1000 mg test item/kg sediment dry weight the recovery was also 90 %. The resulting mortality was not considered to be of biological significance, and was within the range of experimental variation considered acceptable for the control group.

- No. of offspring produced: All female nematodes in the control as well as at the limit test item concentration were observed to be fertile. This also applies to the surviving nematodes in the reference item concentrations.
The reproduction in artificial sediment was not statistically significant reduced compared to the pooled control at the limit test item concentration of 1000 mg test item/kg sediment dry weight. The reproduction rates at the reference item concentrations of 10 and 15 mg reference item/L were statistically significant reduced compared to the pooled control by 40.7 % at 10 mg/L and by 97.0 % at 15 mg/L

- Growth: The initial mean size of the introduced juvenile nematodes was 230.4 µm.
In artificial sediment the growth of the adult nematodes did not statistically significantly differ compared to the pooled control at the limit test item concentrations of 1000 mg test item/kg sediment dry weight.
At 10 and 15 mg reference item/L the growth of the nematodes was statistically significantly reduced compared to the pooled control

- Morphological abnormalities: Not observed
- Behavioural abnormalities: Not observed

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- % recovered test organisms in the control: 100%
- Relevant effect levels:


Effects Reference Item Benzyldimethylhexadecylammonium chloride [mg/L]

LOEC (Mortality, Fertility, Reproduction, Growth) 5
NOEC (Mortality, Fertility, Reproduction, Growth) < 5
EC50 (Mortality) 15.0 (14.2 - 15.8)
EC50
(Reproduction) 10.2 (9.75 - 10.9)
EC50
(Size) 15.9 (11.5 - 23.4)

Reported statistics and error estimates:

t-tests were used for LOEC and NOEC calculations. Prior to running the t-tests a Normality and an Equal Variance Test were done. P-values for both Normality and Equal Variance Test are 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

Any other information on results incl. tables

Recovery / Mortality after 4 Days of Exposure to submission substance

Nominal test item concentration [mg test item/kg sediment dry weight]	[%] Recovered test organisms								Mortality [%]
	Replicate						MV	SD
	1	2	3	4	5	6
Liquid Control	100	90	90	100	-	-	95.0	5.77	5.00
Control	90	80	90	100	100	90	90.0	7.39	10.0
Solvent Control	90	80	80	90	90	100
1000	90	90	90	90	100	80	90.0	6.32	10.0

Nematode Reproduction after 4 Days of Exposure to submision substance

Nominal test item concentration [mg test item/kg sediment dry weight]	Number of offspring per inserted nematode
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	38	35	35	45	35	34	36.7	3.61	-
Solvent Control	35	34	32	38	41	38
1000	35	40	34	38	35	32	35.7	2.98	2.72

Nematode Growth after 4 Days of Exposure to submission substance

Nominal test item concentration [mg test item/kg sediment dry weight]	Mean Growth [µm]
	Replicate						MV	SD	Inhibition [%]
	1	2	3	4	5	6
Control	772	802	799	799	816	794	798	11.8	-
Solvent Control	799	796	808	784	810	804
1000	810	804	826	760	802	760	793	27.5	0.62

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study the submission substance did not induce significant mortality of C. elegans after an exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight for 96 hours. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight.
No reduction of growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after four days of exposure to the submision substance compared to the pooled control.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentrations of Pigment Red 187.

Executive summary:

Effects of the submission substance on the bacterivorous nematode Caenorhabditis elegans in a sediment system were determined. The study was carried out according to the Draft guideline ISO/DIS 10872 (2008). Test duration was 96 hours after insertion of the test organisms. The study was performed by spiking the test item into the sediment with the limit test item concentration of 1000 mg test item/kg sediment dry weight. Six replicates per control, solvent control and limit test item concentration were set up.

After four days of exposure to the limit test item concentration of 1000 mg test item/kg sediment dry weight the submission substance did not induce biologically significant mortality of C. elegans. Also, adult nematode fertility as well as nematode reproduction was not affected at 1000 mg test item/kg sediment dry weight.

Accordingly, no statistically significant reduction of growth was observed at the limit test item concentration of 1000 mg test item/kg sediment dry weight after an exposure of 96 hours to the submission substance.

Thus, the Lowest Observed Effect Concentration (LOEC) concerning all test parameters was set at > 1000 mg test item/kg sediment dry weight. The Overall No Observed Effect Concentration (NOEC) was determined to be 1000 mg test item/kg sediment dry weight. All effect levels given are based on the nominal concentration of the submission substance.

Summary of all Effects and resulting LOEC, NOEC based on nominal concentrations

Effects	Submission substance
	[mg test item/kg sediment dry weight]
Mortality	> 1000
Fertility	> 1000
Reproduction	> 1000
Growth	> 1000
LOEC (Mortality, Fertility, Reproduction, Growth)	> 1000
NOEC (Mortality, Fertility, Reproduction, Growth)	1000