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

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-21 to 2009-01-16

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:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

DIN ISO 11268-2 and BBA Guideline

Deviations:

yes

Remarks:

The room temperature decreased three times to about 17.5 °C and increased three times to about 21 °C . The soil moisture of all test vessels deviated by more than 10 %. Deviations considered to have no impact on study quality and integrity.

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetzt 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

Details on sampling:

No analytical monitoring

Test substrate

Vehicle:

yes

Details on preparation and application of test substrate:

- Method of mixing into soil (if used): The respective test item concentration was weighed out, dissolved in acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the treated quartz sand was given to the artificial soil. Demineralised water was added to adjust the humidity of the artificial soil to a moisture of 52 % of the maximum water holding capacity.
Subsequently, the test medium was mixed with a long handled blender for at least three minutes and filled into the test vessels.
- Controls: Artificial soil with addition of quartz sand moistened with demineralised water without test item was used as control medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Quartz sand treated with acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.5 mL per replicate
- Evaporation of vehicle before use: Yes

Test organisms

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida SAVIGNY 1826, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.54 +/- 0.04 g


ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study design

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Post exposure observation period:

No

Test conditions

Test temperature:

Room Temperature [°C]
nominal 20 +/- 2
actual 17.5 - 21.5*
*) The room temperature decreased three times to about 17.5 deg C for a total of 9 hours and increased three times to about 21 deg C for a total of 12 hours.

pH:

Application rate
[mg test item/kg soil DW] pH-value
day 0 day 56
Control 6.07 6.42
Solv. Control 5.99 6.32
62.5 5.71 6.51
125 5.71 6.65
250 5.71 6.58
500 5.82 6.55
1000 5.69 6.73

Moisture:

Application rate
[mg test item/kg soil DW] Moisture [%] of dry weight

day 0 day 56
Control 33.5 38.3*
Solv. Control 32.3 39.4*
62.5 32.5 39.9*
125 32.5 39.2*
250 32.4 39.3*
500 32.4 37.2*
1000 31.2 38.9*

*) Variation of the water content > 10 %, this deviation is considered to have no impact on the study.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm3, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g DW
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 4



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.36 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 65
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 9 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 614 ± 67.7 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: yes


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2

Nominal and measured concentrations:

nominal concentrations: 62.5 - 125 - 250 - 500 - 1000 mg test item/kg soil DW

Reference substance (positive control):

yes

Results and discussion

Effect concentrations

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

mortality and reduction of body weight

Details on results:

- Mortality at end of exposure period: No mortality was observed in any of the tested test item and control groups
- Total mass of adults at beginning of test: 0.49 - 0.50
- Changes in body weigth of live adults (% of initial weight) at end of exposure period: Control and solvent control: 46.8 +/- 7.47, 62.5 mg/kg soil DW: 46.0 +/- 2.09, 125 mg/kg soil DW: 50.7 +/- 11.3, 250 mg/kg soil DW: 57.1 +/- 8.37, 500 mg/kg soil DW: 56.4 +/- 4.73, 1000 mg/kg soil DW: 57.9 +/- 2.50
- No. of offspring produced: Control AND SOLVENT CONTROL: 235 +/- 39.1, 62.5 mg/kg soil DW: 244 +/- 30.4, 125 mg/kg soil DW: 238 +/- 61.6, 250 mg/kg soil DW: 269 +/- 42.6, 500 mg/kg soil DW: 237 +/-67.5, 1000 mg/kg soil DW: 267 +/- 31.4
- No. of unhatched cocoons: Not determined
- Morphological abnormalities: None
- Behavioural abnormalities: None
- Other biological observations: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
Reference Item (Carben) - Reproduction Rate (Number of Juveniles after 8 weeks)
Application rate
[mg a.i./kg soil dry weight] Rep. Number of juveniles Mean ± SD CV Reduction compared to control [%] Significance*
Control 1 248 272
±
34.8 12.8 - -
2 236
3 305
4 298
1.0 1 224 259
±
47.4 18.3 4.60 No
2 263
3 225
4 325
2.5 1 39 44.8
±
13.8 30.9 83.5 Yes
2 65
3 34
4 41
5.0 1 0 0
±
0.0 - 100 Yes
2 0
3 0
4 0
Rep. = Replicate a.i. = Active ingredient SD = Standard deviation CV = Coefficient of variation

Reported statistics and error estimates:

Differences between the control and the solvent control were statistically analysed using One Way Analysis of Variance (ANOVA). No statistical differences occurred between the controls concerning biomass and reproduction. Thus, both controls were pooled for further statistical analyses.
One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to
the control. A Normality Test and an Equal Variance Test were conducted prior to running the ANOVA. P-values for both Normality and Equal Variance Test are 0.05. The a-value for ANOVA test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05. Since the Normality Test failed using reproduction data, the data were log transformed for ANOVA to obtain a passed Normality Test.

The EC50-value for reproduction was not calculated since no reduction of reproduction occurred at all application rates.

Any other information on results incl. tables

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate [mg test item/kg soil dry weight]	Mortality [%]
	Replicate 1	Replicate 2	Replicate 3	Replicate 4
Control	0	0	0	0
Solvent Control	0	0	0	0
62.5	0	0	0	0
125	0	0	0	0
250	0	0	0	0
500	0	0	0	0
1000	0	0	0	0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate [mg test item/kg soil dry weight]	Effect	Replicate
		1	2	3	4
Control	a)	10/10	10/10	10/10	10/10
Solvent Control	a)	10/10	10/10	10/10	10/10
62.5	a)	10/10	10/10	10/10	10/10
125	a)	10/10	10/10	10/10	10/10
250	a)	10/10	10/10	10/10	10/10
500	a)	10/10	10/10	10/10	10/10
1000	a)	10/10	10/10	10/10	10/10

a) No observation of symptoms or behavioural changes

Body Weight Changes of the Adult Earthworms      

Application rate [mg test item/kg soil dry weight]	Rep.	Mean body weights per replicate		Mean body weight change of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sign.*
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.498	0.723	0.23	45.2	0.23 ± 0.04	46.8 ± 7.47	-
	2	0.520	0.786	0.27	51.2
	3	0.498	0.672	0.17	34.9
	4	0.500	0.720	0.22	44.0
Solvent Control	1	0.474	0.768	0.29	62.0
	2	0.471	0.685	0.21	45.4
	3	0.496	0.716	0.22	44.4
	4	0.502	0.740	0.24	47.4
62.5	1	0.498	0.720	0.22	44.6	0.23 ± 0.01	46.0 ± 2.09	No
	2	0.503	0.744	0.24	47.9
	3	0.499	0.718	0.22	43.9
	4	0.503	0.743	0.24	47.7
125	1	0.465	0.668	0.20	43.7	0.25 ± 0.05	50.7 ± 11.3	No
	2	0.473	0.791	0.32	67.2
	3	0.500	0.744	0.24	48.8
	4	0.499	0.715	0.22	43.3
250	1	0.471	0.703	0.23	49.3	0.28 ± 0.04	57.1 ± 8.37	No
	2	0.467	0.789	0.32	69.0
	3	0.499	0.776	0.28	55.5
	4	0.503	0.778	0.28	54.7
500	1	0.466	0.754	0.29	61.8	0.27 ± 0.02	56.4 ± 4.73	No
	2	0.470	0.739	0.27	57.2
	3	0.502	0.784	0.28	56.2
	4	0.501	0.753	0.25	50.3
1000	1	0.469	0.727	0.26	55.0	0.28 ± 0.02	57.9 ± 2.50	No
	2	0.470	0.736	0.27	56.6
	3	0.501	0.804	0.30	60.5
	4	0.504	0.803	0.30	59.3

Rep. = Replicate; SD = Standard deviation; Sign. = Significance; * Significance compared to pooled control

Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg soil DW]	Rep.	Number of juveniles	Mean ± SD	CV               [%]	[%] of pooled control	Significance compared to pooled control
Control	1	227	235 ± 39.1	16.7	-	-
	2	184
	3	203
	4	275
Solvent Control	1	219
	2	211
	3	260
	4	298
62.5	1	279	244 ± 30.4	12.5	104	No
	2	212
	3	258
	4	226
125	1	284	238 ± 61.6	25.8	102	No
	2	165
	3	294
	4	210
250	1	238	269 ± 42.6	15.8	115	No
	2	231
	3	284
	4	322
500	1	182	237 ± 67.5	28.5	101	No
	2	335
	3	209
	4	221
1000	1	299	267 ± 31.4	11.8	114	No
	2	288
	3	236
	4	244

SD = Standard deviation        CV = Coefficient of variation        Rep. = Replicate      

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the submission substance did not induce mortality or pathological symptoms of adult earthworms after an exposure to the test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight for 28 days. Also, the biomass increase of the adult earthworms after 28 days did not statistically significantly differ compared to the pooled control at all test item concentrations. In addition, after 56 days of exposure the earthworm reproduction was not statistically significantly reduced at all applied test item concentrations.

Thus the No Observed Effect Concentration (NOEC) for the submission substance with respect to mortality, body weight and reproduction of Eisenia fetida fetida after 8 weeks of exposure in artificial soil was determined to be >= 1000 mg test item/kg soil dry weight.

Executive summary:

Effects of the submission substance on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Savigny) were determined according to the guidelines DIN ISO 11268-2 (1998), OECD 222 (2004) and BBA (1994) compliant to GLP (reliability category 1). The study was conducted under static conditions over 8 weeks with five different application rates (62.5, 125, 250, 500, and 1000 mg test item/kg soil dry weight), which were mixed into artificial soil containing 10 % peat. 40 test organisms, with an individual weight of 0.30 to 0.60 g, were divided into 4 replicate groups with 10 test organisms per replicate.

After 28 days no mortality as well as no pathological symptoms or changes in the behaviour of adult earthworms were seen in the control, in the solvent control and at all tested application rates (Table 1). The earthworm biomass increased in the control and at all application rates over 28 days without any statistically significant differences compared to the control. After further four weeks the reproduction rate (average number of juveniles) was 235 in the control (control and solvent control) and ranged between 237 and 269 at the test item application rates. Compared to the control, the reproduction rate did not statistically significantly differ at all application rates.

Overall, the NOEC of Pigment Red 187 concerning mortality, biomass and reproduction of earthworms was determined to be >=  1000 mg test item/kg soil dry weight.

All validity criteria recommended by the test guidelines were fulfilled.

Summary of All Observed Effects in the Tested Doses

Effects	Test Item
mg test item/kg soil dry weight	62.5	125	250	500	1000
Mortality of adult earthworms	No	No	No	No	No
Reduction of body weight	No	No	No	No	No
Reduced reproduction rate   (Number of juveniles)	No	No	No	No	No
Summary of all effects	No	No	No	No	No

NOEC and LOEC for Reproduction, Mortality and Body Weight

Test Item
	Application rate
	mg test item/kg soil dry weight
NOECreproduction, mortality, body weight	>=  1000
LOECreproduction, mortality, body weight	> 1000