Reaction mass of 2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2,4-dimethylphenyl)-3-oxobutyramide] and 2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(2-methylphenyl)-3-oxobutyramide] and 2-[[3,3'-dichloro-4'-[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-4-yl]azo]-3-oxo-N-(o-tolyl)butyramide

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-08-27 - 2002-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was performed with GLP compliance following relevant guidelines. The results are plausible and well presented.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Principles of Good Laboratory Practice (GLP) § 19a, German Chemical Act (ChemG), Annex 1 (May 8, 2001)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water is reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.
The tested concentration was visually present as clear solution.
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species : zebrafish, Danio rerio (HAMILTON-BUCHANAN)
Origin : Aventis Pharma Deutschland GmbH, ProTox
Date of hatching : February 28, 2002
Delivery date : May 02, 2002

The fish were kept for 14 days in water for dilution before the start of the study under the following conditions:
Temperature : 22 ± 1 °C
Oxygen content : >= 80 % of the saturation value
Duration of light period : 12 hours daily
Feeding : twice daily ad libitum
Food : Tetra Min, Tetra Werke, Meile (Germany)

The body length of 7 representative fish from each batch was measured:
Batch No.: 2/2002/L2 (31-Aug-2002); n=7; Body length (Variation range): 2.5 to 2.7; (cm) mean: 2.6; s: ± 0.08

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

The total hardness of the dilution water was determined weekly and was at 2.7 mmol Ca2+ + Mg2+/L during the study.

Test temperature:

Temperature (°C)
Concentration group: 21.2 to 21.3
Control group: 21.2 to 21.3

pH:

Concentration group: 7.4 to 7.9
Control group: 7.3 to 7.9

Dissolved oxygen:

Oxygen content (mg/L):
Concentration group:6.3 to 8.5
Control group: 7.0 to 9.0

Salinity:

Water quality was according to ISO/DIS 73461/1

Nominal and measured concentrations:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance, see above.

Details on test conditions:

The study was conducted in a static system. The test chambers, which were calibrated to 4 liters, were made of glass (length 20 cm, width 15 cm, height 20 cm) and stood in a water bath made of Hostalit Z® with a Plexiglas viewing panel. The temperature of the water bath was regulated by a thermostat to 22 ± 1 °C. The chambers were illuminated from above from 06.00 a.m. to 06.00 p.m. The light intensity directly over the chambers was approximately 700 lux.
The test chambers were not aerated during the course of the study.
After the stirring period and the filtration the test preparation was aerated before Insertion of the test fish using glass capillaries with a bubble-frequency of 1 - 3 per second.

Study groups
There were 7 fish in each group (control and nominal test concentration of 100 mg/L).

Test procedure
After the test concentration had been prepared and water parameters recorded, 7 fish were assigned to each test and control chamber. The fish were not fed during the entire study period. Inspection of the fish took place after 3, 6, 24, 48, 72 and 96 hours and involved recording the mortality and visible alterations in appearance and behavior. The water parameters were measured and recorded before study start and after 0, 24, 48, 72 and 96 hours.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Observations
The 100 mg/L group showed no effect in comparison with the control. In the control group no effects were observed.

Mortality
In this 96-hour acute toxicity study of the test substance in zebrafish (Danio rerio) mortality occurred neither in the 100 mg/L-group (nominal concentration) nor in the control group

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test the LC50 of the submission substance after 96 hours was > 100 mg/L (based an nominal concentration) and is above the solubility limit of the test substance in the test water.

Executive summary:

The submission substance was tested in zebrafish (Danio rerio) for 96 hours in a static system.

The test was performed with GLP compliance and following relevant guidelines with reliability category 1.

The nominal concentration tested was 100 mg/L.

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water was reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.

The tested concentration was visually present as clear solution. Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance was below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

In this 96-hour acute toxicity study, the 100 mg/L group showed no effect in comparison to the control. In the control group no effects were observed.

Under the conditions of this test the LC₅₀ of the submission substance after 96 hours was > 100 mg/L (based on nominal concentration) and is far above the solubility limit of the test substance in the test water.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002-08-27 - 2002-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test was performed with GLP compliance following relevant guidelines. The results are plausible and well presented.

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Principles of Good Laboratory Practice (GLP) § 19a, German Chemical Law (ChemG), Annex 1 (May 8, 2001) (implementation of the OECD Principles of Good Laboratory Practice as published on March 23, 1999, Official Journal of the European Union L77/9

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water is reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.
The tested concentration was visually present as clear solution.
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species : zebrafish, Danio rerio (HAMILTON-BUCHANAN)
Origin : Aventis Pharma Deutschland GmbH, ProTox
Date of hatching : February 28, 2002
Delivery date : May 02, 2002

The fish were kept for 14 days in water for dilution before the start of the study under the following conditions:
Temperature : 22 ± 1 °C
Oxygen content : >= 80 % of the saturation value
Duration of light period : 12 hours daily
Feeding : twice daily ad libitum
Food : Tetra Min, Tetra Werke, Meile (Germany)

The body length of 7 representative fish from each batch was measured:
Batch No.: 2/2002/L2 (31-Aug-2002); n=7; Body length (Variation range): 2.5 to 2.7; (cm) mean: 2.6; s: ± 0.08

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

The total hardness of the dilution water was determined weekly and was at 2.7 mmol Ca^2+ + Mg^2+/L during the study.

Test temperature:

Temperature (°C)
Concentration group: 21.2 to 21.3
Control group: 21.2 to 21.3

pH:

Concentration group: 7.5 to 7.9
Control group: 7.3 to 7.9

Dissolved oxygen:

Oxygen content (mg/L):
Concentration group: 6.5 to 8.6
Control group: 7.0 to 9.0

Salinity:

Water quality was according to ISO/DIS 73461/1

Nominal and measured concentrations:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance, see above.

Details on test conditions:

The study was conducted in a static system. The test chambers, which were calibrated to 4 liters, were made of glass (length 20 cm, width 15 cm, height 20 cm) and stood in a water bath made of Hostalit Z® with a Plexiglas viewing panel. The temperature of the water bath was regulated by a thermostat to 22 ± 1 °C. The chambers were illuminated from above from 06.00 a.m. to 06.00 p.m. The light intensity directly over the chambers was approximately 700 lux.
The test chambers were not aerated during the course of the study.
After the stirring period and the filtration the test preparation was aerated before Insertion of the test fish using glass capillaries with a bubble-frequency of 1 - 3 per second.

Study groups
There were 7 fish in each group (control and nominal test concentration of 100 mg/L).

Test procedure
After the test concentration had been prepared and water parameters recorded, 7 fish were assigned to each test and control chamber. The fish were not fed during the entire study period. Inspection of the fish took place after 3, 6, 24, 48, 72 and 96 hours and involved recording the mortality and visible alterations in appearance and behavior. The water parameters were measured and recorded before study start and after 0, 24, 48, 72 and 96 hours.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Observations
The 100 mg/L group showed no effect in comparison with the control. In the control group no effects were observed.

Mortality
In this 96-hour acute toxicity study of the submission substance in zebrafish (Danio rerio) mortality occurred neither in the 100 mg/L-group (nominal concentration) nor in the control group

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test the LC50 of the submission substance after 96 hours was > 100 mg/L (based an nominal concentration) and is above the solubility limit of the test substance in the test water.

Executive summary:

The submission substrance was tested in zebrafish (Danio rerio) for 96 hours in a static system.

The test was performed with GLP compliance and following relevant guidelines with reliability category 1.

The nominal concentration tested was 100 mg/L and a control (0 mg/L).

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water was reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.

The tested concentration was visually present as clear solution. Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance was below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

In this 96-hour acute toxicity study the 100 mg/L group showed no effect in comparison to the control. In the control group no effects were observed.

Under the conditions of this test the LC₅₀ of the submission substance after 96 hours was > 100 mg/L (based on nominal concentration) and is far above the solubility limit of the test substance in the test water.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2002-08-27 - 2002-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was performed with GLP compliance following relevant guidelines. The results are plausible and well presented.

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Yellow 174 (Chapter 13)

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Principles of Good Laboratory Practice (GLP) § 19a, German Chemical Act (ChemG), Annex 1 (May 8, 2001)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water is reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.
The tested concentration was visually present as clear solution.
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species : zebrafish, Danio rerio (HAMILTON-BUCHANAN)
Origin : Aventis Pharma Deutschland GmbH, ProTox
Date of hatching : February 28, 2002
Delivery date : May 02, 2002

The fish were kept for 14 days in water for dilution before the start of the study under the following conditions:
Temperature : 22 ± 1 °C
Oxygen content : >= 80 % of the saturation value
Duration of light period : 12 hours daily
Feeding : twice daily ad libitum
Food : Tetra Min, Tetra Werke, Meile (Germany)

The body length of 7 representative fish from each batch was measured:
Batch No.: 2/2002/L2 (31-Aug-2002); n=7; Body length (Variation range): 2.5 to 2.7; (cm) mean: 2.6; s: ± 0.08

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

The total hardness of the dilution water was determined weekly and was at 2.7 mmol Ca2+ + Mg2+/L during the study.

Test temperature:

Temperature (°C)
Concentration group: 21.2 to 21.3
Control group: 21.2 to 21.3

pH:

Concentration group: 7.4 to 7.9
Control group: 7.3 to 7.9

Dissolved oxygen:

Oxygen content (mg/L):
Concentration group:6.3 to 8.5
Control group: 7.0 to 9.0

Salinity:

Water quality was according to ISO/DIS 73461/1

Nominal and measured concentrations:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance, see above.

Details on test conditions:

The study was conducted in a static system. The test chambers, which were calibrated to 4 liters, were made of glass (length 20 cm, width 15 cm, height 20 cm) and stood in a water bath made of Hostalit Z® with a Plexiglas viewing panel. The temperature of the water bath was regulated by a thermostat to 22 ± 1 °C. The chambers were illuminated from above from 06.00 a.m. to 06.00 p.m. The light intensity directly over the chambers was approximately 700 lux.
The test chambers were not aerated during the course of the study.
After the stirring period and the filtration the test preparation was aerated before Insertion of the test fish using glass capillaries with a bubble-frequency of 1 - 3 per second.

Study groups
There were 7 fish in each group (control and nominal test concentration of 100 mg/L).

Test procedure
After the test concentration had been prepared and water parameters recorded, 7 fish were assigned to each test and control chamber. The fish were not fed during the entire study period. Inspection of the fish took place after 3, 6, 24, 48, 72 and 96 hours and involved recording the mortality and visible alterations in appearance and behavior. The water parameters were measured and recorded before study start and after 0, 24, 48, 72 and 96 hours.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Observations
The 100 mg/L group showed no effect in comparison with the control. In the control group no effects were observed.

Mortality
In this 96-hour acute toxicity study of the test substance in zebrafish (Danio rerio) mortality occurred neither in the 100 mg/L-group (nominal concentration) nor in the control group

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test the LC50 of the submission substance after 96 hours was > 100 mg/L (based an nominal concentration) and is above the solubility limit of the test substance in the test water.

Executive summary:

The submission substance was tested in zebrafish (Danio rerio) for 96 hours in a static system.

The test was performed with GLP compliance and following relevant guidelines with reliability category 1.

The nominal concentration tested was 100 mg/L.

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water was reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.

The tested concentration was visually present as clear solution. Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance was below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

In this 96-hour acute toxicity study, the 100 mg/L group showed no effect in comparison to the control. In the control group no effects were observed.

Under the conditions of this test the LC₅₀ of the submission substance after 96 hours was > 100 mg/L (based on nominal concentration) and is far above the solubility limit of the test substance in the test water.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2002-08-27 - 2002-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The test was performed with GLP compliance following relevant guidelines. The results are plausible and well presented.

Justification for type of information:

see Rationale and Justification for the Analogue Read-Across Approach used for the Registration of Pigment Yellow 174 (Chapter 13)

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Remarks:

Principles of Good Laboratory Practice (GLP) § 19a, German Chemical Law (ChemG), Annex 1 (May 8, 2001) (implementation of the OECD Principles of Good Laboratory Practice as published on March 23, 1999, Official Journal of the European Union L77/9

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water is reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.
The tested concentration was visually present as clear solution.
Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance is below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species : zebrafish, Danio rerio (HAMILTON-BUCHANAN)
Origin : Aventis Pharma Deutschland GmbH, ProTox
Date of hatching : February 28, 2002
Delivery date : May 02, 2002

The fish were kept for 14 days in water for dilution before the start of the study under the following conditions:
Temperature : 22 ± 1 °C
Oxygen content : >= 80 % of the saturation value
Duration of light period : 12 hours daily
Feeding : twice daily ad libitum
Food : Tetra Min, Tetra Werke, Meile (Germany)

The body length of 7 representative fish from each batch was measured:
Batch No.: 2/2002/L2 (31-Aug-2002); n=7; Body length (Variation range): 2.5 to 2.7; (cm) mean: 2.6; s: ± 0.08

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

The total hardness of the dilution water was determined weekly and was at 2.7 mmol Ca^2+ + Mg^2+/L during the study.

Test temperature:

Temperature (°C)
Concentration group: 21.2 to 21.3
Control group: 21.2 to 21.3

pH:

Concentration group: 7.5 to 7.9
Control group: 7.3 to 7.9

Dissolved oxygen:

Oxygen content (mg/L):
Concentration group: 6.5 to 8.6
Control group: 7.0 to 9.0

Salinity:

Water quality was according to ISO/DIS 73461/1

Nominal and measured concentrations:

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance, see above.

Details on test conditions:

The study was conducted in a static system. The test chambers, which were calibrated to 4 liters, were made of glass (length 20 cm, width 15 cm, height 20 cm) and stood in a water bath made of Hostalit Z® with a Plexiglas viewing panel. The temperature of the water bath was regulated by a thermostat to 22 ± 1 °C. The chambers were illuminated from above from 06.00 a.m. to 06.00 p.m. The light intensity directly over the chambers was approximately 700 lux.
The test chambers were not aerated during the course of the study.
After the stirring period and the filtration the test preparation was aerated before Insertion of the test fish using glass capillaries with a bubble-frequency of 1 - 3 per second.

Study groups
There were 7 fish in each group (control and nominal test concentration of 100 mg/L).

Test procedure
After the test concentration had been prepared and water parameters recorded, 7 fish were assigned to each test and control chamber. The fish were not fed during the entire study period. Inspection of the fish took place after 3, 6, 24, 48, 72 and 96 hours and involved recording the mortality and visible alterations in appearance and behavior. The water parameters were measured and recorded before study start and after 0, 24, 48, 72 and 96 hours.

Reference substance (positive control):

no

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

24 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Observations
The 100 mg/L group showed no effect in comparison with the control. In the control group no effects were observed.

Mortality
In this 96-hour acute toxicity study of the submission substance in zebrafish (Danio rerio) mortality occurred neither in the 100 mg/L-group (nominal concentration) nor in the control group

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this test the LC50 of the submission substance after 96 hours was > 100 mg/L (based an nominal concentration) and is above the solubility limit of the test substance in the test water.

Executive summary:

The submission substrance was tested in zebrafish (Danio rerio) for 96 hours in a static system.

The test was performed with GLP compliance and following relevant guidelines with reliability category 1.

The nominal concentration tested was 100 mg/L and a control (0 mg/L).

A nominal concentration of 100 mg/L was used for the preparation of a test solution at maximum saturation of the test substance. After treatment with ultrasonic waves for approx. 30 minutes the preparation was stirred for approximately 4 days to ensure that the solubility limit of the test substance in the test water was reached. The resulting dispersion was pre-filtered through a cellulose filter followed by filtration through a membrane filter of 0.45 µm pore size.

The tested concentration was visually present as clear solution. Analytical monitoring of the concentration of the test substance in the test water was not conducted since no relevant results could be expected. The solubility limit of the test substance was below the analytical detection limit. From the properties of the test substance it could be concluded that the test substance is chemically stable in water over the test interval of 96 hours.

In this 96-hour acute toxicity study the 100 mg/L group showed no effect in comparison to the control. In the control group no effects were observed.

Under the conditions of this test the LC₅₀ of the submission substance after 96 hours was > 100 mg/L (based on nominal concentration) and is far above the solubility limit of the test substance in the test water.

Description of key information

Under the conditions of this test the LC50 of the submission substance after 96 hours was > 100 mg/L (based an nominal concentration) and is above the solubility limit of the test substance in the test water.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

mg/L

Additional information

Two acute toxicity studies of reliability 1 for Pigment Yellow 12, and its structural analogues, Pigment Yellow 83 are available. As could be expected no toxicity towards fish has been found in any of these OECD 203 studies with nominal concentrations well above the solubility limit.