N-(5-chloro-2-methoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

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:

From 02.OCT 1981 to 09. OCT 1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study performed similar to OECD 203, sufficiently well documented, pre-GLP

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A stock solution of the test item was prepared in test water, applied to the test tanks and evenly distributed by a glass rod.

Test organisms (species):

Leuciscus idus melanotus

Details on test organisms:

Breeder: Paul Eggers, 2345 Hohenwestedt
Acclimation: Fish were kept at least for 14 days in test water at 20 deg C before start of the experiment.
Body weight: 1.8 - 2.5 g (arithmetic mean: 2.1 g; sd = 0.26 g; n = 10)
Body length: 5.5 - 6.7 cm (arithmetic mean: 6.3 cm; sd = 0.34 cm; n = 10)
Corpulence factor: 0.8 - 1.08 (arithmetic mean: 0.86; sd = 0.09; n = 10)
Feeding: Tetra Min (Tetra Werke, Melle)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96

Remarks on exposure duration:

h

Hardness:

Total hardness: 9.5 deg dH
Carbonate hardness: 6.4 deg dH

Test temperature:

20 +/- 1 deg C

pH:

7.8 - 8.2

Dissolved oxygen:

> 7 mg/L (7.0 - 8.0 mg/L)

Nominal and measured concentrations:

0, 1, 10, 100, 500 mg/L nominal concentrations

Details on test conditions:

For each concentration group and the control 10 fish were used.
Deionized tap water ( < 5 µS/cm) was supplemted with the following salts and used as test water:
192 mg/L NaHCO3
120 mg/L CaSO4*2H2O
120 mg/L MgSo4
8 mg/L KCl

Test basins made of glass were used (40 x 25 x 30 cm) and filled with 20 L test water. The test temperature of 20 +/- 1 deg C was kept constant by means of a water bath. Test tanks were aerated during the whole test period by glass capillaries with a aeration rate of 100 mL air/min. Day-night regime was 12 hours, illumination strength approximately 700 Lux.

Approximately 65 hours preceding the experiment 10 fish, each were introduced into the test basins (20L).
Immediately preceding test item application and after 2, 24, 48, 72 and 96 hours pH, O2-concentration and temperature were determined.

During the whole experiment fish were observed on a regular basis and their behaviour was recorded.
At the end of the incubation period fish were killed in 0.05% 3-Aminobenzoic acid ethyl ester methanesulfonate, dissected and macroscopically examined.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

containing 40% submission substance, i.e. LC50 > 200 mg/L submission substance

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

containing 40% submission substance, i.e. EC50 > 200 mg/L submission substance

Basis for effect:

behaviour

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

containing 40% submission substance, i.e. LC0 = 200 mg/L submission substance

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

EC0

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

containing 40% submission substance, i.e. EC0 = 200 mg/L submission substance

Basis for effect:

behaviour

Details on results:

Neither for the control nor for the treatment groups any dead fish were observed. No differences in behaviour were observed in the treatment groups compared to the control group. The onl y macroscopic observation made was a slightly pink colouring of the fins at the highest concentration (500 mg/L test material).

Validity criteria fulfilled:

yes

Conclusions:

In an acute toxicity test on the fish species Leuciscus idus similar to OECD 203 the submission substance proved to have no toxic or behavioural effects up to the highest tested concentration of 200 mg/L submission substance (500 mg/L test material).
LC50 (96h) > 200 mg/L (>500 mg/L test material)
LC0 (96h) = 200 mg/L (= 500 mg/L test material).

Executive summary:

In an acute toxicity test on the fish species Leuciscus idus similar to OECD 203 (reliability category 2) the submission substance proved to have no toxic or behavioural effects up to the highest tested concentration of 200 mg/L submission substance (500 mg/L test material). In the test material, the submission substance was present as a stable dispersion miscible with water.

LC50 (96h) > 200 mg/L (>500 mg/L test material)

LC0 (96h) = 200 mg/L (= 500 mg/L test material).

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2000-12-13 to 2001-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to relevant guidelines and compliant to GLP, well documented translation of original report (in Japanese)

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

no

GLP compliance:

yes

Remarks:

The study was conducted in accordance with "Eco-toxicity test Standards" by Japanese Ministry of the Environment.

Analytical monitoring:

yes

Details on sampling:

Samplings were made at the time point of study initiation (0 hr) and immediately prior to water renewal (48 hr), and analyzed by HPLC.

Vehicle:

yes

Details on test solutions:

After weighing of a necessary amount of test substance using an electronic balance, the same amount of hydrogenated castor oil (HCO-40) was added to the test substance as co-solvent and mixed well. Then, the solution was transferred to the test vessel by rinsing with dilution water and adjusted to 5 L.
The prepared test solution showed red color due to the test substance original but no visible precipitate was observed. The solution of control and solvent control was observed to be clear and colorless without visible undissolved test substance.

Test organisms (species):

Oryzias latipes

Details on test organisms:

Species: Japanese killifish
Scientific name: Oryzias latipes
Body length: 2.0 cm (1.7 - 2.1 cm), n = 10
Body Weight: 0.16 g (0.14 - 0.18 g), n = 10
Supplier: Izumoto fish firm (Osaka, Japan)
Date of receipt: December 18, 2000

Acclimation: December 18, 2000 - January 22, 2001
Mortality during 7 days before the initiation of exposure was less than 5%. Visibly healthy and normal fishes were used for the test.
Condition for acclimation
Culturing water: Dilution water
System: Recirculatory filtering system
Temperature: 24 +/- 1 deg C
Lighting: Indoor lighting, 16 hr light / 8 hr dark
Feeding: Commercially available feed (Trade name: TetraMin)
Feed amount: ca. 2% / body weight / day (until the final 24 hours before the initiation of exposure)

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No

Hardness:

Total hardness: 63.1 mg/L

Test temperature:

23.8 - 24.0 deg C

pH:

7.4 - 7.8

Dissolved oxygen:

In mg/L:
Control: 6.7 (old medium, 96 hours) to 8.1
Solvent control: 6.5 (old medium, 96 hours) to 8.1
Exposure group (100 mg/L): 5.4 (old medium, 96 hours) to 8.0

Nominal and measured concentrations:

Nominal limit concentration: 100 mg/L;
Measured concentration test start (new medium): 100 mg/L;
Measured concentration at 48 hours (old medium): 99 mg/L;
Geometric mean measured concentration: 100 mg/L.
The concentration of test substance in the control and solvent control were less than the quantification limit (0.06 mg/L).

Details on test conditions:

Test conditions
Type of exposure: Semi-static (water renewal was made after 48 hr exposure)
Duration of exposure: 96 hr
Volume of test solution: 5 L / vessel
Replication: 1 vessel / test group
Number of organisms: 10 fish / test group
Aeration: None
Temperature: 24 +/- 1 deg C
Lighting: Indoor lightning, 16-hour light / 8-hour dark
Feeding: None
Dilution water: Tap water (Takarazuka city, Hyogo) was dechlorinated with charcoal filter and saturated with oxygen by aeration. Then the water was used as the dilution water after adjusting to the test temperature. The results of water quality measurement are shown in section "Any other information on materials and methods incl. tables". From the results of the water quality measurement and no observed abnormalities in organisms, it was judged that the dilution water can be used as culturing water in the bio-assays.

Test vessels and water bath
Vessel: 5 L volume whole glass aquarium (inside size approximately 21 x 16 x 23 cm)
Water bath: FRP Thermostatic water bath (inside size 300 x 81.5 x 28.5 cm, controlled with warm and cold water)
Dissolved oxygen analyzer: Model 58 (YSI Inc.)

Selection of test concentration
In a range-finding study conducted at the concentration of 10 and 100 mg/L, 0% mortalities were observed even at maximum exposure concentration of 100 mg/L, and no toxicological symptoms were observed. Based on these results, a limit test with 100 mg/L, which was the maximum concentration tested in the range-finding study, was chosen for the definitive study.
As reference, a control group (dilution water only) and a solvent control group, in which co-solvent concentration was 100 mg/L (i.e. the same as for the exposure group) were allocated in the study.

The body weight and body length of the 10 fishes, which were acclimated in the same aquarium used for culturing, were measured before the initiation of exposure in the study.
After the measurement of temperature, dissolved oxygen and pH of test water in each test vessel, ten fishes for each test group were introduced to each test vessel. The temperature, dissolved oxygen and pH were measured every day during the exposure period. In addition, the temperature, dissolved oxygen and pH were also measured before and after water renewal. Fish were not fed during the exposure period.
Mortality and toxic symptoms or abnormity were recorded at 24, 48, 72 and 96 hr after the initiation of the study. The following were the common symptoms and definitions:
Death: No reaction to the spurs was observed.
Abnormal breath: Different movement of gill cover is observed compared to control group.
Abnormal swimming: Different swimming form is observed compared to control group (e.g., slow-moving, hypersensitivity, convulsion and inversion etc.).
Inability to swim: Lack of swimming capability is observed although it is moving at surface or bottom of the water. State of overturn and apparent death are included.

Reference substance (positive control):

yes

Remarks:

copper (II) sulfate pentahydrate

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

EC0

Effect conc.:

100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

& mobility

Details on results:

The measured concentrations of the test substance in the test solution were 100% and 99% of the nominal at the initiation of exposure and 48 hr after the test initiation, respectively.
Since 0% mortality in the control groups was observed during the study, the conditions for the validity of the study were fulfilled.
During the exposure period, toxic symptoms were not observed at 100 mg/L test item concentration. Also for the controls, no abnormal symptoms were observed.
Water temperature during exposure period was between 23.8 and 24.0 deg C, consequently, the temperature of all groups was within a range of pre-set temperature, 24 +/- 1 deg C. Dissolved oxygen level was in the range from 5.4 to 8.1 mg/L and more than 60% of the saturation concentration (8.25 mg/L at 24.0 deg C) were maintained in all groups. The variation of pH values was slight and in the range from 7.3 to 7.8 during the exposure.

Results with reference substance (positive control):

The median lethal concentration (LC50) at 96 hr with a reference substance (copper (II) sulfate pentahydrate, reagent grate, Lot No. PAH2068, Wako Pure Chemical Industries) was 2.3 mg/L, which was comparable to the historical LC50 of 2.3 -5.7 mg/L, (n = 8) obtained at the testing facility since December, 1995.

Reported statistics and error estimates:

No statistical analysis was applied for the calculation of median lethal concentration (LC50), since the study was conducted as a limit test at 100 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

In a reliable 96-hours acute toxicity study on Oryzias latipes performed according to OECD 203 and compliant to GLP (reliability category 1), the test item caused neither lethality nor abnormal behaviour or mobility at the applied limit concentration of nominal 100 mg/L (geometric mean measured concentration: 100 mg/L). Also no lethality nor abnormal behaviour or mobility was observed in the control and the solvent control (containing the amount of HCO-40 used in the treatment group). The following (no)effect concentrations were determined (geometric mean measured concentrations):
LC50 (96 h): > 100 mg/L;
LC0 (96 h): 100 mg/L;
EC0 (96 h, behaviour & mobility): 100 mg/L.

Executive summary:

In a reliable 96-hours acute toxicity study on Oryzias latipes with semi-static test design (medium renewal after 48 hours) performed according to OECD 203 and compliant to GLP (reliability category 1), the test item caused neither lethality nor abnormal behaviour or mobility at the applied limit concentration of nominal 100 mg/L (geometric mean measured concentration: 100 mg/L). Hydrogenated castor oil (HCO-40) was used at equal weight as co-solvent to achieve the limit concentration of the otherwise insoluble test item. Also no lethality nor abnormal behaviour or mobility was observed in the control and the solvent control (containing the amount of HCO-40 used in the treatment group). The following (no)effect concentrations were determined (geometric mean measured concentrations):

LC50 (96 h): > 100 mg/L;

LC0 (96 h): 100 mg/L;

EC0 (96 h, behaviour & mobility): 100 mg/L.

The robust study summary is based on the translation of the original study report performed by the same company involved with performance of the test (Sumika TechnoService Corporation, Japan).

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982-02-15 - 1982-02-19

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Pre-guideline study performed similar to OECD 203 with GLP compliance. The results are plausible and well presented. No analytical confirmation of test item concentration, however test item assumed to be stable.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Remarks:

Principles of Good Laboratory Practice (GLP), German Federal Register, Vol. 43, No. 247, December 22, 1978

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

yes

Details on test solutions:

The test substance-dispersion is miscible with water in every ratio. Starting with a stock solution in dilution water (10% stock solution), the amounts corresponding to the respective test concentrations had been pipetted into the test basins and mixed with a glass bar.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species : zebrafish, Danio rerio (HAMILTON-BUCHANAN)
Origin : West Aquarium, Bad Lauterberg
Date of hatching: 1981-10-09
Date of receipt: 1982-01-20

Until day 7 before start of the experiment, the fish were kept in dechlorinated tap water and further on until transfer into the test basins in dilution water:
Temperature : 23 ± 1 °C
Oxygen content : >= 80 % of the saturation value
Duration of light period : 12 hours daily
Stocking rate: <= 1 g fish/L water
Feeding : twice daily ad libitum
Food : Tetra Min, Tetra Werke, Meile (Germany)

Immediately before start of the experiment the body length of 10 fish representative for the used fish batch were measured. These animals were not used for the experiment:
Batch No.: 11/82 (1982-02-15); n=10; Body length (Variation range): 2.5 to 3.2 cm; mean: 2.8 cm; s: ± 0.24

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

Not applicable

Hardness:

The total hardness of the dilution water: 2.5 mmol Ca^2+ + Mg^2+/L during the study.
The molar ration of Ca++ to Mg++ was 4:1, the molar ratio of Na+ to K+ was 10:1.

Test temperature:

Temperature (°C)
Concentration groups and control group: 22.0 - 22.9

pH:

Concentration groups: 7.5 - 7.9
Control group: 7.6 - 7.8

Dissolved oxygen:

Oxygen content (mg/L):
Concentration groups: 6.2 to 8.6
Control group: 6.4 to 8.6

Salinity:

Water quality was according to OECD 203

Nominal and measured concentrations:

5 test groups with 10 fishes, each, had been evaluated:
Nominal test item concentration (42.8% pigment) [mg/L]
0 / 1 / 10 / 100 / 500

Details on test conditions:

The study was conducted in a static system. The test chambers, which were calibrated to 10 liters, were made of glass (length 29 cm, width 21 cm, height 23 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.
The test substance-dispersion is miscible with water in every ratio. Starting with a stock solution in dilution water, the amounts corresponding to the respective test concentrations had been pipetted into the test basins and mixed with a glass bar.

Study groups
There were 10 fish in each group. Concentration of test item (42.8% pigment) [mg/L]
0 / 1 / 10 / 100 / 500

Test procedure
After the test concentration had been prepared and water parameters recorded, 10 fish were assigned to each test and control chamber stochastically. The fish were not fed during the entire study period. Inspection of the fish took place after 2-4, 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 at the same time points.

At the end of the study, surviving fish were killed with 0.05% MS 222 (3-Aminobenzoic acid ethyl ester, methane sulfonate, Sandoz).

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

LC0

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: corresponding to 214 mg/L in relation to the submission substance (42.8%)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: corresponding to 214 mg/L in relation to the submission substance (42.8%)

Duration:

48 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: corresponding to 214 mg/L in relation to the submission substance (42.8%)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: corresponding to 214 mg/L in relation to the submission substance (42.8%)

Details on results:

Observations
With the exposure groups 10, 100 and 500 mg/L , the heavy coloration of the water by the test substance compromized the observation of symptoms. However, fish of all test groups showed no difference to the control in terms of behaviour.
Fish from exposure groups 100 and 500 mg/L showed a light red coloring at the end of the test.

Mortality
In this 96-hour acute toxicity study of the submission substance in zebrafish (Danio rerio) mortality occurred neither in the control group nor in the treatment groups.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Validity criteria fulfilled:

yes

Conclusions:

The test was performed with GLP compliance and similar to OECD-guideline 203. Using a dispersion of the submission substance as test item (42.8% of submission substance), high concentrations far above water solubility could be tested avoiding formation of any precipitate.
Under the conditions of this test the LC50 of the test item after 96 hours was > 500 mg/L (214 mg/L submission substance) and the LC0 equal to 500 mg/L (214 mg/L submission substance), based on nominal concentrations, each.
As the test item contains a relevant amount of the submission substance, the test results are considered adequate to fulfill the endpoint requirements.

Executive summary:

The test item (42.8% submission substance) was tested in zebrafish (Danio rerio) for 96 hours in a static system. The test item is a dispersion of the submission substance and readily miscible with water. Such, concentrations of the submission substance far above the level of water solubility could be tested avoiding formation of any precipitate. The nominal concentrations tested were 0 / 1 / 10 / 100 / 500 mg/L with 10 fish for each concentration group. Concentration groups 10, 100 and 500 mg/L showed heavy coloration of the water. Analytical monitoring of the concentration of the test substance in the test water was not conducted as the test item is stable in water. In this 96-hour acute toxicity study, no effects in comparison to the control could be observed at any concentration of the test item (1, 10, 100 and 500 mg/L). Neither in the control nor in the concentration groups any mortalities or behavioural abnormalities occurred. Under the conditions of this test the LC50 of the test item after 96 hours was > 500 mg/L and the LC0(96h) = 500 mg/L nominal concentration (corresponding to 214 mg/L submission substance). As the test item contains a relevant amount submission substance, the test results are considered adequate to fulfill the endpoint requirements.

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

Justification for type of information:

Refer to the analogue approach provided in the CSR.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

containing 40% submission substance, i.e. LC50 > 200 mg/L submission substance

Basis for effect:

mortality (fish)

Remarks on result:

other: Hoechst, 1981

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 500 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: corresponding to 214 mg/L in relation to the submission substance (42.8%)

Remarks:

Hoechst. 1982

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other:

Remarks:

Sumika, 2001

Description of key information

LC50 for freshwater fish: > water solubility limit

Additional information

Studies with Pigments Red 22 and Red 146 were performed compliant to GLP, the study on PR 184 is a pre GLP-study but well documented and meeting scientific standards. All three studies were performed according to (PR 22) or similar to OECD 203 (PR 146, PR 184), meeting the validity criteria of the OECD guideline. Analytical monitoring confirmed the nominal limit concentration of 100 mg/L of PR 22 tested on Oryzias latipes (geometric mean measured concentration: 100 mg/L). Hydrogenated castor oil (HCO-40) was used at equal weight as co-solvent to achieve the limit concentration of the otherwise insoluble test item. Semi-static test design (daily renewal) was applied. Neither mortality nor behavioural abnormalities were observed at the limit concentration during the test. 

While analytical monitoring was not performed in the remaining studies on PR 146 and PR 184, both pigments are known to be stable in aqueous solution. In both cases, test items were stable dispersions of the respective pigments and readily miscible with water. Highest tested concentrations were 200 mg/L PR 146 on Leuciscus idus (500 mg/L test material) and 214 mg/L PR 184 on Danio rerio (500 mg/L test material). For both pigments and fish species, neither mortalities nor behavioural abnormalities or other toxic effects were observed during the 96 hours of the test. 

Therefore, Naphtol-AS Pigments should be regarded as acutely non-toxic to freshwater fish up to and exceeding 100 mg/L nominal concentration. This conclusion is based on three different pigments tested on three different freshwater fish species.