Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29-Nov-2010 to 21-Dec-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), C.
Deviations:
no
Principles of method if other than guideline:
A limit test was performed to demonstrate that the test item has no toxic effect on the test fish up to loading rate of 100 mg/L.
GLP compliance:
yes (incl. QA statement)
Remarks:
swissmedic: Date of inspection: 05 to 09-Nov-2007 and 26 to 30-Nov-2007; Date of decision: 2008-04-30, Date of signature: 12-Nov-2008

Test material

Constituent 1
Reference substance name:
Acid Yellow RN 2903
IUPAC Name:
Acid Yellow RN 2903
Constituent 2
Reference substance name:
Vers. Kilo 6, 26.05.2009
IUPAC Name:
Vers. Kilo 6, 26.05.2009
Details on test material:
The test item and the following information concerning the test item were provided by the Sponsor:

Identity: Acid Yellow RN 2903
Batch No.: Vers. Kilo 6, 26.05.2009
Purity: Approx. 65%
Expiration Date: 01-Aug-2015
Storage Conditions: At room temperature at about 20 °C, in the dark
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For the determination of the actual test item concentrations, duplicate samples were taken from each treatment just before test start (Day 0) and on the last day of preparation (Day 3), and at the end of the first and last renewal period (Day 1 and Day 4, respectively).

All samples were taken from the approximate center of each aquarium without mixing the test medium, and were stored deep-frozen (at about -20 °C) immediately after sampling. The concentration of the test item Acid Yellow RN 2903 was determined in all test medium samples.

From the control samples, only one of the duplicate samples was analyzed per sampling time. The analytical procedure and results are described in the attached Appendix I - Analytical Investigations.

Test solutions

Vehicle:
no
Details on test solutions:
At the start of the test and before each test medium renewal, the test medium with the loading rate of 100 mg/L was freshly prepared by completely dissolving 500 mg of the test item in five liter of test water using ultrasonic treatment for 5 minutes and stirring for 15 minutes at room temperature. Then, the dispersion was left undisturbed for 24 hours in the dark to allow precipitation of the test item. After the 24 h resting period, the dispersion was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) just before introduction of the fish (= start of the exposure and prior to each test medium renewal). The undiluted filtrate containing the maximum concentration of dissolved test item was used as the test medium.

The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The study was performed with zebra fish (Brachydanio rerio). The fish were obtained from a breeding culture at Harlan Laboratories. No medication was applied during holding and acclimatization. Prior to test start, the test fish were acclimated for one week to the test water and temperature. During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany). During holding and acclimatization, no fish died in the test fish batch and all fish were healthy.

From the acclimated test fish batch, 10 fish were measured at the start of the test. The mean body length of the fish was 2.97± 0.14 cm (Mean ± SD), the mean body wet weight was 0.17 ± 0.03 g (Mean ± SD).

The test method and test species are recommended by the international test guidelines.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
1.25 mmol/L
Test temperature:
The water temperature was 21 °C during the test (see attached Table 4).
pH:
The pH values in all treatments ranged from 7.4 to 7.5 (see attached Table 2).
Dissolved oxygen:
The oxygen concentration was always 8.1 mg/L or higher (see attached Table 3), and thus higher than 60% oxygen saturation.
Salinity:
according to OECD guideline
Nominal and measured concentrations:
An undiluted filtrate with the mean measured concentration of 12 mg/L was tested as a single concentration of the test item. In the analyzed test medium samples from the start and end of the first and last test medium renewal periods the measured test item concentrations were 13/15 and 9.4/9.3 mg/L, respectively (see attached Appendix I - Analytical Investigations). The reported biological results were related to the mean measured test item concentration of 12 mg/L.
Details on test conditions:
One glass test vessel with four liters of test medium was used for each treatment. The test vessels were labeled with the study number and all necessary additional information to ensure unique identification.

The water temperature in the test vessels was maintained at 21 °C (see attached Table 4). The test vessels were slightly aerated during the test period. A 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period was used light intensity during the light period was approximately within the range of 140 to 480 Lux. The test duration was 96 hours and the fish were not fed during the test.

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the highest concentration which could be dissolved in the test water. Thus, an undiluted filtrate with the mean measured concentration of 12 mg/L was tested as a single concentration of the test item. Additionally, a control was tested in parallel.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
> 12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 12 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
In the analyzed test medium samples from the start and end of the first and last test medium renewal periods the measured test item concentrations were 13/15 and 9.4/9.3 mg/L, respectively. The reported biological results were related to the mean measured test item concentration of 12 mg/L.

The biological results are listed in the attached Table 1. In the control and test medium with the mean measured concentration of 12 mg/L all fish survived until the end of the test and no visible abnormalities were observed at the test fish. Therefore, the 96 hour NOEC and LC0 of Acid Yellow RN 2903 to zebra fish were both determined to be at least 12 mg/L. The 96 hour LOEC, LC50 and LC100 were clearly higher than 12 mg/L.
In conclusion, the test item Acid Yellow RN 2903 had no acute toxic effects on zebra fish up to solubility limit of the test item in the test water.

No remarkable observations were made concerning the appearance of the test medium. It was colored by the test item throughout the entire test duration.
The pH values in all treatments ranged from 7.4 to 7.5 (see attached Table 2). The oxygen concentration was always 8.1 mg/L or higher (see attached Table 3), and thus higher than 60% oxygen saturation. The water temperature was 21 °C during the test (see attached Table 4).
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The NOEC and LC0 were determined directly from the raw data. The LOEC, LC100 and LC50 at the observation times could not be quantified due to the absence of a toxic effect of the test item at the tested concentration.

Any other information on results incl. tables

Sublethal observations / clinical signs:

Attachments:

Appendix I - Analytical Investigations

Appendix II - Tables 1 to 4:

Table 1: Mortality and Visible Abnormalities Observed in the Test Fish

Table 2: pH in the Freshly Prepared and Old Treatment Media

Table 3: Oxygen Concentrations (mg/L) in the Freshly Prepared and Old Treatment Media

Table 4: Temperatures (°C) in the Freshly Prepared and Old Treatment Media

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the test item Acid Yellow RN 2903 had no acute toxic effects on zebra fish up to solubility limit of the test item in the test water.
Executive summary:

The acute toxicity of the test item Acid Yellow RN 2903 to zebra fish (Brachydanio rerio) was determined in a 96‑hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992).

 

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to loading rate of 100 mg/L.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was prepared using ultrasonic treatment for 5 min and intense stirring at room temperature for 15 min. The so prepared dispersion was left undisturbed over 24 hours in the dark.Then, the dispersion was filtered.The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel.

The preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

 

In the analyzed test medium samples from the start and end of the first and last test medium renewal periods the measured test item concentrations were 13/15 and 9.4/9.3 mg/L, respectively. The reported biological results were related to the mean measured test item concentration of 12 mg/L.

 

In the control and at the mean measured concentration of 12 mg/L, no mortality or other visible abnormalities were determined during the test period of 96 hours.

The biological test results (based on mean measured concentration of the test item) were as follows:

 

Mean measured concentration
(mg/L)

– 96-hour LC50:

>12

 

 

– 96-hour LC0:

>=12

 

 

– 96-hour LC100:

>12

 

 

– 96-hour NOEC:

>=12

 

 

– 96-hour LOEC:

>12