Registration Dossier

Toxicological information

Exposure related observations in humans: other data

Administrative data

Endpoint:
exposure-related observations in humans: other data
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP. Read across from a similar substance which has the same main component and with a different counter ion that doesn't influence the characteristics related to the specific end-point
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Occurrence of residues of the veterinary drug malachite green in eels caught downstream from municipal sewage treatment plants
Author:
Andrea Schuetze, Thomas Heberer, Susanne Juergensen
Year:
2008
Bibliographic source:
Chemosphere 72 (2008) 1664–1670
Report Date:
2008

Materials and methods

Type of study / information:
Evaluation of residues of Malachite Green in eels due to the presence of discharges by municipal STPs and evaluation of the human health risk associated with consumption of the eels investigated.
Principles of method if other than guideline:
Evaluation of an exposure scenario: residues of Malachite Green (MG) have been found in wild eels caught from surface waters downstream from the sewers of different municipal sewage treatment plants (STPs). MG and its metabolite Leucomalachite Green (LG) were detected in the tissues of 25 out of 45 eels caught from different lakes, a river and a canal in Berlin, Germany.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Ethical approval:
not applicable
Details on study design:
- Test Animal: Eels
- Source: different lakes (Griebnitzsee, Mueggelsee, Tegler See and Wannsee), a river (Spree river) and a canal (Teltowkanal) in Berlin, Germany
- Acclimation period: between April and June 2007
- N° animals: 45
- weight: 120.1 to 615.0 g per fish
- Sacrifice: Each eel was sacrificed by a neck cut. The head, the skin, and the vitals were removed mechanically. The filet samples including the backbone and the bones were cut into small pieces and stored into evacuated aluminium bags at - 24°C until
analysis.
- Choose of selection: Eels are living close to the ground of their habituating watercourse also taking up sediments with their food.
European eels strongly bioaccumulate organic compounds in their tissues because of their high natural fat content of around 25%.

Sampling:
Three independent filet samples were prepared and analyzed with double HPLC injection for each investigated eel.
The defrosted samples were homogenized in a blender to obtain a fine mash. 2.0 g of each sample were accurately filled into 50 ml screw-capped polypropylene tubes and then fortified with a defined concentration of the internal standard (MG-D5)
- Blank: One out of two homogenized blank samples was spiked with a defined concentration of Malachite Green (MG) and Leucomalachite Green (LG)
- Extraction: The extraction was conducted after incubation for 15 min at room temperature. 2 ml of the Mc Ilvaine solution (pH 3.0), 100 µl of 1M p-TSA and 50 µl of methanolic TMPD were merged and mixed with the samples. Then, 12 ml acetonitrile were added to the samples mixed with a Vortex shaker and extracted for 15 min using a platform shaker at 500 rpm. The resulting suspension was centrifuged at 15 °C for 5 min at 3400 rpm and the supernatants were transferred into 50 ml screw-capped polypropylene tubes. The residuum was extracted again after mixing with 2 ml of a Mc Ilvaine solution (pH 6.0) and 12 ml of acetonitrile. The suspension was extracted for 15 min with a platform shaker at 500 rpm and centrifuged for 5 min with 3400 rpm at 15 °C.
The supernatants were combined and mixed with 6 ml of dichloromethane. The following centrifugation carried out again for 5 min with 3400 rpm at 15 °C. The water phase at the bottom was removed and the sample solutions were transferred into a SPE column pre-conditioned with a mixture of 2 ml acetonitrile and dichloromethane (80/20). The solution was passed through the SPE cartridges at a flow-rate of 0.5 ml/min until the columns were running dry. The SPE cartridges were washed with 1.5 ml of methanol and dried with nitrogen for 10 min. MG, MG-D5 and LG were eluted into a 6 ml screw-capped polypropylene tube by applying 3 ml of the SPE-eluent. The sample extracts were then dried under a gentle stream of nitrogen at room temperature. The remaining residue was dissolved in 500 µl of the final solvent solution (mixture of eluents A and B, 50:50 v/v) and centrifuged for 5 min with 3400 rpm at 15°C. Twenty microlitter of the supernatants were analyzed by HPLC–ESI-MS/MS.
- Other: pH 3.0 was prepared by mixing 18.9 ml of 0.2 M disodium hydrogen phosphate and 81.1 ml of 0.1 M citric acid solution with a pH of 6.0 was obtained by mixing 62.5 ml of 0.2 M disodium hydrogen phosphate and 37.5 ml of 0.1 M citric acid
Exposure assessment:
measured
Details on exposure:
TYPE OF EXPOSURE: released in waste water (German lakes)

TYPE OF EXPOSURE MEASUREMENT: Biomonitoring tissues

EXPOSURE PERIOD: 3 months

Results and discussion

Results:
All residues and all samples have a concentrations under the 2 µg/kg. LG showed a slightly raised incidence of neoplasms at dose equal to 13 mg LG/kg bw. The risk of adverse health effects is very low both for a single and for casual consumption of eels containing the maximum residues detected in this study.

Any other information on results incl. tables

MG and its reduced metabolite LG were detected in the tissues of 25 out of the 45 investigated eels. LG was the dominating

residue in all of the analyzed samples. The ratio measured for LG and MG varied between 5:1 and 7:1. The occurrence of the residues in the eel samples could directly be linked to the presence of purified sewage discharged by the municipal STPs into the receiving surface waters.

MG is a multiple-use compound with various legal applications. Thus, contamination of fish by environmental sources (from uses with ornamental fish, production sites or uses in consumer products) should also be considered and may explain problems with background contaminations that have been observed during analysis of MG residues at trace-level concentrations. It seems very likely that the residues of MG found in the samples of eels living downstream from discharges by municipal STPs originate from legal uses, e.g. by wash off from clothes or paper towels coloured with MG. Additional loads from the legal use of MG as veterinary drug for the treatment of ornamental fish (private aquaria) are possible.

No one of the investigated samples exceeded the MRPL value of 2 µg/ kg (for the sum of MG and LG) set as action limit for internationally traded food consignments by the EU. Thus, an exceedance of this MRPL value in (imported) fish intended for human consumption may not be explained by background contaminations originating from treated municipal sewage effluents.

Human Health:

Residues are occurring in fish and fish products intended for human consumption both due to illegal uses or, as demonstrated in this paper, due to background contamination most likely caused by multiple, but legal uses.

In these studies, LG showed a slightly raised incidence of neoplasms at a dose equal to 13 mg LG/ kg body weight. Therefore, this value was used for a ‘‘case-related” risk assessment on the basis of a MOE approach to evaluate consumer risks posed by contaminated foodstuffs. Even with the assumption of a ‘‘worst case” scenario with a life-time consumption of 300 g eel filet per day (Committee for Medicinal Products for Veterinary Use, 2001), the MOE value is 3.4 million calculated for the highest residue of LG found in this study (0.765 µg/kg). An acute risk assessment conducted on the basis of consumption data obtained from a nutrition study with children aged 2–5 (Banasiak et al., 2005) yielded a MOE of 1.8 million. With regard to consumers health protection only a margin of exposure below 10000 is regarded as not being acceptable for genotoxic carcinogens. This means that the risk of adverse health effects is very low both for a single and for casual consumption of eels containing the maximum residues

detected in this study.

Calculations of the ‘‘margin of exposure” (MOE) using different models for acute and chronic consumption. Intake calculations were based on individual consumption data and the maximum residue found in the investigated samples of eels (= 0.765 lg kg1 fresh weight)

Consumption model/data Calculated intace (µg/kg bw/d) LOEL (mg/kg bw) MOE
VELS: acute consumption data * (child 2-5 years, 16.15 kg/bw, 153.5 g fish/d 0.007224 13 1.80 x 10^6
VELS: chronic consumption data * (child 2-5 years, 16.15 kg/bw, 153.5 g fish/d 0.0002653 13 49.0 x 10^6
CVMP (EMEA): chronic consumption data ** (adult 60 kg bw, 300 g fish/d) 0.003825 13 3.40 x 10^6

*: consumption data optained from the VELS consumption study for children aged between 2 -5 years

**: consumption data from the risk analysis approach for residues of veterinary medicinal products in food of animal original used by the Committee for Medicinal Product for Veterinary Use (2001) of the Europian Medicines Agency

LOEL: obtained from the studies of US National Toxicology Program (2005)

Applicant's summary and conclusion

Conclusions:
All residues and all samples have a concentrations under the 2 µg/kg. LG showed a slightly raised incidence of neoplasms at dose equal to 13 mg LG/kg bw. The risk of adverse health effects is very low both for a single and for casual consumption of eels containing the maximum residues detected in this study.
Executive summary:

Residues of Malachite Green (MG), a veterinary drug illegally used for the treatment of aqua-cultured fish, have been found in wild eels caught from surface waters downstream from the sewers of different municipal sewage treatment plants (STPs). MG and its metabolite Leucomalachite Green (LG) were detected with total concentrations up to 0.765 µg/ kg fresh weight in the tissues of 25 out of 45 eels caught from different lakes, a river and a canal in Berlin, Germany. In all cases, the occurrence of the residues could directly be linked to the presence of discharges by municipal STPs into the receiving surface waters.

MG and LG are regarded as potential genotoxic carcinogens making it impossible to establish a tolerable daily intake. A margin of exposure (MOE) approach was applied to evaluate the human health risk associated with the consumption of the eels investigated in this study. According to European Union law, zero tolerance applies to all residues of MG and LG found in food for human consumption as MG is not registered for use as veterinary drug.