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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Description of key information

Uptake and adsorption of Cholesterol, the main component of Lanolin alcohols is assessed to follow a complex kinetic behaviour. The available dietary study for Cholesterol, needs to be amended, as only first order kinetic was assumed in the evaluation. Results will be integrated in an update dossier as soon as data are available.

Key value for chemical safety assessment

Additional information

Outline of the present study performance and results

Lanolin alcohols (CAS No. 8027-33-6) is a substance of unknown and variable composition (UVCB), containing approximately 30% Cholesterol (CAS No. 57-88-5) and significant fractions of structure-related substances. Cholesterol was therefore chosen for the investigation of bioaccumulation potential of Lanolin alcohols in fish as required under REACh regulation (EC) 1907/2006. Initial attempts were made to perform an aqueous study in accordance with OECD guideline 305. However, no stable test concentration was reached in pre-tests. Thus it was decided to conduct a dietary fish test as outlined in OECD 305.

 

Bluegill sunfish (Lepomis macrochirus) was investigated in a dynamic flow through system. The fish were fed during 14 days in a flow-through system with food containing a target concentration of 1000 µg/g radiolabelled test item. After the exposure, the fish were transferred to flowing untreated water and the depuration of radioactivity was followed for further 28 days. Temperature, pH and oxygen concentration during the whole study were within acceptable limits.Measurements ranged from 21.6-24.8 °C, 8.0-8.5 and 7.2-8.4 mg/L, respectively.

 

By determination of the radioactivity level, the content of the test item in the food was confirmed. A level of 959.1 µg/g food was measured immediately after preparation. A level of 961.0 µg/g food was measured after 22 days of storage at -20 °C. At the end of the feeding period, the test item concentration in fish amounted to 109.362 ± 10.736 µg/g fish. The values remained almost constant at depuration days 1 (115.319 µg/g fish) and 3 (125.763 µg/g fish) and thereafter decreased slowly. At the end of the depuration phase (day 42), the radioactivity concentration was 88.434 µg/g fish. An additional measurement was performed after 3 days of uptake. The mean value of 5 fish amounted to 27.845 ± 6.715 µg/g fish. No difference in the growth rates of control and test group was observed. The growth rate of the test fish was 0.015 day-1. Assuming first order kinetics, the rate constant k2 for the depuration of the radioactivity from the fish was calculated to be 0.011 day-1 (R2 = 0.29). Because kg was bigger than k2, the growth-corrected rate constant k2g could not be calculated. As the growth rates of control and test group were similar a correction for growth is not considered to be relevant. The derived time 0 concentration C0,d was 110.057 µg/g and the content of test item in the diet was 959.1 µg/g. Based on these values and a feeding rate of 0.02 g food/g fish/day, the substance assimilation efficiency alpha was calculated to be 0.4435. Kinetic dietary BMFK and lipid-corrected kinetic dietary BMFKL amounted to 0.7747 and 4.0470, respectively.

 

The test performance of this study is in line with OECD 305 and considered as valid. However, the calculation of BMF values based on a first order kinetic rate constant is not considered to reflect the cell indigenous presence and active regulation of Cholesterol. Therefore the rate constant of Cholesterol (k2) will be newly assessed in a statistical evaluation assuming a more complex kinetic behavior. The result of this new statistical assessment will be implemented in an update dossier as soon as data are available. 

 

Role of Cholesterol homeostasis in animals

Based on the current statistical evaluation the dietary study resulted in a high lipid corrected BMF. These values should however been taken with caution as the predications are conducted based on the assumption that uptake and depuration patterns will follow “first order kinetics”. Cholesterol is a basic structure component of cell membrane and naturally present in all cells of fish body. The available study did not determine the radioactivity based steady-state concentration. But an increased concentration in fish at beginning of depuration phase and the linearity of depuration curve (R2 = 0.29) indicates a more complex depuration pattern than first order kinetics.

Furthermore, as Cholesterol is not treated as an organic xenobiotic in vertebrates when being administered via diet. In fact, cholesterol is a main component of cell membranes and the precursor of steroid hormones and bile acids. Thus, it is clearly essential to life. In healthy organisms an intricate balance is maintained between the biosynthesis, utilization and transport of cholesterol (see also Voet, Voet & Prat, Principles of Biochemistry, 4thedition; p703f).

 

Cholesterol can be uptaken via diet or synthesized de novo. The cholesterol biosynthesis is one of the most strongly regulated pathways in general. The speed of biosynthesis can vary by a factor of 100, depended on how much cholesterol was taken up via nutrition. The main places for cholesterol synthesis are liver and intestines. Extend of synthesis in these organs is strongly dependent on the cholesterol levels in the cells (see also Stryer, Biochemie, 7thedition, p.781)

 

Intracellular homeostasis is regulated by HMG-CoA reductase. In general, a higher uptake from food leads to a net decrease in endogenous production, whereas lower intake from food has the opposite effect. The main regulatory mechanism is the sensing of intracellular cholesterol in the endoplasmic reticulum. Cholesterol synthesis can be completely turned off when intracellular cholesterol levels are high.

 

Equally to all vertebrates Teleost fish are capable of synthesizing cholesterol and as such have no dietary requirement for it. Fish possess many of the same gastrointestinal organs present in humans (e.g. liver, intestine, exocrine and endocrine pancreas and gallbladder) as well as the specialized cell types involved in lipid absorption and processing (e.g. intestinal enterocytes etc.). There is clear evidence that the regulation of intracellular cholesterol content is very similar to those in mammalian cells (e.g. Betancor et al. 2014; Norambuena et al. 2013). For example: In a recent study rainbow trout was fed with two identical vegetable oil based diets but one with additional cholesterol fortification and one without for 12 weeks. There were no effects on growth and feed efficiency. However in fish fed with high Cholesterol diet no biosynthesis of cholesterol and a remarkably decreased apparent in vivo fatty acid beta-oxidation were recorded, whilst in the low cholesterol fed fish, cholesterol was abundantly synthesized and increased beta oxidation was observed (Norambuena et al. 2013). The result of this study is in line with other studies (Anderson et al. 2011; Deng et al. 2013; Leaver et al. 2008; Kim et al. 2012) showing the presence of cholesterol homeostasis in Teleost fish. In fact, Zebra fish (Danio rerio) is already established as new alternative model organism for in vivo cholesterol metabolism studies (e.g. Anderson et al. 2011).

 

Implications of Cholesterol metabolism to present study

Given the role of Cholesterol in animal cells, it was decided in advance to integrate some sort of proof for the existence of Cholesterol homeostasis in Lepomis macrochirus within this test. As an easy performable method a ready to use kit was chosen for total cholesterol content determination (Total Cholesterol Assay Kit, Cell BioLABS Inc., USA). In this way total cholesterol was measured at test start and at the end of the uptake phase (after 14 days). In addition total Cholesterol was measured at the end of the depuration phase.

As result, there were no significant differences in the total cholesterol levels at day 14 between control and test group animals. The result is in line with the observations as given above, i.e. the existence of a regulating mechanism in the present test species, Lepomis macrochirus.

 

Conclusion

Due to the ability of Teleost fish to regulate intracellular levels of Cholesterol und the uncertainness of predication, the estimated high BMF as determined in the present dietary accumulation study is currently considered not sufficient for the assessment of bioaccumulation. The depuration rate constant of Cholesterol (k2) will be newly assessed in a statistical evaluation assuming a more complex kinetic behavior. The result of this new statistical assessment will be implemented in an update dossier as soon as data are available. 

Differently to organic xenobiotics, Cholesterol is integrated into the cellular metabolism. It is questionable, whether an assessment of bioaccumulation of such cell indigenous, actively regulated substances can be based on a standard bioaccumulation test designed for xenobiotics. On the other hand, excess dieting of cholesterol rich feed in long term could impact the cholesterol metabolism and cholesterol regulation in mammals, resulting in cholesterol accumulation and deposition. 

For references please see attached CSR, section 13.