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Basic toxicokinetics

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Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Data from collection of data. Review article.

Data source

Reference
Reference Type:
review article or handbook
Title:
ERUCIC ACID IN FOOD: A Toxicological Review and Risk Assessment. TECHNICAL REPORT SERIES NO.21
Author:
Food Standards Australia and New Zealand
Year:
2003
Bibliographic source:
Food Standard Australia and New Zealand, ISBN 0 642 34526 0

Materials and methods

Test guideline
Qualifier:
no guideline followed
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Any other information on results incl. tables

The human health concern with erucic acid arises from two findings. Firstly, experimental studies have demonstrated an association between dietary erucic acid and myocardial lipidosis in a number of species. Myocardial lipidosis is reported to reduce the contractile force of heart muscle. The occurrence of myocardial lipidosis can be explained by the effect that erucic acid has on the mitochondrial β–oxidation system. Secondly, studies have also demonstrated an association between dietary erucic acid and heart lesions in rats. So far, however, there is no evidence that dietary erucic acid can be correlated to either of these effects in humans. Furthermore, there is no conclusive evidence indicating that the development of myocardial lipidosis is causally linked to the development of myocardial necrosis. However, given what is know about erucic acid metabolism, it seems reasonable to expect that humans would also be susceptible to myocardial lipidosis following exposure to high levels of erucic acid.

All of the available animal studies rely on short term or sub–chronic oral exposure to oils containing various proportions of erucic acid. The most common effect associated with short–term, and to a lesser extent, subchronic exposure to these oils is myocardial lipidosis.

This effect is observed soon after the commencement of oil feeding and appears to be increased in its severity, in a dose–dependent manner, if erucic acid is present. Clinical signs are typically absent; reduced weight gain only occasionally being correlated with erucic acid dose.

Increased myocardial lipidosis is associated with doses of erucic acid at 1500 mg/kg bw/day in rats, although in nursling pigs this occurs at 900 mg/kg bw/day. Nursling pigs appear to tolerate less erucic acid than adult pigs before myocardial lipidosis is evident, suggesting that the immature myocardium and/or liver may be less able to oxidise long–chain fatty acids.

The severity of the observed myocardial lipidosis appears to decline with time. This is most likely due to the induction of the peroxisomal oxidation system in the liver, with subsequent downstream effects on the heart. It is not clear whether this adaptation to the oxidation of long–chain fatty acids by the liver, and possibly also the heart, has any long term adverse consequences.

In pigs and monkeys, there appears to be no other adverse findings that can be associated with erucic acid consumption, other than myocardial lipidosis. In rats, however, the animals typically also develop myocardial necrosis followed by fibrosis, at erucic acid doses of 6600 mg/kg bw/day. It is not apparent from these studies if this necrosis has any long-term effects, although it has been reported that the lifespan of rats exhibiting such lesions is not affected.

The male rat appears to be predisposed to the development of this type of heart lesion, particularly in response to the feeding of oils, with or without erucic acid.

No chronic, genotoxicity or carcinogenicity data are available. A single generation reproductive study was performed in rats and guinea pigs where doses of erucic acid up to 7500 mg/kg bw/day were not associated with any adverse reproductive or developmental effects.

In establishing a NOEL for the effects of erucic acid, short-term studies are considered the most appropriate as myocardial lipidosis appears rapidly after only short exposures, and is at its most severe early in the exposure period. The available sub–chronic studies are inadequate for deriving a no-effect level because of the absence of myocardial lipidosis in many of the studies as well as inappropriate dosing regimes. A NOEL of 750 mg/kg bw/day, based on the occurrence of increased myocardial lipidosis at 900 mg/kg bw/day in nursling

pigs, is considered appropriate.

A number of human epidemiological studies are available which have attempted to establish if there is any association between dietary erucic acid and the occurrence of heart disease, myocardial lipidosis or erucic acid accumulation in the heart. The studies indicate that erucic acid may occur in human heart muscle in geographic areas where vegetable oils containing erucic acid are consumed. However, the available evidence does not indicate an association between myocardial lesions, of the type observed in rats, or significant myocardial lipidosis, and the consumption of rapeseed oil. None of these studies enable a tolerable level for human exposure to be established.

In the absence of adequate human data, the NOEL of 750 mg/kg bw/day, established for pigs, can be extrapolated to humans in order to establish a tolerable level of human exposure. If an uncertainty factor of 100 (10 for extrapolation to humans, 10 for variation within humans) were applied to this NOEL the tolerable level for human exposure would be 7.5 mg erucic acid/kg bw/day, or about 500 mg erucic acid/day for the average adult. This is regarded as the provisional tolerable daily intake (PTDI) for erucic acid.

 

The toxicity of erucic acid is virtually always considered in the context of the toxicity of rapeseed and mustard seed oils, which can contain high levels of erucic acid. Most humans would be exposed to erucic acid by the inclusion of these oils in the diet. This, however, can complicate the interpretation of the study results, making it difficult to ascertain whether the observed effects are directly attributable to erucic acid, or to some other component (or combination of components) in the oil.

Toxic effects of rapeseed oil were reported. Rats were fed rapeseed oils at up to 70% of the calorie content of their diet. The rats were reported to have developed myocarditis. Weanling rats fed high levels of rapeseed oil have also been reported to accumulate fat in the heart muscle after only one day of feeding. The level of fat in the heart muscle of these rats was sometimes found to exceed four times normal values with similar changes also observed in the skeletal muscles. The fat droplets are mainly triglycerides containing a large proportion of erucic acid. The fatty accumulation decreases over time and finally disappears even with continued feeding of rapeseed oil. The fat accumulation is reported to disappear even more quickly if erucic acid is removed from the diet. The physiological repercussions of the myocardial infiltration are not entirely clear but have been reported to reduce the contractile force in the heart through the impairment of mitochondrial function and subsequent reduction in ATP synthesis. In this respect, myocardial lipidosis can be regarded as an adverse effect, although the long–term implications are unclear given that the effect appears to be reversible, even without removal of erucic acid from the diet. This does not exclude the possibility that the adaptation of the liver and/or myocardium to the oxidation of long chain fatty acids will itself produce long term adverse consequences.

The disappearance of fat accumulation has been reported to be followed by mononuclear cell infiltration, focal myocardial necrosis and eventually myocardial fibrosis in the rat. A causal link between myocardial lipidosis and myocardial necrosis, however, has not been conclusively established; it appears that myocardial necrosis occurs spontaneously in male rats in the absence of any observed myocardial lipidosis. Rapeseed oil fed at high levels has also been reported to retard growth in the rat, and when fed throughout the lifespan at such levels, causes a high incidence of degenerative changes in the liver, nephrosis, and smaller size and weight of the litters of these animals. The lifespan of these animals, however, is reported to be unaffected, in spite of these degenerative changes.

It has been suggested that the rat is not an appropriate model for determining whether erucic acid may pose a risk to human health. A number of reasons have been put forward for this. Firstly, most of the rat studies involve feeding oils at a concentration of around 20 % or more by weight in the diet. A level of 20% approximates human lipid consumption. It has been suggested that rats are physiologically incapable of metabolising such concentrations of oil in the diet. Secondly, there is some evidence that fatty acid metabolism in the rat is dissimilar to that of pigs and primates, making the rat highly susceptible to myocardial lipidosis. Lastly, focal myocardial necrosis, followed by reparative fibrosis, is a spontaneous idiopathic lesion in the male rat. The background incidence is reported to be of the order of 17–33% but it has been suggested that this background incidence is under–reported. The incidence and severity of these heart lesions can be influenced by the feeding of various marine and vegetable oils but may not be specifically related to the erucic acid content of the oil.

 

Kinetics and metabolism

Absorption

As erucic acid is a fatty acid, much of this review will concentrate on the general physiological processes for the absorption, digestion and metabolism of lipids and fatty acids.

The digestion of triacylglycerols begins in the small intestine where they are hydrolysed by intestinal lipases to a mixture of free fatty acids and 2–monoacylglycerols. A small fraction of the triacylglycerols remains unhydrolysed. The fatty acids and uncleaved acylglycerols are emulsified by the bile and absorbed by the intestinal cells, where they are largely reassembled into triacylglycerols that enter into the small lymph vessels in the intestinal villi. These highly emulsified triacylglycerols pass from the thoracic duct into the blood via the subclavian vein.

The absorption of fatty acids depends not only on the chain length and degree of saturation but also on the digestibility of the triglyceride molecule into which the fatty acid is incorporated. Digestibility is primarily influenced by the position of the fatty acid in the triglyceride molecule. The absorption of fatty acids is reported to be maximal when they are in position 2 of the triglyceride molecule. Erucic acid, present in high erucic acid rapeseed (HEAR) oil, is nearly always situated in positions 1 and 3 of the triglyceride molecule. Position 2 is mainly occupied by oleic, linoleic or a–linoleic acids.

In humans, the digestibility of erucic acid containing oils is 99%. In the adult female rat, however, the digestibility of HEAR oil is only 77%. The reason for this difference is not apparent but may reflect interspeciesdifferences in the activities of various lipases. In contrast to the rat, HEAR oil is alsoreported to be quite readily digestible in pigs with apparent digestibility values similar tothose observed in humans.

In other animals, such as rabbits and guinea pigs, HEAR oil is reported to have low digestibility, similar to that reported for rats.

 

Distribution

Free fatty acids become bound to serum albumin and are carried via the blood to other tissues such as the heart and skeletal muscles, which absorb and oxidise free fatty acids as their major fuel source. The rate of uptake of free fatty acids by tissues correlates with the concentration of albumin–bound free fatty acids in the circulation. A large proportion of the initial fatty acid metabolism occurs in the liver, which is particularly efficient at fatty acid uptake from the circulation.

In experiments with rats fed on erucic acid containing oils, the greatest accumulation of erucic acid was noted in the myocardium; the amount of erucic acid being proportional to the amount of erucic acid in the diet. In rats fed 30% of their calorie intake from HEAR oil (containing 47% erucic acid), about 34% of the erucic acid was found in the myocardial lipids after 7 days on the diet. This had declined to about 20% after 28 days on the diet. Significant accumulation of erucic acid was also noted in the reserve fatty tissue, the adrenal lipids and in the blood (each containing between 5–15% of the total erucic acid).

After long–term feeding of HEAR oil to rats, the amount of erucic acid in the myocardial lipids decreased gradually and at 12 months was reduced to 4.2%. Much less erucic acid (about 2%) was found in the hepatic lipids, suggesting that erucic acid may be more readily metabolised in liver cells.

Metabolism

Almost all cells are capable of metabolising fatty acids. Fatty acids are delivered into cells from two sources. One source is the free fatty acids that arrive via the blood, bound to serum albumin. The other source is from the breakdown of cell triaclglycerols by the action of lipases.

The degradation and oxidation of fatty acids occurs primarily in the mitochondria. The first step in their metabolism is their transport into the mitochondria. This is a carrier–dependent process using carnitine. Fatty acid molecules are degraded in the mitochondria by progressive release of two–carbon segments in the form of acetyl coenzyme A (acetyl–CoA).

This process is known as β–oxidation. The peroxisomes are also capable of performing β- oxidation.

Erucic acid is poorly oxidised by the mitochondrial β–oxidation system. This is because a number of enzymes involved in β-oxidation are inhibited by, or have low activity for, erucic acid. In humans, it has been shown that isolated heart mitochondria metabolise erucic acid more slowly than oleic acid,confirming that rates of erucic acid oxidation are decreased in humans, similar to experimental animals. Not only do the individual enzymes of the mitochondrial β–oxidation pathway have low affinity for erucic acid as a substrate, but the overall β-oxidation rates of other fatty acids are also reduced in the presence of erucic acid. This inhibition of fatty acid oxidation is not unique to erucic acid, but rather is a feature common to very long chain monoenoic fatty acids. The inhibitory effect on overall β-oxidation rates is thought to be responsible for the accumulation of lipids in the heart, and to a lesser extent in the liver, following the feeding of HEAR oils.

In liver, the presence of erucic acid appears to induce the peroxisomal β-oxidation system. This leads to greater overall rates of erucic acid oxidation relative to the shorter chain fatty acids, such as oleic and palmitic acid. Oleic and palmitic acid are more easily metabolised by the mitochondrial β-oxidation. Cardiac tissue, on the other hand, is less able than the liver to oxidise erucic acid, leading to greater relative accumulation of erucic acid in this tissue. The reasons for this are not readily apparent but could be due to a reduced capacity for peroxisomal oxidation in heart tissue.

As the erucic acid concentration in hepatic tissue decreases with induction of the peroxisomal β-oxidation system, the inhibitory effects on the oxidation of other fatty acids decreases.

Increased rates of hepatic oxidation by the peroxisomes is thought to lead to a reduced influx of long chain monoenoic fatty acids into the heart, leading to a gradual decline in the lipid accumulation that occurs in that tissue. In this respect, it appears as though the liver, and possibly also the myocardium, can adapt to the oxidation of long– chain fatty acids. It is not clear if this metabolic adaptation, itself, has any long-term consequences on the physiology of either the liver or the heart.

Significant interspecies differences are reported to exist in the relative and absolute rates of oxidation of different chain length fatty acids by heart muscle.

For example, mitochondria isolated from pig heart had threefold greater erucic acid oxidation rates than mitochondria isolated from rat. Similar findings have been found between monkey and rat hearts. These findings are probably due to interspecies differences in the enzymes of fatty acid β-oxidation, where differences in physiochemical and chain length specificities of particular enzymes isolated from various species, have been observed.

Excretion

It was demonstrated experimentally that erucic acid accounts for 75–77% of all fatty acids recovered from faeces, where it is excreted as free acid, monoglyceride,or diglycerides. Other experiments have shown that after oral administration of erucic acid in the form of rapeseed oil, or as the ethyl ester, it is eliminated with the faeces over a period of 5 days.

Applicant's summary and conclusion