Registration Dossier

Administrative data

Link to relevant study record(s)

Description of key information

Short description of key information on bioaccumulation potential result: 
Please view expert statement regarding toxicokinetic behaviour given under "Toxicokinetics, metabolism and distribution".

Key value for chemical safety assessment

Additional information

Toxicokinetic assessment of oleamide:

Possible ways for the uptake of fatty acid amides, like oleamide, stearamide and erucamide are the oral and the dermal route due to their use as slipping agents in the production of plastic articles and films, which might also be used as beverage containers (Cooper and Tice, 1995). Therefore, oleamide will most likely enter the body via the oral route as a result of foodstuff contamination, although usually it represents a proportion of less than 1% of the plastic (Hiley and Hoi, 2007).

 

Directive 90/128/EEC, relating to plastic materials and articles intended to come into contact with foods, was amended in 1994/1995 to cover plastic additives. At that time, migration data with food simulants for the fatty acid amides had been provided to the Scientific Committee for Foods (SCF), but only insufficient toxicological data was available. Therefore, the SCF requested that either further toxicological data was provided or studies were conducted demonstrating the full hydrolysis of fatty acid amides in simulated body fluids. Guidelines for the conduction of such hydrolysis studies had been provided by the SCF (Cooper, 1994; Cooper, 1995).

The SCF had granted the option to provide data on only one of a group of three primary fatty acid amides, consisting of oleamide, stearamide and erucamide, as being representative for all three. At that time behenamide (C22 saturated fatty acid amide) had already been qualified as being metabolised into ammonia and behenic acid by the SCF; therefore, as the longest saturated fatty acid amine among the four had already been tested, the study sponsor chose to investigate the hydrolysis of oleamide, the shortest unsaturated fatty acid amide among the four, as a representative for the mentioned group, as testing the C18 unsaturated fatty acid amide was considered to give more overall reassurance that these compounds all behave in a similar way.

 

The hydrolysis fluid compositions commonly used at that time were believed not to be entirely representative of the in vivo gastrointestinal fluids by the study sponsor and, in case of the simulated intestinal fluid, the pancreatin to be used was not considered adequately specified. Therefore, the hydrolysis measurements have been performed with the simulated gastrointestinal fluids as specified by the SCF and additionally with slightly modified preparations regarded to be more representative of the in vivo fluids. The modifications included the addition of bile salts, testing of various substrate:enzyme ratios and use of pancreatin purified from inherent fatty acids, which interfered with the stoichiometric determination of the metabolite formed by enzymatic hydrolysis.

 

For the determination of hydrolysis levels an oleamide starting concentration of ~5 mg/L (4.788 mg/L) in the hydrolysis mixture was chosen in order to achieve a concentration after hydrolysis of 95% (i.e. 0.25 mg/L) which could be determined with sufficient precision (± 0.02 mg/L). Furthermore, oleamide levels chosen for hydrolysis testing were based on worst case migration levels of fatty acid amides observed from those plastics containing the highest levels of fatty acid amides typically used in food packaging (Cooper and Tice, 1995). The highest migration level of oleamide of about 3 mg/L(kg) was found from polyethylene (LDPE) into the aggressive fat simulant olive oil, but no detectable levels (<0.05 mg/kg) were determined with aqueous food simulants (Cooper and Tice, 1995). For most foods containing substantial quantities of water, migration levels significantly less than 3 mg/L(kg) food were expected by Cooper and Tice. Therefore, the authors considered it very unlikely that human intake will ever exceed the value of 5 mg/L in real life, and this value can thus be considered as worst case.

 

The addition of bile salts to the intestinal fluid preparations with 8 x USP activity enhanced oleamide hydrolysis significantly from 66% to 93% after 4 hours at 37 °C (Cooper, 1994; Cooper, 1995). Use of pancreatin of 8 x USP activity led to significantly higher levels of oleamide hydrolysis than 1 x USP grade. It was demonstrated that about 95% of oleamide at a concentration of ~5 mg/L hydrolyses after 4 hours in simulated gastrointestinal fluids containing 4 x USP or 8 x USP pancreatin and bile salts. Actual levels of oleamide hydrolysis were even considered to be slightly underestimated in this experiment due to a small interfering peak with the same retention time as oleamide, for which no correction had been made, and possible small loss of the internal standard in processing of the samples. In one of the first experiments described in this study hydrolysis levels of oleamide were measured after different incubation times in intestinal fluid preparations with 4 x USP activity and bile salts; the results showed hydrolysis levels of 65, 82, 94 and 96% after 1, 2, 4 and 6 h, respectively, demonstrating the influence of time on hydrolysis (Cooper, 1994; Cooper, 1995).

According to Barlow hydrolysis can be considered as complete if 95%, or more, loss of the starting compound is found (Barlow, 1994).

Oleamide was therefore considered unlikely to pose a threat to the health of the consumer if ingested at the very low levels expected to migrate into most foodstuffs from food packaging.

Other unsaturated fatty acid amides, such as erucamide (CAS No. 112-84-5), are expected to hydrolyse to a similar degree as oleamide (Cooper, 1994; Cooper, 1995).

 

 

Organic chemistry textbooks describe the products formed on acid and alkaline hydrolysis of fatty acid amides to be ammonia, forming ammonium salts, and the corresponding fatty acid, i.e. oleic acid in case of oleamide. In principle, the hydrolysis mechanisms of fatty acid amides correspond to those of fatty acid esters (Vollhardt, 1st ed., 1990, Christen, 4th ed., 1977). However, these reactions only take place under very harsh reaction conditions; under physiological conditions they are catalysed by respective enzymes, e.g. those included in pancreatin which is a complex mixture of active ingredients and substances derived from the pancreas of animals (usually pigs). The approaches to determine the quantitative formation of oleic acid by hydrolysis of oleamide in simulated intestinal fluid by gas chromatography were initially impaired by the presence of inherent fatty acids in the pancreatin. Therefore, further experiments were conducted using, on the one hand, a higher starting concentration of oleamide (~50 mg/L) and, on the other hand, pancreatin which had been purified from fatty acids by room temperature extraction with pentane. An additional experiment demonstrated that the extraction procedure had only little effect on the ability of pancreatin to hydrolyse oleamide.

These hydrolysis experiments demonstrated stoichiometric formation of oleic acid and, by inference, ammonium salts. The loss of oleamide found in these studies was slightly lower at 89%, compared to 95% without purification of the pancreatin as described above. This lower result was considered to be the consequence of a small detrimental effect of pentane extraction on enzyme activity. The determined quantitative formation of oleic acid at 86% correlated well with the observed loss of oleamide in these experiments (Cooper, 1994; Cooper, 1995).

 

The described hydrolysis study demonstrates the complete hydrolysis of fatty acid amides into the corresponding fatty acids in the lumen of the gut. Thus, the same fate for fatty acids derived from ingested fatty acid amides and those derived from nutritional triglycerides can be concluded. In the aqueous milieu of the gut the triglycerides are mechanically emulsified and hydrolysed by lipases of the pancreatic juice to 2-mono-glycerides and free fatty acids. Spontaneous micelle formation of these hydrolysis products with the involvement of bile salts is an important prerequisite for their absorption. In this form they reach the microvili of the gut mucosa where they are taken up into the mucosa cells by a passive process (Silbernagl & Despopoulos, 4th ed., 1991). Furthermore, the bile salts aid in the emulsification of triglycerides and activate digestive enzymes (Pschyrembel, 263rd ed., 2012). The relevance of bile salts for both the hydrolysis of fatty acid amides and triglycerides in the small intestines justifies the assumption of a similar function and a comparable mechanism and, considering the chemical structures of fatty acid amides and triglycerides, the assumption of the same hydrolysis products, i.e. fatty acids.

On the one hand the dependence of fatty acid amide hydrolysis on the presence of bile salts, which aid in the formation of micelles, demonstrates that the small intestines are the main sites of fatty acid amide hydrolysis. In thein vitrohydrolysis study by Cooper, complete degradation of oleamide was already observed within 4 hours with simulated intestinal fluid containing bile salts, which was considered to be comparable to the conditions in the human gutin vivo. Furthermore, the stoichiometric formation of the corresponding fatty acid, oleic acid, was demonstrated (Cooper, 1994; Cooper, 1995). On the other hand, the chemical characteristics of the fatty acid amides suggest that their absorption in the gut might be possible.

 

However, considering the normal retention/passage times in the human small intestines (7-9 hours) of food/substances taken up by oral ingestion (Silbernagl & Despopoulos, 4thed., 1991) there is ample time to expect an even higher degree of hydrolysis under physiological conditionsin vivo.

Fatty acid amides and fatty acids share many structural similarities; both feature a carbonyl group, in which the carbon atom occurs in sp2-hybridisation, both have a substituent at the carbonyl C-atom which exerts a –I-effect due to the electronegativity of the oxygen and the nitrogen atom in the hydroxy and the amine group, respectively, while both substituents have a +M-effect, as both are able to provide a lone pair for resonance structures (Mortimer, 6th ed., 1996; Vollhardt, 1st ed., 1990; Christen, 4th ed., 1977). In conclusion, both feature a hydrophobic alkyl chain and a hydrophilic amide or carboxy group, which are stabilised by resonance (Vollhardt, 1st ed., 1990).

Due to the described chemical similarities between fatty acid amides and fatty acids, a minor amount of fatty acid amides, which might escape hydrolysis, will also be included in the spontaneously forming micelles consisting otherwise of mono-glycerides, free fatty acids and bile salts. They will be transported and taken up into the mucosa cells of the small intestines in analogous fashion to their fatty acid derivatives oleic acid, stearic acid and erucic acid because they are also able to penetrate biomembranes due to their poor water and high fat solubility.

 

The principle of hydrolysis of fatty acid amides to fatty acid and ammonia has already been acknowledged by the Commission of The European Communities, Directorate General for Industrial Affairs and Internal Market, Brussels, Belgium, in 1994 for behenamide (C22-saturated fatty acid amide), which had already been a List 3 substance of Annex I (Cooper, 1994). This is a list of “Substances for which an ADI or a TDI could not be established, but where the present use could be accepted. Some of these substances are self-limiting because of their organoleptic properties or are volatile and therefore unlikely to be present in the finished product. For other substances with very low migration, a TDI has not been set, but the maximum level to be used in any packaging material or a specific limit of migration is stated. This is because the available toxicological data would give a TDI which allows that a specific limit of migration or composition limit could be fixed at levels very much higher than the maximum likely arising from present uses of the monomer.” Annex I lists “Substances for which the Committee was able to express an opinion” (described in the “Reports of the Scientific Committee for Food, 30th series, Third addendum to the first report of the Scientific Committee for Food on certain monomers and other starting substances to be used in the manufacturing of plastic materials intended to come into contact with foodstuffs”; SCF 30th series, 1991).

 

At that time, erucic acid had already been included in List 3, as well, and oleic and stearic acid in List 1 (“Substances, for which an ADI, a temporary ADI (t-ADI), a MTDI, a PMTDI, a PTWI or the classification “acceptable” has been established by this Committee or by JECFA”) of Annex I (SCF 30th series, 1991; included since “Reports of the SCF, 25th series”, 1990).

 

Subsequently, erucamide, oleamide, and stearamide had also been included in SCF-List 3 prior to publication of the 2007 "Inventory List for Coatings intended to come into Contact with Food - Compiled Lists approved by the Council of Europe" (CEPE, 2007). That means their uses at that time were accepted, although an ADI or a TDI could not be established, and no maximum migration levels or other restrictions have since been introduced, either, as demonstrated by the list included in Commission Regulation (EU) No 10/2011, which came into force in 2011 (CEPE, 2007; Commission Regulation EU No 10/2011, 2011).

 

Oleamide is known to have endogenous functions as signalling molecule (Cravatt, 1995) and belongs to the emerging class of lipid signalling molecules called primary fatty acid amides (Driscoll, 2007). Nevertheless, although unlikely due to the presence of effective endogenous degradation mechanisms as described, the theoretically possible influence of the substance on those signalling pathways is discussed below.

 

The fatty acid amides are a large and diverse class of lipid transmitters that include the endogenous cannabinoid anandamide (a fatty acid amide identified as the endogenous ligand for the cannabinoid receptor), and the sleep-inducing substance oleamide itself (Lerner, 1994; Cravatt, 1995). Both molecules have messenger functions in the human CNS (Fedorova, 2001; Bisogno, 2002). Naturally occurring oleamide was reported to be one of five primary fatty acid amides present in human plasma; endogenous levels of up to 31.7 mg/L have been reported in 10 of 16 tested individuals. However, it should be noted that this value represents a case observation in a single person, representative for those individuals demonstrating detectable endogenous fatty acid amide levels in their plasma, but without the manifestation of effects; in the other 6 individuals investigated in the same study plasma levels of fatty acid amides were much lower or not detectable (Arafat, 1989). In conclusion, this study demonstrates that there is a possible range of oleamide plasma concentrations which do not induce adverse effects; a plasma level of 31.7 mg/L should be tolerated without effects.

 

Oleamide could be isolated from the cerebrospinal fluid of sleep-deprived cats (Lerner, 1994; Cravatt, 1995). It was demonstrated to be a predominant element of the human meibomian gland secretions, where it is speculated to have functions in ocular surface signalling or the maintenance of the complex tear film (Nichols, 2007).

Oleamide participates in the biochemical mechanisms underlying sleep regulation (Lerner, 1994; Cravatt, 1995), locomotor activity, antinociception and thermoregulation (Fedorova, 2001). Oleamide treatment was shown to reduce locomotor activity and body temperature, and to have an analgesic effect. However, these effects were demonstrated to be only of short duration and to abate within 60 minutes after intraperitoneal application.

After 8 days of repeated intraperitoneal treatment twice daily with a dose of 30 mg/kg bw the animals developed tolerance against oleamide, as the subsequent application of an oleamide challenge dose failed to induce the previously described effects. Oleamide was further demonstrated to be a poor inducer of physical dependence; animals spontaneously withdrawn from oleamide after a 10-day period of application failed to show typical signs of withdrawal behaviour (Fedorova, 2001).

The observed effects have to be considered with reservation, as oleamide was applied intraperitoneally in all of these experiments; due to this application technique the main site of oleamide hydrolysis, the small intestines, had been circumvented by this route, and thus, an unphysiologically high dose of oleamide was introduced into the organism of the experimental animals. Therefore, correlation of those results with typical occupational or general exposure conditions is not justified; intraperitoneal application is no relevant route of exposure, neither for workers, nor for the general population. In conclusion, the health hazards caused by exposure to oleamide are considered as very low.

 

Oleamide was even demonstrated to be endogenously synthesised, the source of the oleamide synthesising activity isolated from rat kidney tissue could be identified as cytochrome c (Driscoll, 2007). Another hypothesis is the synthesis from oleoylglycine via the actions of the peptide amidating enzyme, peptidylglycine alpha amidating monooxygenase (PAM) (Merkler, 2004). This hypothesis is supported by the existence of an enzyme generating oleoylglycine (Hiley and Hoi, 2007). A possible candidate for this task is BACAT (bile acid-CoA:amino acid N-acyltransferase) which can conjugate oleoyl-CoA to glycine although it prefers saturated acyl-CoAs (O’Byrne, 2003).

 

 

The magnitude and duration of fatty acid amide signalling are controlledin vivoby enzymatic hydrolysis (Wei, 2006). Both oleamide and anandamide are substrates of the fatty acid amide hydrolase (FAAH), originally named oleamide hydrolase (Patterson, 1996; Day, 2001; Bisogno, 2002; Cravatt, 1996), which, apart from anandamide and oleamide, is able to hydrolyse a wide range of other fatty acid amides (Boger, 2000; Cravatt, 1996). In case of oleamide the hydrolysis into its fatty acid derivative oleic acid is catalysed, which, at the same time, leads to inactivation of the endogenous signalling molecule. FAAH is an integral membrane protein (Day, 2001) widely distributed in mammalian tissues and belongs to a large family of enzymes that share a highly conserved ~130 amino acid motif designated the “amidase signature” (AS) sequence. Blockade of FAAH activity leads to highly elevated endogenous levels of fatty acid amides in the nervous system and peripheral tissues (Wei, 2006).

 

Lately, a second gene for an enzyme with fatty acid amide hydrolase activity has been discovered which has been designated FAAH-2, referring to the original AS enzyme as FAAH-1 (Wei, 2006). Interestingly, it is expressed in primates (which also include humans) and a variety of distantly related vertebrates, but not in murids, i.e. mice and rats. The exclusive expression of the FAAH-2 gene in primates suggests even more possibilities for the metabolisation of fatty acid amides in humans compared to rats, for example.

These enzymes exhibit overlapping but distinct tissue distribution, substrate selectivity, and inhibitor sensitivity profiles. Whereas only FAAH-1 is expressed in brain, small intestine and testis, only FAAH-2 is expressed in the heart. Both enzymes are abundantly expressed in kidney, liver, lung and prostate, both are able to hydrolyse a wide variety of fatty acid amides (Wei, 2006). The expression of one or both FAAH isoforms in tissues known to be closely associated with absorption, metabolism and excretion of chemicals in the body, like small intestine, liver and kidney, is a reasonable argument for assuming their contribution also in the metabolism of ingested fatty acid amides to their respective metabolites.

FAAH-1 is reported to have a KM-value of 5 ± 2 µM for oleamide (Patterson, 1996); this value is quite low compared to common ranges of KM-values reported for other enzymes and their respective substrates (10 mM – 1 µM) (Karlson, 14th ed., 1994), demonstrating a high affinity of FAAH-1 for oleamide. A hydrolysis rate for oleamide of 0.526 nmol/min/ng FAAH has been reported in the literature (Boger, 2000). Relative rates for hydrolysis compared to that of oleamide (oleamide representing 1.0) of 3.11 for arachidonamide to 0.52 for 9E-octadeceneamide (9-trans-oleamide) have been reported for 32 fatty acid amides, among them erucamide, palmitamide and stearamide with relative hydrolysis rates of 0.83, 0.72 and 0.69, respectively (Boger, 2000).

 

These relative hydrolysis rates demonstrate that FAAH-1 will hydrolyse more of a given dose of oleamide per minute than of erucamide, for example; hydrolysis of erucamide by FAAH-1 will be slower than that of oleamide, which would lead to a higher systemic availability of erucamide compared to oleamide under comparable experimental conditions. Therefore, conduction of tests with erucamide and read-across from these studies will represent a worst case approach, as erucamide, like oleamide, is known to have endogenous functions, as well; for instance, an angiogenic effect was demonstrated (Wakamatsu, 1990). Besides, adverse effects of its hydrolysis product erucic acid are widely reported. In detail, an association between dietary erucic acid and myocardial lipidosis has been shown in rats and nursling pigs, and an association between dietary erucic acids and heart lesions has been demonstrated in rats, although at very high daily exposure levels. However, there is no evidence that dietary erucic acid can be correlated to either of these effects in humans; nevertheless, concerning what is known about erucic acid metabolism a possible susceptibility of humans to myocardial lipidosis following high levels of erucic acid cannot be completely excluded (Food Standards Australia New Zealand, 2003). This has led to the development of rape plants producing only low amounts of erucic acid by selective breeding; up to the beginning of the 1970s rapeseed oil had a relatively high content of erucic acid (40-50% in high erucic acid rapeseed oil), but by genetic selection of plants for breeding this was almost eliminated (< 2%, the corresponding oil referred to as low erucic acid rapeseed oil); at the same time the oleic acid content increased. This low erucic acid oil is utilised for food uses in Europe, the US, Australia and New Zealand (Food Standards Australia New Zealand, 2003; Beare-Rogers, 2001). In 1976 the maximum level of erucic acid in edible oils, fats and mixtures thereof was limited to 5% in EC Council Directive 76/621/EEC, and this level was ordered to enter into force from 1 July 1979 at the latest. This maximum level had been established in the Directive in order to protect consumers from the potential cardiac effects associated with high dietary exposure to erucic acid.

 

The two human FAAH enzymes share 20% of sequence identity and hydrolyse fatty acid amide substrates (e.g. oleamide) at equivalent rates (Wei, 2006). Thus, the total rate of fatty acid amide hydrolysis will be increased in humans compared to rats, which only express the FAAH-1 isoform. The presence of the second FAAH isoform will result in a relatively lower systemic availability of fatty acid amides in humans compared to rats. Therefore, testing in rats has to be considered as worst case scenario, as well; the corresponding systemic availability of fatty acid amides in humans will be much lower than in the experimental animals, and possible effects, if occurring at all, would be overestimated from results of animal experiments.

 

 

As described above, degradation of 95% of oleamide under conditions comparable to those in the gastrointestinal tract was demonstrated. Therefore, although degradation is considered as complete, in a worst case anticipation a maximum of only 5% of a reasonable and realistic dose of orally administered fatty acid amides, which might have escaped hydrolysis in the gut, could be available for absorption. In case of complete absorption of those remaining fatty acid amides via the epithelium of the small intestines, only 5% of the originally administered dose could enter the organism, at all.

 

All lipids, including fatty acids and similar substances, which also include fatty acid amides, are absorbed in the small intestines as described previously (Silbernagl & Despopoulos, 4th ed., 1991). Due to their high level of physicochemical similarity it is conceivable that the fatty acid amides are taken up into the mucosa cells in an analogous fashion to fatty acids. However, the small intestine is one of the organs with abundant expression of FAAH-1 (Wei, 2006). Therefore, hydrolysis of a great part of absorbed fatty acid amides into their corresponding fatty acids by FAAH-1 in the small intestine is expected, again reducing the amount of fatty acid amides potentially available for systemic distribution. Subsequently, only those fatty acid amides escaping from this second hydrolysis step have to be considered for further systemic availability.

Short-chain fatty acids are able to enter the blood of the portal vein as free fatty acids due to their good solubility in water; long-chain fatty acids, like oleic acid, and mono-glycerides cannot be transported via that way due to their poor water solubility. They are reassembled to triglycerides in the smooth endoplasmatic reticulum of the mucosa cells of the small intestine. As these triglycerides are insoluble in water they are embedded into the core of lipoproteins, the chylomicrons, in the Golgi apparatus and released into the lymphatic system of the gut. Other non-polar molecules like cholesterol esters and lipid-soluble vitamins are also transported included in chylomicrons (Silbernagl & Despopoulos, 4th ed, 1991). Therefore, the transport of hydrophobic fatty acid amides, like oleamide or erucamide, in the hydrophobic core of the chylomicrons is expected. However, this is a hypothesis which seems to have never been investigated, as fatty acid amides are commonly regarded as being completely hydrolysed in the gut. It should be noted that it is discussed here only in order to consider possible routes of systemic exposure to ingested fatty acid amides; in reality, exposure via these routes is unlikely.

 

Chylomicrons are transported in the lymph via the thoracic duct which drains into the bloodstream at the left venous angle (Pschyrembel, 263rd ed., 2012), which is demonstrated by clouding of the plasma after ingestion of food containing high amounts of fat (Silbernagl & Despopoulos, 4th ed., 1991). In the blood the triglycerides in the chylomicrons are hydrolysed by endothelial lipoprotein lipases (Silbernagl & Despopoulos, 4th ed., 1991; Stryer, 2nd ed., 1994; Pschyrembel, 263rd ed., 2012). Finally, the cholesterol-rich rest of the chylomicrons, the chylomicron remnants, reach the liver, are taken up in a receptor-mediated process and are completely degraded (Stryer, 2nd ed., 1994; Doenecke, 15th ed., 2005; Pschyrembel, 263rd ed., 2012).

The liver is one of the organs with the highest FAAH expression, both forms of fatty acid amide hydrolase (FAAH-1 and FAAH-2) are expressed here (Wei, 2006). Therefore, the final and complete hydrolysis of those minor amounts of fatty acid amides, which might have escaped hydrolysis in the lumen and the cells of the small intestine so far, by the liver FAAH enzymes is anticipated.

 

 

However, even if no further hydrolysis mechanisms were present in the human body in addition to that observed in the lumen of the small intestines, adverse effects should not be expected based on exposure estimations.

The following hypothetical calculation can be performed. As described above a human plasma level of 31.7 mg/L was tolerated without effects (Arafat, 1989). The human body contains about 60% of water, and the blood plasma represents a proportion of about 4.5% of the complete body weight (corresponding to 0.045 L/kg bw) (Silbernagl & Despopoulos, 4thed., 1991); this equals 3.15 L (kg) for a person with a body weight of 70 kg.

Based on this, a total amount of 99.86 mg oleamide would have been available in the plasma of that person if a body weight of 70 kg is anticipated (the actual weight was not reported in the study). If a worst case scenario is assumed with 95% hydrolysis in the lumen of the small intestines, complete absorption of the hypothetically remaining 5% in the small intestine, no hydrolysis after absorption, and distribution of 50% (arbitrarily chosen value just as an example for calculation) of absorbed fatty acid amides into other compartments than plasma, the ingested amount would have had to be 3994 mg of oleamide (equivalent to 57.06 mg/kg bw). However, considering a worst case migration level of 3 mg/L from food containers, which was only observed with aggressive fat simulants from that material reported to contain the highest amount oleamide of all types of plastic (Cooper and Tice, 1995), the respective person would have had to ingest about 1331 L of contaminated food in order to take up an amount of 3994 mg of oleamide, resulting in a plasma oleamide level of 31.7 mg/L.

This is regarded to be an unrealistic scenario.

 

The unintentional ingestion of approximately 4.0 g of oleamide by workers has to be considered as very unlikely, as well. Therefore, the risk imposed by oral uptake of oleamide is regarded as very low.

 

Dermal exposure is unlikely due to risk mitigation measures (protective gloves must be worn) being in place.

 

Due to the presence of adequate and effective hydrolysis enzymes for fatty acid amides in the mammalian and especially the human organism, and the characteristics of the metabolites, i.e. fatty acids, rapid and effective absorption, distribution and metabolism of the hydrolysis products are very likely. In case of oleamide the corresponding metabolite is oleic acid. Oleic acid is the most common mono-unsaturated fatty acid and is present in nearly all naturally occurring animal and vegetable oils and fats, including almond (69.6%), avocado (64.0%), cashew (68-75%), corn (20.0-42.2%), hazelnut (77.8%), olive (56-83%), pecan (64%), peanut (36.4-67.1%), rape seed (8.0-66.9%) sesame (35.9-42.3%), sunflower (19.6-45.3%) and tomato seed oil (16.0-25.0%), cocoa butter (32.6%), lard (35.0-62.0%), chicken fat (~35%), turkey fat (20.7-30.4%), beef fat (45.8%) and milk (26.6%), to name only a few, which are common elements of our daily nutrition. It is even included in human milk to a minor extent (3.6%) (Beare-Rogers, 2001).

 

Fats are hydrolysed and absorbed in the gastrointestinal tract and are transported in form of lipoproteins and chylomicrons via blood and lymph, respectively. Most of the absorbed triglycerides are used by muscle and fat tissue, but also the liver is one of the target organs, where fatty acids can be either used to generate energy, or where they can be resynthesised to triglycerides if there is an excess supply of fatty acids. A large proportion of the initial fatty acid metabolism occurs in the liver, which is particular efficient at fatty acid uptake from the circulation (Food Standards Australia New Zealand, 2003). Fatty acids are separated from triglycerides by lipases. They are absorbed and transported in the blood and in the lymph as described above. Fatty acids can penetrate the plasma membranes and enter the cells of their target tissues by a passive process due to their poor water solubility and their high fat solubility (Silbernagl & Despopoulos, 4th ed., 1991). In the cytoplasm they are activated by coupling to coenzyme A and are transported into the mitochondria by a transporter system after coupling to L-carnitine. Inside the mitochondria they are again coupled to coenzyme A before they enter the cycle of β-oxidation.

 

As described in common biochemistry textbooks, which still reflect the recent state of science, in this process an acyl-CoA is degraded by progressive release of two-carbon segments in the form of acetyl coenzyme A (acetyl-CoA) in a repeating sequence of four reactions. The first step is the oxidation of acyl-CoA by acyl-CoA dehydrogenase. The enzyme catalyses the formation of a trans-double bond (trans-Δ2-enoyl-CoA) between C-2 and C-3; the releasing hydrogen atoms are transferred to FAD. The second step is the stereospecific hydration of the trans-Δ2-double bond between C-2 and C-3 by enoyl-CoA hydratase, followed by the third step, the oxidation of L-3-hydroxyacyl-CoA by L-3-hydroxyacyl-CoA dehydrogenase, converting the hydroxy group into a keto group. The released hydrogen atoms are transferred to NAD+. Both FADH2and NADH H+are dehydrogenated in the process of oxidative phosphorylation, leading to the production of ATP. The final step of each degradation round is the cleavage of 3-ketoacyl-CoA by the thiol group of another molecule of CoA; this reaction is catalysed by β-ketothiolase. The shortened acyl-CoA enters another cycle, and the process continues until the entire chain is cleaved into acetyl-CoA units (Stryer, 2nd ed., 1994).

 

A special case is the degradation of unsaturated fatty acids like oleic acid and erucic acid with a cis-double bond in Δ9- and Δ13-position, respectively. After 3 and 5 rounds, respectively, of regular degradation cis-Δ3-enoyl-CoA is formed, which is no substrate for enoyl-CoA hydratase. The double bond between C-3 and C-4 prevents the introduction of another double bond between C-2 and C-3. This issue is circumvented by the contribution of another enzyme, an isomerase (cis-Δ3-enoyl-CoA-isomerase) which converts the cis-Δ3-double bond into a trans-Δ2-double bond, the regular substrate of enoyl-CoA hydratase. The following steps are identical to the oxidation of a saturated fatty acid (Stryer, 2nd ed., 1994).

 

Therefore, the degradation of both oleamide and erucamide, comprising hydrolysis to the corresponding fatty acids and their subsequent degradation into acetyl-CoA, is more or less identical for both molecules; both have the same functional group (amide group), both feature a cis-double bond at an odd-numbered carbon atom (cis-Δ9and cis-Δ13, respectively). The only difference is the requirement of two additional degradation rounds of erucic acid in the process of β-oxidation for complete breakdown into acetyl-CoA due to four additional CH2-units in the carbon chain of erucamide. Considering the breakdown by the same pathway and the generation of the same breakdown products the use of erucamide as source substance for read-across is scientifically justified.

 

Acetyl-CoA enters the citric acid cycle and is used for the chemical reduction of further molecules NAD+and FAD, which in turn are used for the production of ATP in the process of oxidative phosphorylation. In case of this C18-compound 146 molecules of net ATP are produced, which serve as energy source for the cells of the organism. On the contrary, acetyl-CoA can also be used for the endogenous synthesis of fats which can be stored in fat cells and serve as energy reservoirs that can be mobilised in times of low carbohydrate availability (Stryer, 2nd ed., 1994). Oleic acid was shown to be the most abundant fatty acid in human adipose tissue; it is present in quantities of 47.3 to 52.0%, depending on the anatomical site, age and race of the individual (Kokatnur, 1979).

 

The oxidation of odd-numbered fatty acids will not be addressed; the fatty acid amides discussed here are derived from fatty acids from vegetable origins which exclusively comprise even-numbered acyl chains.

 

 

Conclusion:

Considering the important endogenous functions of the fatty acid amides, the presence of effective and fast metabolic pathways for their inactivation, the fact that their metabolites are educts for the most effective processes of energy production and storage, and the abundant endogenous availability of their metabolites even in the human body, adverse effects after application of fatty acid amides via physiologically relevant routes are not to be expected.

 

However, the hydrolysis rate of erucamide by endogenous FAAH is lower than that of oleamide, and oxidation of erucic acid by the mitochondrialb-oxidation system is worse, in contrast to oleic acid, as a number of enzymes involved in this process 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, similar to experimental animals. On the one hand the individual enzymes of the mitochondrial β-oxidation pathway have low affinity for erucic acid as a substrate; on the other hand the overall β-oxidation rates of other fatty amides are also reduced in the presence of erucic acid (Food Standards Australia New Zealand, 2003).

 

These findings support the opinion of the registrant that adverse effects, if to occur at all, are rather to be expected from erucamide than from oleamide, based on the experiences with their respective metabolites.

 

 Therefore, read-across from erucamide as source substance is regarded as worst case approach and will rather lead to an overestimation of the hazards possibly caused by oleamide. The conduction of analogous vertebrate tests with oleamide in addition to those tests already proposed for erucamide (sub-chronic toxicity 90 days, OECD 408 and prenatal developmental toxicity, OECD 414, in particular) will neither provide new aspects to the hazard assessment of oleamide, nor will it increase the safety of its applications. Therefore, a one-to-one read-across from the source substance erucamide was used to close the identified data gaps, especially for repeated dose toxicity and prenatal developmental toxicity, of oleamide with the respective results of the proposed tests. For the sake of animal welfare the conduction of such tests with oleamide, in addition to those proposed for erucamide, is considered ethically not acceptable.

Discussion on bioaccumulation potential result:

Considering the important endogenous functions of the substance itself, the presence of effective metabolic pathways, the fact that its metabolites are educts for the most effective processes of energy production and storage, and their abundant endogenous availability even in the human body, adverse effects after application are not to be expected.