Health & Gastronomy 29/06/2015

Tetrahydrocannabinol in milk and other food of animal origin

Tetrahydrocannabinol is derived from the hemp plant Cannabis sativa. The exposure to via consumption of milk and dairy products, resulting from the use of hemp seed-derived feed materials at the reported concentrations, is unlikely to pose a health concern

Tetrahydrocannabinol, more precisely delta-9-tetrahydrocannabinol (Δ9-THC) is the most relevant constituent derived from the hemp plant Cannabis sativa. Four stereoisomers of Δ9-THC are possible, with (-)-trans-Δ9-THC being the only naturally occurring isomer. delta-9-tetrahydrocannabinolic acid, which is found in the growing and harvested plant, is the biosynthetic precursor of Δ9-THC. Besides 2-COOH-Δ9-THC, which in this opinion is referred to as Δ9-THCA-A, another positional isomer, 4-COOH-Δ9-THC, denoted as Δ9-THCA-B, may also occur in the hemp plant. In fresh plant material of C. sativa, up to 90 % of the ‘total’ Δ9-THC is present as the non-psychoactive precursor Δ9-THCA-A. The rate and extent of transformation of the precursor acids into Δ9-THC in the plant material is dependent on physical effects, in particular temperature. In addition to Δ9-THC, C. sativa preparations may contain at least 60 other cannabinoids, several of which are biologically active. This risk assessment does not evaluate the exposure and associated risks of cannabis used as a medicinal drug or for recreational purposes, but as requested in the terms of reference (ToR) addresses the potential risks caused through human dietary exposure from food of animal origin. Cannabinoids other than Δ9-THC are also beyond the ToR and are not considered in the opinion, unless there is potential for interaction with Δ9-THC. Therefore, this assessment concentrates on the primary psychoactive cannabinoid Δ9-THC, in particular (-)-trans-Δ9-THC.

In the European Union (EU), the cultivation of C. sativa varieties is granted provided they are listed in the EU’s ‘Common Catalogue of Varieties of Agricultural Plant Species’ and the THC content does not exceed 0.2 %. The hemp plant is a multi-purpose crop grown both for its fibres – principally for industrial purposes - and seed for the production of feeding stuffs. For example, hemp seed cake (the residue of the seed after oil extraction) may be used to replace – or be fed in conjunction with – oilseed meals, such as rape seed meal, in diets for cows, pigs and horses. A less common practice is the use of leaves, flowers and stalks (the by-product of hemp production for fibres and seeds) as feed for ruminants and pigs.

The determination of Δ9-THC in hemp plants used/grown for feed is prescribed by Commission Regulation (EC) No 1122/2009. The official method based on capillary gas chromatography with flame ionisation detection (GC-FID) is not able to differentiate between the psychoactive Δ9-THC and its non-psychoactive precursor acids. Capillary gas chromatography coupled with mass spectrometry (GC-MS) following liquid-liquid extraction or solid phase microextraction is the method of choice for the determination of Δ9-THC and other cannabinoids in hemp-containing food products. A separate determination of Δ9-THC and its precursor acids is possible by derivatization, such as silylation of the extract prior to gas chromatography (GC) analysis or by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

As high temperatures are not generated during de-hulling and oil extraction, these processes are not expected to result in increased levels of Δ9-THC in the oil or meal resulting from decarboxylation of precursor acids. Although most milk is subjected to pasteurisation prior to marketing and consumption, the temperatures used are unlikely to result in decarboxylation of any non-psychoactive precursors that may be present as a result of transfer from feed.

Following a call for data, by the end of June 2014 a total of 603 analytical results on cannabinoids from eight different countries were available in the EFSA database, 281 on food and 322 on feed. Only very few samples of animal origin were reported, i.e. two samples of cheese, and two samples reported as meat products. The CONTAM Panel decided not to use the 603 available analytical results on cannabinoids to estimate dietary exposure to Δ9-THC. This decision was based on the fact that in many cases the data represented the sum of Δ9-THC and its precursor acids since they were analysed by GC-MS or by GC-FID each without prior derivatization, and in other cases the presence of Δ9-THC was not confirmed.

Individual data on levels of THC in hemp plants used in the EFSA FEEDAP Scientific Opinion (2011) were also available, together with some aggregated data on THC in hemp plants collected between 2009-2012 and provided by the EC. As the data did not differentiate between Δ9-THC and its precursor acids, they were not considered suitable for the exposure assessment.

At a later stage, additional analytical data on levels of different cannabinoids in food and feed, were provided by the European Industrial Hemp Association (EIHA) and the Swiss Federal Food Safety and Veterinary Office (FSVO). These data included clearly defined levels of Δ9-THC on hemp seed-derived feed materials and were considered in the different scenarios used to carry out dietary exposure assessment.

Acute dietary exposure to Δ9-THC was estimated combining different scenarios for the presence of ∆9-THC in hemp seed-derived feed materials, the transfer rate from feed to milk, the daily milk yield (L/cow), the daily feed consumption and the human consumption of milk and dairy products.

In adults, acute exposure to Δ9-THC through the consumption of milk and dairy products ranged between 0.001 µg/kg body weight (b.w.) per day and 0.03 µg/kg b.w. per day, the latter obtained under the scenario 20 litres (L) milk yield and 11 mg/kg of Δ9-THC in hemp seed-derived feed materials. In toddlers, acute exposure estimations to Δ9-THC through the consumption of milk and dairy products ranged between 0.006 µg/kg b.w. per day and 0.13 µg/kg b.w. per day, the latter obtained under the scenario 20 L milk yield and 11 mg/kg of Δ9-THC in hemp seed-derived feed materials.

Due to the lack of Δ9-THC data on whole hemp plant-derived feed materials a dietary exposure assessment, assuming a potential transfer of Δ9-THC from feed to milk, was not possible.

As pregnant women are not considered different from other population groups with respect to consumption of dairy products, the CONTAM Panel concluded that a separate exposure scenario was not needed.

There is substantial uncertainty associated with the exposure estimates obtained under the different scenarios. This uncertainty is mainly due to the limited number of available analytical data on ∆9-THC, and the lack of information on the fate of Δ9-THC and its precursor acids in the rumen of the cow and during food processing.

Dietary exposure to Δ9-THC via consumption of animal tissues and eggs could not be estimated, due to a lack of data on the potential transfer and fate of Δ9-THC.

After oral exposure, Δ9-THC is slowly and incompletely absorbed from the gastrointestinal tract. The oral bioavailability is lower compared to inhalation. In humans following oral administration of Δ9-THC, levels in plasma of the inactive metabolite 11-nor-9-carboxy-Δ9-THC are higher and measurable for longer compared to the parent compound and the active metabolite 11-OH-Δ9-THC. Thus, 11-nor-9-carboxy-Δ9-THC is a good indicator of the oral exposure to Δ9-THC irrespective of the source of Δ9-THC. Δ9-THC is highly bound to plasma proteins (97–99 %) and exhibits extensive tissue distribution, rapidly entering highly vascularised tissues resulting in a quick decrease in plasma concentration. Due to its lipophilic nature, Δ9-THC is accumulated in adipose tissue and may be released back to other tissues including the brain. Studies in both rats and humans indicate that the in vivo conversion of ∆9-THCA-A to Δ9-THC does not occur. The main oxidative biotransformation pathways occur in the liver. Extrahepatic biotransformations resulting in hydroxylation of the pentyl side chain have been documented in brain microsomes from mice, rats, guinea pigs, and rabbits. The main metabolic pathways are cytochrome P450 (CYP)-mediated with generation of the psychoactive 11-OH-Δ9-THC, a further oxidation to the inactive carboxylic acid 11-nor-9-carboxy-Δ9-THC and the glucuronidation of the parent compound and both metabolites. CYP3A4 and CYP2C9 enzymes are primarily involved in Δ9-THC metabolism in humans. Polymorphisms of the CYP2C9 enzyme have been shown to alter the kinetics of Δ9-THC. Oral administration of Δ9-THC results in a higher extent of metabolism of Δ9-THC to the psychoactive metabolite 11-OH-Δ9-THC compared to exposure from smoking. Δ9-THC and its metabolites are slowly excreted via the faeces mainly, and also in urine; an active entero-hepatic cycle has been described. Milk excretion of Δ9-THC has been documented in humans and in a number of laboratory animals.

No data were identified on the fate of Δ9-THCA-A in farm animals, in particular whether or not it is converted to Δ9-THC. The milk excretion of Δ9-THC, 11-OH-Δ9-THC and 11-nor-9-carboxy-Δ9-THC has been documented in ruminants. Tissue distribution in intravenous (i.v.) dosed pigs reflects that described in experimental animals.

In dairy cows, limited data indicate that the transfer rate of Δ9-THC to milk is in the range of 0.10–0.15 %. No appropriate studies could be identified to derive a transfer rate into other animal products.

The major effects of Δ9-THC are mediated by the cannabinoid receptor system. The presence of specific polymorphisms of genes in the endocannabinoid system (CNR1, FAAH and COMT Val158Met) may affect the response to Δ9-THC.

In experimental animal studies, acute exposure to doses up to 3 000 and 9 000 mg Δ9-THC/kg in dogs and monkeys, respectively, were not lethal. The oral LD50 for rats and mice were 666 mg Δ9-THC/kg and 482 mg Δ9-THC/kg, respectively. In repeated dose toxicity studies in rats, findings included mortality attributable to treatment at the high dose (500 mg/kg b.w. per day), decreases in body weight, nervous system effects, decreases in epididymal weights and sperm motility and increases in abnormal sperm, lower uterus weight, increase in oestrus cycle length. Histopathological findings included atrophy of the testes and ovarian and uterine hypoplasia. In repeated dose toxicity studies in mice, decreases in body weight, nervous system effects, lower sperm concentration, increase in oestrus cycle length, lower uterus weight, hyperplasia of thyroid gland follicular cells and of the forestomach were noted. Δ9-THC exposure affects various components of the immune system in mice, i.e. natural killer cells, macrophages, dendritic cells. Resistance to influenza virus was shown to be affected. Perinatal exposure of mice to Δ9-THC caused fetal thymic atrophy and T cell dysfunction, postnatally. There is no evidence of teratogenicity following exposure to Δ9-THC in rodent studies. Other findings from reproduction studies in rats and mice include decreases in the number of viable pups, an increase in fetal mortality and early resorptions, in the presence of maternal toxicity. Δ9-THC and other cannabinoids may affect the hypothalamic-pituitary-gonadal axis mainly via the interaction with cannabinoid receptor CB1 found in the hypothalamus, mainly resulting in a depression of the reproductive hormones, prolactin, and growth hormone. The major neurotoxic effects of Δ9-THC in experimental animals include alterations in locomotor activity, reduced social interactions and impaired learning. These behavioural alterations have been observed following acute or chronic administration of Δ9-THC, and in adult animals that were exposed to Δ9-THC during development. The developing brain appears to be sensitive to Δ9-THC at doses that have no adverse effects in the adult brain (1 mg/kg b.w.). Although Δ9-THC has shown some limited DNA damage in vitro, the available evidence indicates that it is not genotoxic in vivo. There was equivocal evidence of carcinogenic activity of Δ9-THC in male and female mice based on increased incidences of thyroid gland follicular cell adenomas at 125 mg/kg b.w. per day.

From human data, observed central nervous system (CNS) effects, as well as the increase in heart rate are considerable at low Δ9-THC levels and are both relevant for the risk assessment. Since these effects occur within a short time after administration, the CONTAM Panel concluded that it was appropriate to establish an acute reference dose (ARfD) for Δ9-THC. From the reported human data, the CONTAM Panel further concluded that adverse effects on the human CNS, such as mood alteration and sedation, are the most sensitive endpoint and thus, these dose-response relationships are most suitable for the derivation of the ARfD for Δ9-THC. The CONTAM Panel concluded that for the purpose of this risk assessment 2.5 mg Δ9-THC/day, corresponding to 0.036 mg Δ9-THC/kg b.w. per day for a person with a body weight of 70 kg, may be regarded as the lowest-observed-adverse-effect level (LOAEL) in both single and repeated uses. By applying an overall uncertainty factor (UF) of 30 (using an UF of 3 for extrapolation from the LOAEL to a no-observed-adverse-effect level (NOAEL) and a UF of 10 for interindividual differences) an ARfD of 1 μg Δ9-THC/kg b.w. was established by the CONTAM Panel. From repeated dose toxicity data in rodents, the CONTAM Panel identified a lowest 10 % lower confidence limit of the benchmark dose (BMDL10) of 0.73 mg Δ9-THC/kg b.w. per day for the increased length in oestrus cycle as a possible chronic reference point (RP) for the establishment of a tolerable daily intake (TDI). The CONTAM Panel noted that a difference of approximately 700 times is present between the chronic RP calculated from experimental animal studies and the established ARfD of 1 μg Δ9-THC/kg b.w. Therefore, the CONTAM Panel concluded that ensuring exposure is below the ARfD would also protect against possible effects of repeated exposure and establishing a TDI was not necessary.

Due to the limited data on Δ9-THC in samples of animal origin, the characterisation of the acute risks for human health is based on the estimates of dietary exposure to Δ9-THC by means of different scenarios, which consider the consumption of milk and dairy products resulting from the use of hemp seed-derived feed materials at the reported Δ9-THC concentrations These exposure estimates are at most 3 % and 13 % of the ARfD of 1 µg/kg b.w. in adults and toddlers, respectively. The CONTAM Panel concluded that the estimates of dietary exposure to Δ9-THC, by means of different scenarios that consider the consumption of milk and dairy products resulting from the use of hemp seed-derived feed materials at the reported concentrations, are unlikely to pose a health concern.

Due to the lack of representative occurrence data for Δ9-THC in whole hemp plant-derived feed materials, the CONTAM Panel considered a risk assessment of dietary exposure to Δ9-THC via milk and dairy products resulting from the use of these feed materials currently not feasible. Therefore, the CONTAM Panel could not conclude on the possible risks to public health from the use of whole hemp plant-derived feed materials for livestock.

Due to the lack of data on the potential transfer and fate of Δ9-THC in animal tissues and eggs, scenarios considering the exposure via other food of animal origin resulting from the use of hemp-derived feed materials could not be performed. Therefore, the CONTAM Panel did not conclude on the possible risks to public health from the consumption of these food commodities.

The main sources of uncertainty are associated with the dietary exposure estimates obtained under the different scenarios (as described previously). Other sources of uncertainty are that information is limited or lacking on the fate of Δ9-THC and its precursor acids in food producing animals, and on the impact of food processing. There are limited or insufficient data on co-occurrence of Δ9-THC with other cannabinoids and possible interactions of cannabinoids. The LOAEL was derived from human studies using pure Δ9-THC.

The CONTAM Panel recommended that analytical methods for the analysis of hemp plants and hemp derived products should be implemented to differentiate between the psychoactive compound Δ9-THC and their non-psychoactive precursor acids. Data on occurrence of Δ9-THC, its precursors and other cannabinoids in hemp-derived feed materials for food-producing livestock are needed. Further studies on the transfer rate of Δ9-THC, its metabolites, as well as other cannabinoids, and in particular on those that are known to be psychoactive, into animal products intended for human consumption are needed. More information is also needed on the fate of Δ9-THC and its precursor acids in food-producing animals, especially ruminants, and in food processing. Data on occurrence of cannabinoids in food, in particular on those that are known to be psychoactive, and those that have the potential to interact with Δ9-THC are needed.

di S. C.