Coumarin is a naturally occurring flavouring substance. The Scientific Committee for Food (SCF) delivered an opinion on coumarin in 1994 (SCF, 1997) in order that the Commission could consider whether the limits for coumarin in food set out in Annex II of the flavourings Directive 88/388/EEC (EEC, 1988) needed to be amended. The SCF concluded that coumarin was a carcinogen in rats via the oral route and possibly in mice, noting that adenomas and carcinomas of the liver and bile ducts and adenomas of the kidney have been observed in rats and adenomas and carcinomas of the lung and liver adenomas in mice. The Committee noted that a key issue in assessing the risk of coumarin to humans was deciding whether or not coumarin was genotoxic and that particularly strong reassurance was needed that coumarin was not genotoxic in vivo since, in addition to positive results from in vitro genotoxicity studies, an epoxide had been postulated as a metabolic intermediate. The available in vivo mutagenicity studies, while negative, were not of a high enough standard to provide sufficient reassurance that coumarin was not active in vivo. A further key consideration was whether the carcinogenicity seen in rats and mice, if due to an epoxide, was relevant to humans. The SCF concluded that the epoxide route could not be ruled out in humans and need only be a minor pathway for genotoxic/carcinogenic effects to be of concern.
The SCF in 1994 considered that further research, especially on the genotoxicity of coumarin, would be desirable, particularly if any proposals to raise the general limits from that then recommended (the lowest achievable limit of detection) were to be considered.
The SCF considered coumarin again in 1999 (SCF, 1999). Consideration of new data on liver metabolism did not reassure the Committee in 1999 that the epoxide-forming pathway was so minor in humans that no further concern with respect to genotoxicity was warranted. On the contrary, the new data on liver metabolism further supported the conclusions drawn in the opinion of the Committee in 1994 (SCF, 1997). Data from therapeutic use of coumarin also suggested that hepatotoxicity may occur in humans following coumarin treatment. No new data on genotoxicity as requested by the Committee in 1994 had become available, but data on the influence of human genetic polymorphism in the metabolism of coumarin reaffirmed concerns that a toxic epoxide intermediate may be produced in a significant proportion of the human population. Thus further information on genotoxicity was requested and the SCF considered that a study of in vivo DNA-adduct formation in rats in the relevant target organs, liver and kidney would be appropriate.
The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food has now been asked to review the toxicity in the light of the latest studies relevant to the safety assessment of coumarin in food products, particularly the specifically requested studies on possible DNA-adduct formation in kidney and liver, and to consider whether the previous opinions of the SCF need to be updated.
The results of the requested study on DNA-adduct formation in kidney and liver of rats demonstrate that coumarin does not bind covalently to DNA, supporting a non-genotoxic mode of action for tumour induction. The data now available allow the derivation of a Tolerable Daily Intake (TDI).
In addition to the requested studies, a number of recent publications relevant to the metabolism of coumarin in different species including humans were submitted. Studies on lung metabolism and morphology indicate that humans are unlikely to be susceptible to coumarin lung toxicity. Recent in vitro and in vivo studies show that the hepatotoxicity seen in rats is related to the 3,4- coumarin epoxide pathway, which is not a major metabolic pathway in humans. Originally it was thought that coumarin 3,4-epoxide was responsible for the liver toxicity, but in the mouse comparable levels of the epoxide are found but hepatotoxicity is negligible. From these comparative studies the Panel concluded that liver toxicity is not directly correlated to 3,4 coumarin epoxide/ortho-hydroxy phenyl acetic acid, but rather the balance between bioactivation and detoxification likely dictates the susceptibility of the animal species to coumarin-mediated liver toxicity.
Comparative studies in humans from South Europe and Asia show that a considerable number of individuals exhibit polymorphism, in that they have a considerable reduction in the 7-hydroxy coumarin pathway, which is normally the major metabolic route in humans that does not lead to the formation of 3,4 coumarin epoxide. However, it is not known what this reduction means with respect to the involvement of other metabolic pathways for coumarin. The Panel therefore concluded that hepatotoxic responses should be taken into account in setting a TDI and that in applying safety factors to the no-observed- adverse-effect level (NOAEL) for hepatotoxicity, it would be prudent to use a factor of 10 for potential interspecies variation, together with a factor of 10 for potential individual differences between humans. The overall NOAEL for liver toxicity in the most sensitive animal species, based on hepatotoxicity in a two year dog study, was 10 mg coumarin/kg bw/day. Applying a safety factor of 100, a TDI of 0 - 0.1 mg coumarin/kg bw can be established.
Conservative estimates of intake based on current maximum permitted concentrations in foodstuff suggest that present dietary intakes do not exceed the TDI.