scientific opinion on the risk to public health related to the presence of high levels of dioxins...

EFSA Journal 2011;9(7):2297

Suggested citation: EFSA Panel on Contaminants in the Food Chain (CONTAM); Scientific Opinion on the risk to public health related to the presence of high levels of dioxins and dioxin-like PCBs in liver from sheep and deer. EFSA Journal 2011;9(7):2297. [71 pp.] doi:10.2903/j.efsa.2011.2297. Available online: www.efsa.europa.eu/efsajournal © European Food Safety Authority, 2011

SCIENTIFIC OPINION

Scientific Opinion on the risk to public health related to the presence of high levels of dioxins and dioxin-like PCBs in liver from sheep and deer1

EFSA Panel on Contaminants in the Food Chain (CONTAM Panel)2, 3

European Food Safety Authority (EFSA), Parma, Italy

ABSTRACT EFSA was asked by the European Commission to deliver a scientific opinion on the risk to public health related to the presence of high levels of dioxins and dioxin-like PCBs in liver from sheep and deer. The opinion should also explore possible reasons for these high findings. Moreover, EFSA was asked whether dioxin and polychlorinated biphenyl (PCB) levels for liver should better be expressed on fresh weight rather than on a fat basis. The Panel on Contaminants in the Food Chain (CONTAM Panel) evaluated dioxin and PCB results from 332 sheep liver, 175 sheep meat and 9 deer liver samples submitted by eight European countries and estimated the exposure through consumption of sheep liver for adults (consumers only) and children. Regular consumption of sheep liver would result on average in an approximate 20 % increase of the median background exposure to dioxins and dioxin-like PCBs (DL-PCBs) for adults. On individual occasions, consumption of sheep liver could result in high intakes exceeding the tolerable weekly intake (TWI). The CONTAM Panel concluded that the frequent consumption of sheep liver, particularly by women of child-bearing age and children, may be a potential health concern. Additional intake of non dioxin-like PCBs (NDL-PCBs) from consumption of sheep liver does not add substantially to the total dietary intake. The range of fat content in sheep liver is considerably narrower than for a number of other food categories regulated in Regulation (EC) No 1881/2006. Therefore, the CONTAM Panel sees no need to change the basis for expression of results and maximum levels solely for liver from fat weight to fresh weight basis. A lower activity of CYP1A enzymes in sheep than in cattle was identified as a possible reason for higher dioxin and DL-PCB levels in sheep liver.

© European Food Safety Authority, 2011

KEY WORDS Dioxins, PCBs, sheep liver, occurrence, consumption, exposure, risk assessment

1 On request from the European Commission, Question No EFSA-Q-2010-00959, adopted on 5 July 2011. 2 Panel members: Jan Alexander, Diane Benford, Alan Raymond Boobis, Sandra Ceccatelli, Bruce Cottrill, Jean-Pierre

Cravedi, Alessandro Di Domenico, Daniel Doerge, Eugenia Dogliotti, Lutz Edler, Peter Farmer, Metka Filipič, Johanna Fink-Gremmels, Peter Fürst, Thierry Guérin, Helle Katrine Knutsen, Miroslav Machala, Antonio Mutti, Martin Rose, Josef Rudolf Schlatter and Rolaf van Leeuwen. Correspondence: [email protected]

3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Dioxins in Sheep Liver: Jean-Pierre Cravedi, Alessandro Di Domenico, Peter Fürst, Carlo Nebbia and Dieter Schrenk for the preparatory work on this scientific opinion and EFSA staff: Alessandro Carletti, Gina Cioacata and Luisa Ramos Bordajandi for the support provided to this scientific opinion for the support provided to this scientific opinion. The CONTAM Panel acknowledges all the European countries that provided dioxin and PCB occurrence data in food and supported the consumption data collection for the Comprehensive European Food Consumption Database.

Dioxins and dioxin-like PCBs in liver from sheep and deer

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SUMMARY Following a request from the European Commission, the Panel on Contaminants in the Food Chain (CONTAM Panel) was asked to deliver a scientific opinion on the risk to public health related to the presence of high levels of dioxins and dioxin-like polychlorinated biphenyls (PCBs) in liver from sheep and deer. The opinion should also explore possible reasons for the findings of high levels of dioxins and PCBs in liver from sheep and deer. Finally, the question was raised whether dioxin and PCB levels for liver, as for fish, should better be expressed on a fresh weight basis rather than on a fat basis.

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are two groups of tricyclic planar compounds that often are together referred to as “dioxins”. Unless otherwise stated, in this opinion the term “dioxins” refers to PCDDs and PCDFs together. Dependent on the number of chlorine atoms (1-8) and their positions in the rings, 75 PCDDs and 135 PCDFs, termed “congeners”, can occur. Dioxins are poorly soluble in water but highly soluble in lipids. Due to their lipophilic properties they accumulate in the food chain and are stored in fatty tissues. The toxicity of the various congeners depends on the chlorine substitution. Of special importance are those congeners that are substituted in the 2,3,7,8-positions and have at least one vicinal hydrogen atom. These are highly toxic, only sparsely degradable in the environment and have long biological half-lives.

Dioxins have no technological or other use, but are generated in a number of thermal and industrial processes as unwanted and often unavoidable impurities or by-products. Dioxins are generally not generated as single congeners but mostly as complex mixtures which are often characteristic of the source. Due to the numerous sources, dioxins are ubiquitous. However, due to a number of regulatory measures since the 1980s the emission of dioxins into the environment has considerably decreased.

PCBs are a group of organochlorine compounds that are synthesised by catalysed chlorination of biphenyl. Depending on the number of chlorine atoms (1-10) and their position at the two rings, 209 different compounds, also termed “congeners” are possible. In contrast to dioxins, PCBs had widespread use in numerous industrial applications, generally in the form of technical mixtures with various chlorine content. They were massively produced for over four decades, from 1929 until they were banned, with an estimated total world production of 1.2-1.5 million tonnes. Due to their physicochemical properties, such as non-flammability, chemical stability, high boiling point, low heat conductivity and high dielectric constants, PCBs were widely used in a number of industrial and commercial closed and open applications. According to Directive 96/59/EC Member States should have taken the necessary measures to ensure that used PCBs were disposed off and equipment containing PCBs were decontaminated or disposed off at the latest by the end of 2010. In fires and other thermal events, PCBs can be converted to PCDFs and other products.

Based on structural characteristics and toxicological effects, PCBs can be divided into two groups. One group consists of 12 congeners that easily can adopt a coplanar structure and show toxicological properties similar to dioxins. This group is therefore called “dioxin-like PCBs” (DL-PCBs). The other PCBs do not show dioxin-like toxicity and have a different toxicological profile. This group is called “non dioxin-like PCBs” (NDL-PCBs).

Investigations of the different pathways have indicated that dietary exposure represents the main route of dioxin and PCB exposure to humans, generally contributing more than 90 % of total dioxin and PCB exposure. Because of the lipophilic properties and the high accumulation potential, products of animal origin are of special importance.

Comprehensive monitoring programmes conducted worldwide during the past two decades showed that human exposure to dioxins and PCBs has decreased significantly over time. The monitoring programmes also demonstrated that certain food commodities, such as sheep liver and deer liver, can have high dioxin levels even when not affected by specific contamination sources. In 2008/2009 a


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number of sheep samples were analysed in Germany for dioxins and PCBs. Most sheep meat samples were below the respective maximum levels set by Regulation (EC) No 1881/2006. However, the corresponding liver samples from the same sheep in almost all cases exceeded the respective maximum levels considerably. Investigations indicated that these high levels were not due to poor husbandry practices or high localised contamination but were much more likely to be associated with the physiology of the animals.

For grazing ruminants, such as sheep, the involuntary intake of soil can occur through particles deposited on vegetables or directly when feeding on pasture herbage close to ground surface. Cattle normally feed on vegetation above 5 to 10 cm from the ground surface; sheep are anatomically able to nip closer to ground surface. The grazing surface is generally wider for sheep as flocks tend to change pasture fields more frequently than cattle, thus increasing the probability of coming into contact with land variably contaminated. The quantity of soil ingested depends on pasture abundance and quality, and on the density of animals at pasture. As the former decrease and/or the latter increases, the animals tend to graze the vegetation closer to ground surface, thereby taking up greater amounts of soil. Soil intake is highly variable and strongly seasonal: in sheep, a median soil intake has been reported in the order of 8 % of dry matter intake. On the whole, soil intake might be expected to contribute to sheep’s exposure to dioxins and PCBs to a non-negligible extent. A number of feed contamination incidents have occurred in recent years, potentially adding to the exposure to dioxins and PCBs.

EFSA evaluated the dioxin and PCB results from 332 sheep liver, 175 sheep meat and 9 deer liver samples submitted by eight European countries. More than 60 % of the results were from Germany. For sheep liver the mean upper bound concentrations for dioxins and the sum of dioxins and DL-PCBs (expressed as WHO-TEQ1998) were 14.9 pg WHO-TEQ/g fat (range: 0.27-116) and 26.1 pg WHO-TEQ/g fat (range: 0.47-279), respectively. The corresponding levels in sheep meat were considerably lower: 0.70 pg WHO-TEQ/g fat (range: 0.08-5.1) and 2.0 pg WHO-TEQ/g fat (range: 0.16-11.9), respectively. The mean value for the sum of dioxins and DL-PCBs in deer liver was almost 2.5-fold the mean value in sheep liver.

Occurrence data for NDL-PCBs were submitted by eight European countries for 257 sheep liver, 146 sheep meat and 9 deer liver samples. For sheep liver and sheep meat the mean upper bound concentrations for the sum of the six PCBs (PCB-28, -52, -101, -138, -153 and -180) identified as indicator-PCBs by the CONTAM Panel in 2005, were 26.8 ng/g fat (range: 0.41-350) and 13.1 ng/g fat (range: 0.51-162), respectively. The mean values for sheep and deer liver were comparable.

In addition to higher levels of dioxins in sheep liver, also a distinct difference was observed in the relative contributions of PCDD, PCDF and DL-PCBs to the total TEQ. In general, the relative contribution of PCDFs in sheep liver was considerably higher compared to sheep meat.

Consumption data on sheep liver are scarce in Europe. Data extracted from the EFSA’s “Comprehensive European Food Consumption Database” indicate that only a very small fraction of the European population consumes sheep liver. The available data for adults from six countries show that less than 3 % are consumers of “mutton and lamb liver”. The methodology used to collect the food consumption data differ between surveys affecting comparability. In three cases the surveys reported data taken on a 7-day food record basis, while the remaining three are either reported as 24 hour recall or as a 3-day food record, with consumption data reported either “as raw”, “as consumed” or “as cooked”. Consequently, calculation of average consumption of sheep liver expressed on a weekly basis for the small number of sheep liver consumers in the total adult population is subject to a high degree of uncertainty.

A viable alternative is to use the portion size distribution assuming that an arbitrary frequency of one eating occasion in a week can be taken as a conservative estimate.


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For adults, the results show an average sheep liver portion size of 106 g (or 1.5 g/kg b.w.) across six countries with the highest average value being in the UK of 141 g (or 1.9 g/kg b.w.). The average sheep liver portion size recorded in the UK has been used for the exposure assessment with the assumption that it represents a weekly amount. Assuming that sheep liver consumption in children is similar to consumption of “all liver”, the average weekly amount of 2.8 g/kg b.w. from a Bulgarian survey was selected as a conservative estimate for the exposure assessment for children.

Because of the small fraction of the European population that consumes sheep liver, the CONTAM Panel did not evaluated the exposure through consumption of sheep liver for the total European population but for “consumers only”. As it was assumed that sheep liver is not consumed daily, the evaluation was based on a potential weekly intake. Two different concentrations for the sum of dioxins and DL-PCBs in sheep liver were used in this evaluation. These are the mean levels calculated from the results reported by the submitting European countries and the maximum level laid down in Regulation (EC) No 1881/2006. The dietary intake was estimated using upper bound (UB) concentrations as UB and lower bound (LB) values were coinciding when rounded to the first decimal. An average fat content of 5.1 % for sheep liver was calculated from the submitted occurrence data and used in the exposure assessment.

The average weekly exposure to dioxins and DL-PCBs based on the mean concentration calculated from the occurrence data submitted by the European countries is 2.5 pg WHO-TEQ/kg b.w. for adults (consumers only). When using the maximum level laid down in Regulation (EC) No 1881/2006 the average exposure is 1.2 pg WHO-TEQ/kg b.w. For children, the average weekly exposure is 3.7 pg WHO-TEQ/kg b.w. and 1.7 pg WHO-TEQ/kg b.w. considering the occurrence data submitted by the European countries and the maximum level in the legislation, respectively.

For NDL-PCBs, the exposure estimations were performed with the mean concentration for the sum of the six indicator NDL-PCBs (PCB-28, -52, -101, -138, -153 and -180) calculated from the occurrence data submitted by the eight European countries. Harmonized maximum levels for the sum of these six congeners in various food categories are foreseen to be set soon. The average dietary exposure for adults (consumers only) is 2.6 ng/kg b.w. For children, the average weekly exposure to NDL-PCBs based on the consumption value derived by the “generic liver” data is 3.8 ng/kg b.w.

In order to characterize the risk of chronic consumption of sheep liver, calculations were made to compare how much the consumption of sheep liver would add to the total human exposure and how this compares with the tolerable weekly intake (TWI) for dioxins and DL-PCBs. As TWI the CONTAM Panel used the value of 14 pg TEQ/kg b.w. per week established by the SCF in 2001.

As a starting point, a human background daily intake was derived from the literature. The median dietary intake of dioxins and DL-PCBs across European countries for which data were reported as WHO1998-TEQs is 1.53 (0.51-3.2) pg/kg b.w. per day or converted to a weekly basis around 11 (3.6-23) pg WHO-TEQ/kg b.w. The CONTAM Panel noted that the available data on dietary exposure was only available from a limited number of European countries and might not reflect the most recent exposure. The data based on the WHO-TEF1998s were used because a considerable number of occurrence data was only reported as TEQs calculated with these TEFs without giving the raw data which made a conversion with the most recent WHO-TEF2005s impossible. Applying the latter TEFs may lead to 10-15 % lower values.

For adults, consumption of about 140 g (or 1.9 g/kg b.w.) sheep liver with the mean value of 26.1 pg WHO-TEQ/g fat would result in a weekly intake of 2.5 pg WHO-TEQ/kg b.w. and in a total weekly intake of 13.5 pg WHO-TEQ/kg b.w., taking a median background exposure of 11.0 pg WHO-TEQ/kg b.w. into account. This is 96 % of the TWI. As a number of sheep liver samples showed considerably higher concentrations for dioxins and DL-PCBs, it can be assumed that frequent consumers may be exposed on individual occasions to much higher values.


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Due to lack of data, the consumption of sheep liver for children was based on an age range up to 18 years old. As respective current data on background exposure to dioxins and DL-PCBs for children are sparse, the CONTAM Panel decided not to estimate a median dietary background exposure for children. The assessment indicates that for children the exposure to dioxins and DL-PCBs through consumption of sheep liver is approximately 50 % higher as compared to adults because of the higher food consumption relative to body weight.

Assuming a weekly background exposure of 35-161 ng/kg b.w. for the sum of the six indicator NDL-PCBs, the additional contribution through consumption of sheep liver for adults amounts to 1.6-7.4 %. The respective evaluation for children would result in an additional weekly intake between 2.2 and 11 %

In conclusion, regular consumption of sheep liver would result on average in an approximate 20 % increase of the background exposure to dioxins and DL-PCBs. On individual occasions, consumption of sheep liver could result in high intakes exceeding the TWI. The CONTAM Panel concluded that the frequent consumption of sheep liver, particularly by women of child-bearing age and children, may be a potential health concern.

For deer liver only nine results were reported on concentrations for dioxins, DL-PCBs and NDL-PCBs. This number of samples is too small to perform a meaningful risk assessment. However, as the reported concentrations for dioxins and DL-PCBs were generally higher than the concentrations for sheep liver (with an almost 2.5-fold higher mean value), the CONTAM Panel concluded that frequent consumption of deer liver, especially for high consumers, may be of health concern.

The European Commission also asked EFSA to provide scientific elements on the appropriateness to establish in future regulatory levels in liver on a product basis rather than on a fat basis. In general, the expression of results on a product basis would be preferable from a dietary exposure point of view as this would better reflect the exposure to the consumed products. The CONTAM Panel noted that the fat content of sheep liver reported in literature and submitted by the European countries generally range from 3 to 8 % fat with a mean content of 5.1 %. Comparable fat contents are found for liver samples of other terrestrial animals, such as bovine, pigs and chicken. These ranges of fat content are considerably narrower than for a number of other food categories regulated in Regulation (EC) No 1881/2006 such as dairy products which cover a range from 1 to >80 %. Even if there would be a possible hepatic sequestration and the dioxins and PCBs would not be totally associated with the fat fraction of the liver, this would have no influence on the result, whether based on lipid or fresh weight basis, as all dioxins and PCBs are extracted during the analytical procedure irrespective of the liver compartment where they are present. Therefore, the CONTAM Panel sees no need to change the basis for expression of results for liver only. A change of the expression of maximum levels seems only meaningful if all food categories would be considered.

The CONTAM Panel also explored possible reasons for the findings of high levels of dioxins and PCBs in liver from sheep and deer. Similar to other mammalian species, sheep are able to metabolize DL-PCBs and probably dioxins to hydroxy-derivatives, very likely through cytochrome P450 (CYP) 1A enzymes. Studies in vitro and in vivo with prototype substrates for CYP1A enzymes indicate a lower CYP1A1 activity in sheep than in cattle and suggest that differences in metabolism might be possible explanations for the marked differences in the liver storage of dioxins and related compounds between the two species. The differences in liver CYP1A expression between the two species, however, remain to be confirmed. Furthermore, as demonstrated in rodents, it cannot be excluded that other mechanisms such as the sequestration of dioxins and dioxin-like compounds by hepatic CYP1A2 or their biotransformation by other enzymes, may affect their accumulation in the liver of ruminants.


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TABLE OF CONTENTS Abstract .................................................................................................................................................... 1 Summary .................................................................................................................................................. 2 Table of contents ...................................................................................................................................... 6 Background as provided by the European Commission ........................................................................... 7 Terms of reference as provided by the European Commission ................................................................ 7 Assessment ............................................................................................................................................... 8 1. Introduction ..................................................................................................................................... 8

1.1. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) .. 8 1.2. Polychlorinated biphenyls (PCBs) .......................................................................................... 9 1.3. The toxicity equivalent (TEQ) system .................................................................................. 10 1.4. Previous risk assessments ..................................................................................................... 12

2. Legislation ..................................................................................................................................... 14 2.1. Expression of maximum levels on fat or fresh weight basis ................................................. 15

3. Sampling and methods of analysis ................................................................................................ 16 4. Sources and environmental fate with special emphasis on sheep’s potential contamination ........ 17 5. Occurrence and patterns on dioxin and dioxin-like PCBs in liver and meat from sheep and deer 20

5.1. Previously reported literature data on dioxin and dioxin-like PCBs in liver and meat from sheep, deer and other farm animals .................................................................................................... 20 5.2. Current occurrence of dioxins and DL-PCBs in liver and meat from sheep and deer .......... 24

5.2.1. Summary of data collected ............................................................................................... 24 5.2.2. Distribution of samples reported for sheep liver, sheep meat and deer liver .................... 25 5.2.3. Contribution of dioxins and DL-PCBs to total TEQ in sheep liver and meat .................. 26 5.2.4. Occurrence data reported for dioxins and PCBs in sheep and deer .................................. 30

6. Food consumption ......................................................................................................................... 33 7. Human exposure assessment ......................................................................................................... 35

7.1. Human exposure to dioxins and PCBs via consumption of sheep liver ............................... 35 7.2. Previously reported literature data on dietary intake of dioxins and DL-PCBs .................... 37

8. Hazard identification and characterization .................................................................................... 40 8.1. Toxicokinetics in ruminants .................................................................................................. 40

8.1.1. Absorption ........................................................................................................................ 40 8.1.2. Distribution ....................................................................................................................... 40 8.1.3. Metabolism ....................................................................................................................... 40 8.1.4. Elimination ....................................................................................................................... 43 8.1.5. Transfer and accumulation ratios ...................................................................................... 44

8.2. Effects of dioxins and PCBs on ruminants, particularly in sheep ......................................... 44 8.3. Toxicological end-points for dioxins and DL-PCBs ............................................................. 45

8.3.1. Laboratory animals ........................................................................................................... 45 8.3.2. Adverse effects in humans ................................................................................................ 47

9. Risk characterization ..................................................................................................................... 48 10. Uncertainty ................................................................................................................................ 52

10.1. Assessment objectives ........................................................................................................... 52 10.2. Exposure scenarios/Exposure model .................................................................................... 52 10.3. Model input (parameters) ...................................................................................................... 52 10.4. Summary of uncertainties ..................................................................................................... 53

Conclusions and recommendations ....................................................................................................... 53 References .............................................................................................................................................. 57 Appendix ................................................................................................................................................ 69 A. Average Dietary intake of dioxins and DL-PCBs for children reported in the literature for different EU countries and its relationship to adult average dietary exposure ....................................... 69 Abbreviations ......................................................................................................................................... 70


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BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The Scientific Committee on Food (SCF) established a group tolerable weekly intake (TWI) of 14 pg WHO-TEQ/kg body weight (b.w.) for 2,3,7,8-TCDD, all 2,3,7,8-substituted PCDDs and PCDFs and the dioxin-like PCBs in 2001.4 Due to the very long half-lives in humans the tolerable intake was expressed on a weekly rather than on a daily basis.

Commission Regulation (EC) No 118/2006 of 19 December 20065 lays down maximum levels for certain contaminants in foodstuff. For liver of terrestrial animals (bovine animals, sheep, poultry and pigs) and derived products thereof, a maximum level of 6.0 pg WHO-TEQ per g fat for dioxins and 12.0 pg WHO-TEQ per g fat for the sum of dioxins and dioxin-like PCBs apply. The maximum level is not applicable for food containing less than 1 % fat.

Recently, high levels of dioxins and dioxin-like PCBs were reported for a significant proportion of sheep and venison liver (in some cases levels higher that 100 pg WHO-TEQ per g fat). The generic term, sheep’s liver, encompasses the liver of lambs, sheep and wethers, further investigations suggest that these high levels are not due to poor husbandry practices or high localised contamination but are much more likely to be associated with the physiology of the animals.

It is appropriate to consider on the basis of these findings the need for possible additional measures for the protection of public health and/or the possible need to change the current provisions on maximum levels for dioxins and PCBs in liver and derived products, including the consideration to establish in future the maximum level in liver on a product basis rather than on a fat basis, as is currently the case.

TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION In accordance with Art. 29 (1) (a) of Regulation (EC) 178/2002 the Commission asks EFSA to assess the risk for public health of the levels of dioxins and dioxin-like PCBs found in liver of sheep and deer. In particular the opinion should indicate if there is a potential increase in consumer health risk for subgroups of the population consuming such products (e.g. high consumers, people following specific diets, etc). The opinions should also explore possible reasons for the findings of high levels of dioxins and PCBs in liver from sheep and deer, and to provide scientific elements on the appropriateness to establish in future regulatory levels in liver on a product basis rather than on a fat basis.

4 Scientific Committee on Food, 2001. Opinions of the on the risk assessment of dioxins and dioxin-like PCBs in food (update based on the new scientific information available since the adoption of the SCF opinions of 22 November 2000. Adopted by the SCF on 30 May 2001). Report CS/CNTM/DIOXIN/20 final. Available at URL: http://europa.eu.int/comm/food/fs/sc/scf/out90_en.pdf 5 OJ L 364, 20.12.2006, p 5-17.


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ASSESSMENT

1. Introduction

1.1. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs)

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are two groups of tricyclic planar compounds (Figure 1) that often are together referred to as “dioxins”. Unless otherwise stated, in this opinion the term “dioxins” refers to PCDDs and PCDFs together. Dependent on the number of chlorine atoms and their positions at the rings 75 PCDDs and 135 PCDFs, termed “congeners” can occur.

OO

O1

2

3

46

7

8

9 1

2

3

46

7

8

9

Clx y ClCl Clx y

Figure 1: Structure of PCDDs and PCDFs. Clx + Cly = 1-8.

Dioxins are poorly soluble in water but highly soluble in lipids. Due to their lipophilic properties they accumulate in the food chain and are stored in fatty tissues. Dioxins are highly resistant against acids and bases, possess a low vapour pressure and are thermally stable below 600 °C. The toxicity of the various congeners depends on the chlorine substitution. Of special importance are those congeners that are substituted in the 2,3,7,8-positions and have at least one vicinal hydrogen atom. These are highly toxic, only sparsely degradable in the environment and have long biological half-lives. The best known and most intensively studied congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which has gained special public and scientific attention as an impurity in “Agent Orange”, a mixture of the two herbicides 2,4-D and 2,4,5-T which was applied during the Vietnam War as a defoliant (Hay, 1978). The Seveso incident in 1976 was characterized by the release of considerable amounts of TCDD into the environment following a runaway reaction in a trichlorophenol-producing chemical plant (Hay, 1976, 1977; di Domenico et al., 1990). Although a detailed analysis of the environmental impact of the Seveso incident is beyond the scope of this opinion, it is worthwhile to recall that domestic animals and some wildlife started dying a few days after the incident.

In 1980, Bumb et al. (1980) reported on the presence of PCDDs in particles from a variety of combustion sources, and in dust and soil near combustion sources. Along with the data they obtained from the study, they hypothesized that PCDDs can in general result from trace chemical reactions occurring in fire provided some chlorine is available. There is evidence that dioxins were present in the environment and biosphere long before human activities started to become environmentally relevant (Schecter et al., 1988; Ligon et al., 1989; Green et al., 2004). However, other studies show that the majority of dioxins is today of anthropogenic origin, largely linked to the upsurge of the chemistry of chlorine during the 20th century (Czuczwa et al., 1984; Czuczwa and Hites, 1984, 1985; Smith et al., 1992).

PCDDs PCDFs


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Dioxins have no technological or other use, but are generated in a number of thermal and industrial processes as unwanted and often unavoidable impurities or by-products. Important emission sources are, inter alia, metal production and processing, waste incineration and domestic furnaces. Dioxins are generally not generated as single congeners but mostly as more or less complex mixtures which are often characteristic of the source. Due to the numerous sources, dioxins are ubiquitous. However, due to a number of regulatory measures since the 1980s the emission of dioxins into the environment has considerably decreased.

Investigations of the different pathways have indicated that dietary intake represents the main route of dioxin exposure to humans, generally contributing more than 80 % of total dioxin exposure. Because of the lipophilic properties and the high accumulation potential, products of animal origin are of special importance. Such food samples, especially if gained from mammals, show characteristic dioxin profiles in which the toxic 2,3,7,8-chlorine substituted congeners predominate. In contrast, foodstuffs of plant origin generally contain only low dioxin concentrations mostly in the range of the limit of detection (LOD).

Because of the reduced emissions and the declining levels in the environment also the dioxin concentrations in feed and food have decreased. Comprehensive monitoring programs conducted worldwide during the past two decades showed that the human exposure to dioxins has decreased significantly over time. However, these programs also detected a number of major contamination incidents resulting in withdrawal and destruction of thousands of tons of food and feed. This caused huge economic damage (with dioxin analyses only being a small portion).

The monitoring programmes also demonstrated that certain food commodities can have high dioxin levels although not being affected by specific contamination sources. In 2008/2009 a number of sheep samples were analysed in Germany for dioxins and polychlorinated biphenyls (PCBs). Most sheep meat samples were below the respective maximum levels set by Regulation (EC) No 1881/2006. However, the corresponding liver samples from the same sheep in almost all cases exceeded the respective maximum levels considerably. These findings triggered extensive monitoring programmes on the contamination of sheep meat and liver with dioxins and PCBs not only in Germany and led to the question of whether consumption of sheep liver constitutes a health risk for the European consumer. In this opinion, the generic term, sheep liver, comprises the liver of lambs, sheep and wethers.

1.2. Polychlorinated biphenyls (PCBs)

PCBs are a group of organochlorine compounds that are synthesised by catalysed chlorination of biphenyl. Depending on the number of chlorine atoms (1-10) and their position at the two rings, 209 different compounds, also termed “congeners” are possible. Figure 2 shows the structure of PCBs and the numbering of the carbon atoms in the two rings.

2 3

4

56

2'3'

4'

5' 6' Cl xCl y

Figure 2: Structure of PCBs. Cly + Clx = 1-10.

In contrast to dioxins, PCBs had widespread use in numerous industrial applications, generally in the form of complex technical mixtures. They were massively produced for over four decades, from 1929


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until they were banned, with an estimated total world production of 1.2-1.5 million tonnes (Fiedler, 2001; Holoubek, 2001; WHO, 2003). Due to their physicochemical properties, such as non-flammability, chemical stability, high boiling point, low heat conductivity and high dielectric constants, PCBs were widely used in a number of industrial and commercial closed and open applications. The technical PCB mixtures were mobile oils, viscous liquids or sticky resins depending on the degree of chlorination (between 21 and 68 % chlorine) (Hutzinger et al., 1974). According to Directive 96/59/EC6 Member States should have taken the necessary measures to ensure that used PCBs are disposed off and equipment containing PCBs are decontaminated or disposed off at the latest by the end of 2010.

In fires and other thermal events, PCBs can be converted to PCDFs and other products (Erickson, 1989). As PCBs are often mixed with polychlorobenzenes (for instance, in mixtures for dielectric fluids), their thermal degradation may also be associated with a relevant production of PCDDs (De Felip et al., 1994).

As a result of their widespread use, leakages and improper disposal practices, PCBs (like dioxins) also have a global distribution in the environment where they are persistent because they are poorly degraded and thus they are bioaccumulated in the food chain. Like dioxins, PCBs belong to the initial list of 12 persistent organic pollutants (POPs) that are regulated under the Stockholm Convention on POPs. The main pathway of human exposure for the majority of the population is via food consumption with the exception of specific cases of accidental or occupational exposure.

Based on structural characteristics and toxicological effects, PCBs can be divided into two groups. One group consists of 12 congeners that easily can adopt a coplanar structure and show toxicological properties similar to dioxins. This group is therefore called “dioxin-like PCBs” (DL-PCBs). The other PCBs do not show dioxin-like toxicity and have a different toxicological profile. This group is called “non dioxin-like PCBs” (NDL-PCBs).

In its risk assessment related to the presence of non dioxin-like PCBs in food and feed, the CONTAM Panel decided to use the sum of the six PCB congeners -28, -52, -101, -138, -153 and -180 as the basis for their evaluation, because these congeners are appropriate indicators for different PCB patterns in various sample matrices and are most suitable for a risk assessment of NDL-PCB on the basis of the available data. The Panel noted that the sum of these six indicator PCB represents about 50 % of total NDL-PCB in food (EFSA, 2005).

1.3. The toxicity equivalent (TEQ) system

As dioxins and DL-PCBs are generally not generated or released into the environment as single compounds but as complex mixtures with varying composition dependent on the respective source, an estimation of the toxic potential can not be performed by summing the concentrations of the determined congeners. In order to compare the toxicity of a mixture of congeners, the concept of toxicity equivalents (TEQs) based on different toxicity equivalency factors (TEFs) for the toxic congeners was introduced. Thereby it is assumed that the dioxins and dioxin-like compounds behave similarly by binding to the intracellular aryl hydrocarbon receptor (AhR), however with different affinity. It is assumed that the effects are additive. By definition, the most toxic congener TCDD is assigned a value of 1 and the TEFs for the other 16 toxic dioxins and 12 toxic DL-PCBs are between 0 and 1. Thus, a TEF indicates an order of magnitude estimate of the toxicity of a dioxin-like compound relative to TCDD. To calculate the total TEQ value of a sample, the concentration of each congener is multiplied with its TEF and they are then added together. The resulting TEQ value expresses the toxicity of dioxins and DL-PCBs in a complex sample in terms of the most toxic congener TCDD.

6 Council Directive 96/59/EC of 16 September 1996 on the disposal of polychlorinated biphenyls and polychlorinated terphenyls (PCB/PCT). OJ L 243, 24.9.1996, p. 31-35.


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The current TEF values were proposed by the World Health Organization (WHO) in 1997 and 2005 and termed WHO-TEF1998 (van den Berg et al., 1998) and WHO-TEF2005 (van den Berg et al., 2006) based on the year of publication (Table 1). In the European legislation all maximum levels for food and feed are presently expressed as TEQs using the WHO-TEFs1998. The potential impact of a change of the European legislation from the current WHO-TEFs1998 in relation to the more recent TEFs proposed by WHO in 2005 was assessed by EFSA in 2010 (EFSA, 2010). The report concluded that changing the basis for calculating TEQs to the new recommendations issued by WHO in 2005 will on average result in 14 % lower values with the extent of the difference highly variable across food and feed groups.

According to the European legislation all occurrence data on dioxins and PCBs in sheep and deer samples were reported on the basis of the WHO-TEFs1998. As for a considerable number of samples only the TEQ values but not the raw data were reported, a conversion with the WHO-TEF2005 was not possible. Thus, throughout this opinion the WHO-TEFs1998 were used. Taking the before mentioned EFSA report into account, the application of the WHO-TEF2005 would not significantly impact the final result and conclusion of this risk assessment.


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Table 1: Toxicity equivalency factors (TEFs) proposed by WHO.

Congener WHO-TEF1998 WHO-TEF2005 PCDDs

2,3,7,8-TCDD 1 1 1,2,3,7,8-PeCDD 1 1 1,2,3,4,7,8-HxCDD 0.1 0.1 1,2,3,6,7,8-HxCDD 0.1 0.1 1,2,3,7,8,9-HxCDD 0.1 0.1 1,2,3,4,6,7,8-HpCDD 0.01 0.01 1,2,3,4,6,7,8,9-OCDD 0.0001 0.0003

PCDFs 2,3,7,8-TCDF 0.1 0.1 1,2,3,7,8-PeCDF 0.05 0.03 2,3,4,7,8-PeCDF 0.5 0.3 1,2,3,4,7,8-HxCDF 0.1 0.1 1,2,3,6,7,8-HxCDF 0.1 0.1 2,3,4,6,7,8-HxCDF 0.1 0.1 1,2,3,7,8,9-HxCDF 0.1 0.1 1,2,3,4,6,7,8-HpCDF 0.01 0.01 1,2,3,4,7,8,9-HpCDF 0.01 0.01 1,2,3,4,6,7,8,9-OCDF 0.0001 0.0003

Non-ortho PCBs PCB-77 0.0001 0.0001 PCB-81 0.0001 0.0003 PCB-126 0.1 0.1 PCB-169 0.01 0.03

Mono-ortho PCBs PCB-105 0.0001 0.00003 PCB-114 0.0005 0.00003 PCB-118 0.0001 0.00003 PCB-123 0.0001 0.00003 PCB-156 0.0005 0.00003 PCB-157 0.0005 0.00003 PCB-167 0.00001 0.00003 PCB-189 0.0001 0.00003

1.4. Previous risk assessments

TCDD was evaluated as carcinogenic to humans (group 1 carcinogen) by the International Agency for Research on Cancer (IARC) in 1997. This classification was based on the assumption that, (i) TCDD is a multi-site carcinogen in experimental animals that has been shown by several lines of evidence to act through a mechanism involving the AhR, (ii) the AhR is highly conserved in an evolutionary sense and functions the same way in humans as in experimental animals and (iii) tissue concentrations are similar both in heavily exposed human populations in which an increased overall cancer risk was observed and in rats exposed to carcinogenic regimens in bioassays.


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Other dioxin congeners were not classifiable as to their carcinogenicity to humans (group 3) due to inadequate data.

In the IARC (1997) evaluation, the strongest evidence for the carcinogenicity of TCDD from epidemiological studies of high-exposure cohorts was for all cancers combined, rather than for any specific site.

In 1998, the WHO established a tolerable daily intake of 1-4 pg TEQ/kg body weight (b.w.) for dioxins and DL-PCBs based on effects of maternal TCDD exposure on the development of the offspring in rats.

The Scientific Committee on Food (SCF) revisited the WHO evaluation in 2000/2001, giving special attention to whether a weekly rather than a daily tolerable intake should be considered given the cumulative nature of the substances, and established a tolerable weekly intake (TWI) of 14 pg WHO-TEQ/kg b.w. (SCF, 2001).

In 2001, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) performed an updated comprehensive risk assessment of dioxins and DL-PCBs. The JECFA concluded that a tolerable intake could be established for dioxins on the basis of the assumption that there is a threshold for all effects, including cancer. The long half-lives of PCDDs, PCDFs and DL-PCBs mean that each daily ingestion has a small or even a negligible effect on overall intake. In order to assess long- or short-term risks to health due to these substances, total or average intake should be assessed over months, and the tolerable intake should be assessed over a period of at least one month. As a result, JECFA established a provisional tolerable monthly intake (PTMI) of 70 pg WHO-TEQ/kg b.w. per month.7

A risk assessment related to the presence of NDL-PCBs in feed and food was performed by the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) in 2005 (EFSA, 2005). It concluded that no health based guidance value for humans can be established for NDL-PCB because simultaneous exposure to NDL-PCB and dioxin-like compounds hampers the interpretation of the results of the toxicological and epidemiological studies, and the database on effects of individual NDL-PCB congeners was rather limited. However, there were indications that subtle developmental effects, caused by NDL-PCB, DL-PCB or dioxins, alone or in combination, may occur at maternal body burdens that are only slightly higher than those expected from the average daily intake in European countries. Because some individuals and some European (sub)-populations may be exposed to considerably higher average intakes, a continued effort to lower the levels of NDL-PCB in food was warranted.

In 2009, the German Federal Institute for Risk Assessment (BfR) carried out a health risk assessment of sheep liver related to the high levels of dioxins and PCBs (BfR, 2009). The mean (range) concentration of dioxins and PCBs in 140 samples of sheep liver from six different federal states was 41 (1.5-502) pg WHO-TEQ/g fat, and 94 % of the samples exceeded the maximum levels established by the EU Legislation. The assessment concluded that sheep liver with dioxin and PCBs concentrations below the maximum limit established in the EU Legislation is safe for consumption. However, the weekly consumption of 250 g of sheep liver with concentrations above the maximum EU level would considerably exceed the TWI of 14 WHO-TEQ/kg b.w. established by the SCF in 2001. For precautionary reasons, the BfR health assessment advised against the consumption of a food like sheep liver with such a high level of contamination. In contrast to sheep liver, the assessment concluded that there are no health concerns about the consumption of lamb or mutton meat, since far lower levels of dioxins and PCBs accumulate in muscle meat.

7 http://whqlibdoc.who.int/trs/WHO_TRS_909.pdf.


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2. Legislation

In order to protect public health, Article 2 of Council Regulation (EEC) No 315/938 of 8 February 1993 laying down Community procedures for contaminants in food stipulates that, where necessary, maximum tolerances for specific contaminants shall be established. Subsequently, a number of maximum levels for dioxins as well as for the sum of dioxins and DL-PCBs for various foodstuffs of animal origin are laid down in the Annex, Section 5 of Commission Regulation (EC) No. 1881/20069 of 19 December 2006 setting maximum levels (MLs) for certain contaminants in foodstuffs. Currently the MLs for dioxins and the sum of dioxins and DL-PCBs in food are under discussion between the EU Commission and the Member States with the intention of considerably reducing the levels.

Maximum contents for dioxins and the sum of dioxins and DL-PCBs for feed are regulated in Council Directive 2002/32/EC10 on undesirable substances in animal feed. The maximum levels for dioxins and the sum of dioxins and DL-PCBs in food and feed are both expressed as TEQs using the WHO-TEFs1998. All maximum levels are set as upper bound concentrations (UB). These are calculated on the assumption that all values of the different congeners below the limit of quantification (LOQ) are equal to the LOQ. Except for fish and fish products all maximum levels for foodstuffs of animal origin are given on a fat basis.

The current maximum levels for meat and meat products (excluding edible offal) of bovine animals and sheep are 3.0 pg WHO-TEQ/g fat and 4.5 pg WHO-TEQ/g fat for dioxins and the sum of dioxins and DL-PCBs, respectively. For meat and meat products of poultry the respective maximum levels are 2.0 pg WHO-TEQ/g fat and 4.0 pg WHO-TEQ/g fat. The lowest maximum levels in the food category “meat and meat products” apply for pigs with 1.0 pg WHO-TEQ/g fat and 1.5 pg WHO-TEQ/g fat for dioxins and the sum of dioxins and DL-PCBs, respectively. While the maximum levels for meat and meat products of the above animals differ, the same maximum levels of 6.0 pg WHO-TEQ/g fat and 12.0 pg WHO-TEQ/g fat for dioxins and the sum of dioxins and DL-PCBs, respectively apply to liver and derived products from these terrestrial animals.

Foodstuffs have to comply with the maximum levels for dioxins and for the sum of dioxins and DL-PCBs. However, these maximum levels are not applicable for foodstuffs containing < 1 % fat.

In addition to maximum levels, the European Commission has set action levels for dioxins and DL-PCBs as an early warning tool through Commission Recommendation 2006/88/EC11 and Directive 2002/32/EC in food and feed, respectively. Due to the fact that their sources are generally different, separate action levels for dioxins and DL-PCBs were established. The action levels for meat and meat products of bovine animals and sheep are 1.5 pg WHO-TEQ/g fat and 1.0 pg WHO-TEQ/g fat for dioxins and DL-PCBs respectively. The respective levels for meat of poultry and game are each 1.5 pg WHO-TEQ/g fat both for dioxins and for DL-PCBs. For meat and meat products of pigs action levels of 0.6 pg WHO-TEQ/g fat and 0.5 pg WHO-TEQ/g fat apply for dioxins and for DL-PCBs, respectively. The action levels for liver and derived products of terrestrial animals are each 4.0 pg WHO-TEQ/g fat both for dioxins and for DL-PCBs.

In cases where levels of dioxins and/or DL-PCBs in excess of the action levels are found, it is recommended that Member States, in co-operation with operators, initiate investigations to identify the

8 Council Regulation (EEC) No 315/93 of 8 February 1993 laying down Community procedures for contaminants in food. OJ L 37, 13.2.1993, p. 1-3. 9 Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. OJ L 364/5, 20.12.2006, p. 5-24. 10 Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed. OJ L 140/10, 30.5.2002, p. 10-21. 11 Commission Recommendation of 6 February 2006 on the reduction of the presence of dioxins, furans and PCBs in feedingstuffs and foodstuffs. s. OJ L 42/26, 14.2.2006, p. 26-28.


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source of contamination, take measures to reduce or eliminate the source of contamination and check for the presence of NDL-PCBs.

In view of disparities caused by different national maximum levels for NDL-PCBs in several Member States, the setting of respective harmonized MLs for the sum of the six indicator-PCBs (PCB-28, -52, -101, -138, -153, and -180) in various food categories is foreseen to be set soon.

Harmonized maximum values for dioxins and PCBs in soil do not exist in the European Union. Instead, several Member States have set national Regulations, reference values and recommendations for dioxin concentrations in soils depending on the type of use. A comprehensive overview on Member States Legislation is summarized in a report produced for the European Commission DG Environment (EC, 1999).

2.1. Expression of maximum levels on fat or fresh weight basis

The European Commission asked EFSA to provide scientific elements on the appropriateness to establish in future legislation levels in liver on a product basis rather than on a fat basis.

Dioxins and PCBs are lipophilic compounds that accumulate in the food chain and are stored in fatty tissues of animals and humans. Consumption of food of animal origin is generally the main route of human exposure to dioxins and PCBs. Due to their accumulation in the fat of the food of animal origin, the European Commission has set the maximum levels (MLs) for dioxins and DL-PCBs generally on a fat basis. The MLs for muscle meat of fish, fishery products and products thereof constitute an exemption because of the wide range of fat content of different fish species. As all ingested dioxins and PCBs are stored and concentrated in the fatty tissue of the fish, species with a low fat content, such as cod with around 0.4 % fat, could have extremely high levels if the concentrations of the lipophilic compounds was expressed on a fat basis. As a consequence, a considerable number of low-fat fish species would exceed MLs on a fat basis, while fish species with high fat content of 20 % and more, such as eel or herring would seem favourable because of the greater dilution of the lipophilic compounds in the higher fat amount. However, it is not the isolated fat but the muscle meat of fish that is consumed and thus the maximum levels based on a fresh weight basis allow a better estimation of the importance of the concentrations for human exposure and their potential health impact.

The CONTAM Panel ascertains that in general the expression of results on a product basis would be preferable from a dietary exposure point of view as this would better reflect the exposure to the consumed products. However, the Panel sees no justification to change the basis in future Regulation from fat basis to fresh weight basis only for liver of terrestrial animals for the following reasons. The fat content of sheep liver reported in literature and submitted by the European countries generally range between 3 and 8 % fat with a mean content of around 5.1 %. Comparable fat contents are found for liver samples of other terrestrial animals, such as bovine, pigs and chicken. These ranges of fat content is considerably narrower than for a number of other food categories regulated in Regulation (EC) No 1881/2006, such as dairy products which cover a range from 1 to >80 %. Moreover, even if there would be a possible hepatic sequestration and the dioxins and PCBs would not be totally associated with the fat fraction of the liver, this would have no influence on the result, whether based on lipid or fresh weight basis, as all dioxins and PCBs are extracted during the analytical procedure irrespective of the liver compartment where they are present.

Therefore, the CONTAM Panel sees no need to change the basis for expression of results for liver of terrestrial animals only. A change of the expression of maximum levels seems only meaningful if all food categories would be considered.


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3. Sampling and methods of analysis

Detailed requirements for methods of sampling and analysis for the official control of levels of dioxins and DL-PCBs in certain foodstuffs are laid down in Commission Regulation (EC) No 1883/2006.12 This Regulation contains inter alia a number of provisions concerning methods of sampling depending on the size of the lot, packaging, transport, storage, sealing and labelling.

Regarding analytical methods for the determination of dioxins and DL-PCBs in food and feed, the EU generally follows the “criteria approach”. This means that no fixed methods are prescribed but detailed and strict performance criteria are established by the European Commission which have to be fulfilled. As long as it can be demonstrated in a traceable manor that these performance criteria are fulfilled and the method is fit for purpose the analysts can apply their method of choice. The respective performance criteria are laid down in Commission Regulation (EC) No 1883/2006. As a basic requirement for acceptance of analytical procedures the sensitivity for dioxins and non-ortho-PCB must be in the picogram TEQ range. According to this Regulation, monitoring for the presence of dioxins in foodstuffs may be performed by a strategy involving a screening method in order to select those samples with levels of dioxins and dioxin-like PCBs that are less than 25 % below or exceed the maximum level. Screening methods may comprise bioassays and GC/MS methods. The concentration of dioxins and the sum of dioxins and DL-PCBs in those samples with significant levels needs to be determined/confirmed by a confirmatory method. Confirmatory methods are based on gas chromatography/high resolution mass spectrometry (GC/HRMS). The criteria for confirmatory methods concerning trueness and precision (relative standard deviation calculated from results generated under reproducibility conditions, RSDR) are given as -20 % to +20 % and <15 %, respectively. Further criteria concern addition of isotope-labelled standards, gas chromatographic separation of congeners, maximum tolerances for retention time and isotope ratios based on US-EPA method 1613 B, reporting of results and others.

Comparable criteria for the official control of levels of NDL-PCBs in food are foreseen to be set soon by the European Commission. In general, the extraction and clean-up of the samples can follow the same approach as for dioxins and DL-PCBs. However, as NDL-PCBs are normally present at considerably higher concentrations, an analytical determination using GC/HRMS is not compulsory. Instead use of analytical systems, such as GC with electron capture detection (GC-ECD), GC-low resolution mass spectrometry (GC-LRMS) or GC/tandem mass spectrometry (GC-MS/MS) are often sufficient provided it can be demonstrated that they are able to unequivocally detect the compounds at the level of interest.

In order to contribute to a high quality and uniformity of analytical results, an analytical network of a European Reference Laboratory (EU-RL), National Reference Laboratories (NRL) and Official National Laboratories (OFL) were designated in the past for dioxins and PCBs. The activities of reference laboratories cover all areas of feed and food law, in particular those areas where there is a need for precise analytical results. For example, the EU-RL for dioxins and PCBs organizes annual proficiency tests with different matrices for NRLs and OFLs. Since the year 2000 the Norwegian Institute of Public Health has offered interlaboratory comparison studies on various POPs in food. Generally more than 100 laboratories from all over the world participate in the studies on the determination of dioxins and PCBs in non-spiked food specimens.13 The results indicate that most of the participating laboratories, although applying different GC/HRMS methods, are capable of reliably analysing dioxins and PCBs at the level of interest.

12Commission Regulation (EC) No 1883/2006 of 19 December 2006 laying down methods of sampling and analysis for the official control of levels of dioxins and dioxin-like PCBs in certain foodstuffs. OJ L 364/32, 20.12.2006, p. 32-43. 13 www.fhi.no.


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A specific certified reference material (CRM) for dioxins and PCBs in sheep liver is not available. However, the determination of dioxins and PCBs in liver samples from other animals was part of proficiency tests.

4. Sources and environmental fate with special emphasis on sheep’s potential contamination

Emissions from thermal and combustion sources, followed by aerial distribution and deposition, have been and are the principal cause of diffuse environmental contamination, which may ultimately affect dioxin levels in agricultural products (SCF, 2000). At a local level, the quality of such products may be influenced by a number of agricultural practices - including disposal of farming waste by on-site burning (“backyard emissions”) and use of accidentally contaminated agricultural fertilizers, such as sewage sludge and compost (Jones and Sewart, 1997; EC SCAN, 2000; Brambilla et al., 2004; Rideout and Teschke, 2004; Umlauf et al., 2011) - which may also be responsible for some PCB contamination. Backyard emissions and minor open-air combustions are estimated to be major contributors to the ubiquitous complex mixtures of environmental dioxins (US-EPA, 2001, 2009).

Direct or indirect release of PCBs into the environment has resulted from their uses, inappropriate disposal practices, accidents, leakages from industries and manufacturing facilities, open-air waste incineration (backyard emissions), diffuse emission from PCB-containing materials (e.g. paints, sealants, coatings, plastics), and PCB out-gassing from buildings (Erickson, 2001; Holoubek, 2001). Aerial transport and deposition have been and are an important cause of diffuse environmental contamination. As a consequence of their widespread use and physicochemical properties - high stability, persistency and high bioaccumulation potential - PCB presence in the environment and biosphere appears to be ubiquitous. Cases of severe food and feed contamination by PCBs have been reported on several occasions (SCF, 2000; Holoubek, 2001; EFSA, 2005). Here, it is sufficient to mention three major events, such as the corruption of rice oil in Japan in 1968 (Masuda, 2003) and in Taiwan in 1979 (Guo et al., 2003), both resulting in severe health effects, and of the food chain in Belgium in 1999 (Bernard et al., 1999, 2002). In all cases, PCBs were the major contaminant and the vehicle for the highly toxic PCDFs and, to a lesser extent, PCDDs.

Soil and particles suspended in water (in particular the world’s oceans), are natural reservoirs of dioxins and PCBs (Fries, 1995; EC SCAN, 2000; Holoubek, 2001; Fiedler, 2003; WHO, 2003). Soil and sediments tend to accumulate these chemicals. Except for several members of the cucumber family (cucurbitaceae), in general soil-to-plant transfer of dioxins via the root is of minor importance. Because of this, aerial distribution and deposition are the main factors of dioxin contamination of edible herbage and leafy vegetables (Fries, 1995; Jones and Sewart, 1997). Aerial fall-out is also an important mechanism for PCB contamination of vegetation, although plant uptake of PCBs from soil has a role more relevant than for dioxins (WHO, 1993; Liu and Schnoor, 2008). The uptake magnitude and the extent of within-plant translocation appear to be dependent on plant species and positively correlate with PCB concentration in soil and a decrease of chlorination degree, the latter generally entailing an increase of chemical water solubility and mobility in aqueous carriers.

Sheep grazing habits and soil ingestion

For sheep, regardless of whether production is for meat or milk, grazing is a primary mechanism for intake of dioxins and PCBs (EC SCAN, 2000). For grazing ruminants, the involuntary intake of soil can occur through dust deposited on vegetables or directly when feeding on pasture herbage close to ground surface. Cattle normally feed on vegetation above 5 to 10 cm from ground surface, but sheep and deer, with their narrow mouths and highly curved incisor arcades, are anatomically able to nip closer to ground surface, possibly at a distance as small as ca. 3 cm (SNH, online; Menneer et al., 2004; Rook et al., 2004; Celaya et al., 2007), thereby increasing the likelihood of ingesting soil relative to cattle (Abrahams and Thornton, 1994; Abrahams and Steigmajer, 2003). The grazing surface is generally wider for sheep as flocks tend to change pasture fields more frequently than cattle,


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thus increasing the probability of coming into contact with land variably contaminated. In addition, sheep often graze on marginal lands (Celaya et al., 2007) with possible problems of poor environmental quality due to illegal waste disposal and burning, both representing important sources of contamination.

The quantity of soil ingested depends on pasture abundance and quality, and on the density of animals at pasture. As the former decrease and/or the latter increases, the animals tend to graze the vegetation closer to ground surface, thereby taking up greater amounts of soil. Although the bioavailability of dioxins and PCBs from soil is likely to be lower than that from a number of other sources (Fries, 1995), soil intake may contribute to a non-negligible extent to chemical body load or milk contamination, depending on the extent of soil contamination. Forages are either directly eaten by grazing ruminants, or cropped and preserved in a dry form (hay) or silage for a later use. When sewage sludge is spread on forages, some may adhere on vegetation while most will settle on soil surface. Livestock exposure to contaminants is potentially increased, although increases in livestock dioxin intake due to routine sludge applications appear to be of limited importance, unless soil or sludge are ingested at an unusually high rate (Jones and Sewart, 1997; EC SCAN, 2000). Dioxins and PCBs are poorly soluble in water. However, they are adsorbed onto suspended particles, and therefore ruminants can be exposed to these contaminants in water from rivers or ponds (EC SCAN, 2000). However, dioxin and PCB intakes may be expected to be limited unless unusual conditions occur.

The average weight of a sheep (ewe or ewe lamb) of commercial interest is in the range of 35-80 kg. Relative to body weight, the corresponding daily intakes of dry matter are estimated in the order of 1.9-3.5 % for non-lactating ewes in the first 15 weeks of pregnancy, 2.4-3.8 % for sheep in the last four weeks of pregnancy, and 3.2-5.2 % for lactating ewes during the first six to eight weeks after delivery (US NRC, 1985). DM intake may be season-dependent and, in European countries, lower in winter than in summer months (SNH, online). Despite its relevance, data on soil ingestion in sheep are available from only very few studies. According to Abrahams and Steigmajer (2003) and Smith et al. (2009), who studied soil ingestion of sheep grazing on various sites in a mid-Wales floodplain over several months in 1999 and 2000, the median soil intake was estimated as 7.6 % of dry matter intake (full range, 0.1-81.8 %). From the more extensive investigation of 2009, which was in agreement with the earlier work, it is clear that the soil intake rate is remarkably variable and strongly seasonal, with inter-season variations between one and two orders of magnitude, the highest rates being recorded in winter months and the lowest in summer months.

On the whole, and in spite of the limited bioavailability of dioxins and PCBs in soil (Fries, 1995), it can reasonably be assumed that soil intake might contribute to sheep’s exposure to the aforesaid chemicals to a non-negligible extent.

Feed contamination

Feed contamination by dioxins and/or PCBs may occur at different levels of the feed chain and have various origins. What follows is a brief description of relevant cases that have taken place during the last 15 years.

In 1997, the US-FDA identified a clay material - “ball clay”, a technological additive used as an anti-caking agent in feeds - as the source of elevated PCDD concentrations in US poultry and catfish samples (Rappe et al., 1998; Hayward et al., 1999; Ferrario et al., 2000). Similarly, in 1999 a PCDD contamination was detected in Europe in some kaolinitic clay used for feeds. Primeval biogenic and abiotic formation processes were proposed to be the origin of contamination (Ferrario et al., 2000; Rappe and Andersson, 2000; Eljarrat et al., 2002; Holmstrand et al., 2006; Gu et al., 2008; Horii et al., 2008).

In 1998, citrus pulp pellets from Brazil were found to contain high levels of dioxins due to an accidental production process treatment with highly contaminated lime (calcium hydroxide) from


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industrial or waste recycling (Malisch, 2000). Citrus pulp pellets are used as filler in beef or milk cattle feed.

In 1999, grass meal was found to be highly contaminated by dioxins in the German state of Brandenburg (EC SCAN, 2000). The contamination was due to the drying process for which different types of wood were burnt, including waste wood treated with paints and/or preservatives.

In 1999, in Belgium recycled fat destined for the production of a variety of feeds was severely contaminated by a discharge of a waste PCB mixture containing high levels of dioxins (Bernard et al., 1999, 2002; Hoogenboom et al., 1999; van Larebeke et al., 2001; Covaci et al., 2008).

In 2000, feed pre-mixtures, produced in Spain and containing pro-vitamin choline chloride, were found to be contaminated by dioxins (Llerena et al., 2003). The choline chloride carrier, declared as corn cob meal, also contained rice husks and/or sawdust presumably treated with pentachlorophenol (PCP), a wood preservative generally containing PCDDs as impurities. PCP has been responsible over the years for several cases of contamination of the food chain (Fries et al., 1999; Brambilla et al., 2004, 2009; Fochi et al., 2006; de Filippis et al., 2008). In 2010-2011 contaminated fatty acids originating from the production of biodiesel from used cooking fats illegally entered the feed chain. How the fatty acids were contaminated with dioxins is still unclear. Most likely chlorophenols-containing waste was dumped into the used cooking oils (Fürst, 2011).

The occasional presence of dioxins and possibly PCBs in mineral feed supplements such as copper sulphate, magnesium oxide, and zinc oxide, has also been reported as the outcome of production processes (Ferrario et al., 2003; US FDA, 2003; Huwe and Smith, 2005; Kim et al., 2011). It may be observed that zinc is a particularly important feed ingredient for sheep.

Sheep exposure to dioxins and PCBs from feed

The relationship between contaminated feed and feed components and dioxin and/or PCB presence in food-producing animals has been investigated in several cases including large and small ruminants, poultry, and pigs, and their produce (namely eggs, meat, and milk). Most ruminant studies have involved cattle (Travis and Arms, 1988; Willett et al., 1990; McLachlan, 1993; Slob et al., 1995; Schuler et al., 1997; McLachlan and Richter, 1998; Thomas et al., 1998; Fries et al., 1999; Sweetman et al., 1999; Traag et al., 1999; Malisch, 2000; Birak et al., 2001; Richter and McLachlan, 2001; Thorpe et al., 2001; Hoogenboom, 2004; Huwe and Smith, 2005; Schulz et al., 2005; Rychen et al., 2008). Only few studies have been published involving small ruminants (Schulz et al., 2005; Costera et al., 2006; Rychen et al., 2008). Of these studies only one deals shortly with dioxin concentrations in sheep liver and other tissues (Schulz et al., 2005), and shows the dioxin content in the liver to have reached the highest concentration of 20.7 pg WHO-TEQ/g fat - several times higher than that in muscle - after a four-week grazing on flooding areas.

Different measures have been used to quantify the transfer to animal products, as indicated in the references mentioned above. All the expressions reported apply when a steady state is reached, a condition for which up to a few months may be required (EC SCAN, 2000). Carry-over ratios or rates (CORs) are expressed as percentages of the dose that is transferred to milk fat on a daily basis. By comparing the CORs of different dioxin congeners, it may be concluded that their magnitude depends on a range of factors, including the source of contamination (or the correlated contaminated matrix), the nature of contaminant carrier or the matrix ingested, the congener, and the animal species. In general, low COR values characterize dioxin congeners with seven and eight chlorine atoms, whereas those with four to six chlorine atoms may reach CORs as high as 50-80 %. The data available for PCBs are too limited for an evaluation. On the whole, PCB congeners with a lower degree of chlorination seem to have rather low CORs (< 20 %), whereas other congeners with a medium-to-high chloro-substitution degree have quite greater CORs (> 40 %). In all cases, the available COR estimates exhibit a remarkable level of variability, to the extent that dioxin and PCB transfer from feed to food


EFSA Journal 2011;9(7):2297 20

should basically be viewed as a congener-specific phenomenon. Limited data are available as to transfer to sheep liver (see Section 8.1.5).

5. Occurrence and patterns on dioxin and dioxin-like PCBs in liver and meat from sheep and deer

The following section summarizes occurrence data on dioxins and PCBs in liver and meat of sheep and other animals. Except for a few cases, no information was retrieved whether the meat samples of sheep were compliant with the legislation if the levels in liver exceeded the maximum levels.

5.1. Previously reported literature data on dioxin and dioxin-like PCBs in liver and meat from sheep, deer and other farm animals

The levels of dioxins and DL-PCBs in liver of sheep, deer and other farm animals reported in the literature are shown in Table 2.

Levels of dioxins in muscle, liver and fat were measured in five sheep within one herd (Schulz et al., 2005). Two sheep were slaughtered to test the original dioxin concentrations in the tissues, and the other three were brought to graze on flooding areas and the associated dikes. After 4, 8 and 16 weeks of grazing one sheep was slaughtered and the tissues analysed. Before and after the trial the levels in liver were at least 10 times higher (4.92-20.7 pg WHO-TEQ/g fat, in 4 out of the 5 sheep the levels were > 6 pg WHO-TEQ/g fat) than those in muscle (0.47-1.58 pg WHO-TEQ/g fat) and fat tissue (0.47-1.82 pg WHO-TEQ/g fat). In cows, even if fed contaminated grass silage, the levels in muscle and fat tissue were similar to those in sheep (1.11 pg WHO-TEQ/g fat) while the concentrations in liver were at least 2-fold lower (maximum level 2.27±1.3 pg WHO-TEQ/g fat).

The Food Safety Authority of Ireland (FSAI) carried out a surveillance study of the levels of dioxins and PCBs in food supplements, milk and offals available on the Irish market (FSAI, 2005). A total of 11 lamb livers were analysed with mean (range) UB concentrations of 3.43 (0.50-6.76), 0.80 (0.15-1.58) and 4.23 (0.75-8.34) pg WHO-TEQ/g fat for dioxins, DL-PCBs and the sum of both, respectively. Dioxins were the major contributors to the total WHO-TEQ values. In one porcine liver analysed, the levels were lower with a UB concentration of 1.44, 0.22 and 1.67 pg WHO-TEQ/g fat for dioxins, DL-PCBs and the sum of both, respectively.

Lindström et al. (2005) studied the profile of dioxins and non-ortho PCBs in muscle, fat and liver of sheep feeding at an old PCP contaminated sawmill site and compared it to that of the top soil from the pastureland. According to the authors, the exposure to dioxins was due to perinatal (transplacental and lactational) transfer as well as exposure by grazing on the contaminated soil. The UB dioxin level in the top soil was reported to be 86 pg WHO-TEQ/g. The UB concentrations in liver (49 and 13 pg WHO-TEQ/g fat for dioxins and non-ortho PCBs, respectively) were much higher than those found in fat (3.7 and 4.1 pg WHO-TEQ/g fat, respectively) and in muscle (3.2 and 3.9 pg WHO-TEQ/g fat, respectively). The UB concentration for the sum of dioxins and non-ortho PCBs in liver was 61 pg WHO-TEQ/g fat, while for fat and muscle the levels were lower and similar between them (7.8 and 7.1 pg WHO-TEQ/g fat, respectively). The reported level in liver exceeded by approximately 5-fold the EU maximum level of 12 pg WHO-TEQ/g fat for the sum of dioxins and DL-PCBs.

Lund et al. (2008) reported the content of dioxins and DL-PCBs in four different matrices (liver, leaf fat, flank and shank) from the same sheep (n=5) from farms in Denmark. The levels of dioxins and DL-PCBs in the liver ranged from 6.8-16.7 pg WHO-TEQ/g fat and 1.0-3.0 pg WHO-TEQ/g fat, for dioxins and DL-PCBs, respectively. The levels in the two meat cuts (shank and flank) were lower and ranged from 0.26-1.9 pg WHO-TEQ/g fat and 0.18-0.48 pg WHO-TEQ/g fat, respectively. The contribution of the different congeners to the total TEQs was different for liver compared to that found in the meat cuts and fat. While 2,3,4,7,8-PeCDF was reported to account for more than 50 % to the total TEQs for liver, it accounted for 20 % of the three other matrices. On the other hand,


EFSA Journal 2011;9(7):2297 21

1,2,3,7,8-PeCDD and PCB-126 accounted for around 10 % of the total TEQs in the liver and 20 % in the other three matrices.

Bruns-Weller et al. (2010) investigated the occurrence of dioxins and DL-PCBs in 77 livers of sheep from Lower Saxony (Germany). The median (range) concentrations reported were 18.3 (4.8-161.2), 14.66 (2.00-133.3) and 33.89 (7.22-203.7) pg WHO-TEQ/g fat for dioxins, DL- PCBs and the sum of both, respectively. Seventy two out of the 77 liver samples analysed exceeded the EU maximum level for the dioxin concentration, while 71 exceeded the maximum level established for the sum of dioxins and DL-PCBs. No significant correlation was found between the levels of dioxins or PCBs and the fat content, and no correlation was found between the age of the animals and the concentration in liver. No significant differences were observed between the levels in females and males, and almost no differences were observed between samples from rural and municipal areas or between areas located near or apart from waterways. PCDFs were reported to be the most abundant congeners in the sheep liver samples, while PCB-118 and -156 dominated the profile of DL-PCBs.

In the UK, Rose et al. (2010) and Fernandes et al. (2010) carried out two studies in liver from various species in order to investigate the preferential accumulation of dioxins and PCBs in liver, particularly sheep and venison. Rose et al. (2010) analysed 22 lamb liver and 10 venison liver samples (largely from red deer) obtained from randomly selected retail outlets throughout the UK in 2005. The levels in lamb liver were between 0.24 and 25 pg WHO-TEQ/g fat, while the levels found in venison liver were higher and ranged from 3.7 to 50 pg WHO-TEQ/g fat. The authors reported no correlation between the fat content in the lamb livers and dioxin concentration. However, it was not discounted that the levels could reflect different ages or husbandry systems of livestock. The average ratio of dioxin to DL-PCBs in the lamb samples was 4 (range: 1.2-11.8) and a similar situation was found for venison liver samples. According to the authors, this high ratio might indicate differences in source contamination, in husbandry or in lipid metabolism of these two species compared to others. The authors discounted poor husbandry practices and high localized contamination as the causes of these high levels, and suggested it to be associated with the physiology of the animals.

Fernandes et al. (2010) carried out an investigation on the levels of dioxins and PCBs in commonly consumed offals (n=173) including lamb, deer, ox, and pig’s liver, kidney, tongue and heart, and offal products such as pâté, haggis, tripe and black pudding. The samples were obtained from a range of retail outlets across the UK in 2005. In general, the offal found to have the highest levels was liver, and in particular the liver of grazing animals such as lamb, deer and oxen. The average concentration for the sum of dioxins and dioxin-like PCBs reported in lamb liver (n=19) was 8.36 pg WHO-TEQ/g fat and for deer liver (n=10) was 68.2 pg WHO-TEQ/g fat. These values were higher than those reported for pig liver (n=20, average 2.83 pg WHO-TEQ/g fat) and poultry liver (n=14, 0.55 pg WHO-TEQ/g fat). The most significant contributors to the total WHO-TEQ value were 2,3,4,7,8-PeCDF, 1,2,3,7,8-PeCDD and PCB-126.

At the Chemical and Veterinary Analytical Institute in Münster (Germany), samples of both meat and liver from 43 sheep were analysed in 2009 for dioxins and DL-PCBs (CVUA-MEL, 2010). For meat, median concentrations of 0.64 pg WHO-TEQ/g fat (range: 0.24-3.9) and 1.62 pg WHO-TEQ/g fat (range: 0.38-8.34) were found for dioxins and the sum of dioxins and DL-PCBs. The respective median concentrations in liver from the same sheep were 11.72 pg WHO-TEQ/g fat (range: 4.48-68.0) and 20.28 pg WHO-TEQ/g fat (range: 8.47-110.7). While the median contribution of dioxins to total TEQ amounted to 35.8 % in meat, the respective median contribution in liver was found to be 61.5 %. The median ratio of dioxin TEQ between liver and meat was calculated as 20.1 (range: 5.3-41.8). For the sum of dioxins and DL-PCBs the median TEQ ratio was 12.6 (range: 3.1-27.6). These ratios differ considerably from respective ratios derived from meat and liver samples from bovines analyzed at the CVUA-MEL in 2010. Based on the analysis of both meat and liver from 27 bovine animals, the median ratio of dioxin TEQ between liver and meat was 5.4 (range: 1.4-8.2). For the sum of dioxins and DL-PCBs the median TEQ ratio was 2.8 (range: 1.2-4.8).


EFSA Journal 2011;9(7):2297 22

Fernandes et al. (2011) designed a study to investigate the transfer and uptake of dioxins and PCBs into conventionally reared farm animals, including sheep. A total of 16 sheep (8 pregnant lowland and highland sheep carrying twins) were studied and the levels of dioxins and DL-PCBs analysed in meat (n=16) and in liver taken from the oldest lambs (n=2). The concentrations in meat ranged from 0.51 to 3.21 pg WHO-TEQ/g fat, while in the two liver samples the levels were 8.21 and 19.55 pg WHO-TEQ/g fat. In addition to the higher levels found in liver compared to meat, the PCB contribution to the total TEQ was lower in liver with a predominance of hepta- and octaCDD/Fs. This different contribution of dioxins and PCBs to the total TEQ was also observed for pigs. According to the authors, these differences in contaminant concentrations between meat and liver are influenced by the physiological function of the liver more than other parameters, e.g. difference in environmental conditions and diet.

Results from different studies need to be compared with caution due to different methodologies, different exposure types and congeners analysed. Despite this, all studies in sheep reported higher levels in liver than in meat. While dioxin concentrations in liver ranged from 0.24 to 161.2 pg WHO-TEQ/g fat, the levels in meat ranged from 0.24 to 3.9 pg WHO-TEQ/g fat (Table 2). The concentration in liver for the sum of dioxins and DL-PCBs were in some cases up to 203.7 pg WHO-TEQ/g fat. The values reported in liver of deer were in the same range as those reported for sheep (range between 13-109 pg WHO-TEQ/g fat for dioxins).


EFSA Journal 2011;9(7):2297 23

Table 2: Dioxin and DL-PCB concentrations (pg WHO-TEQ/g fat) in liver and meat of sheep, deer and other ruminants or farm animals reported in the literature.

n.a.: not analysed. n.r.: not reported. (a): Concentration reported for cows fed dioxin-contaminated grass silage during the dry period and uncontaminated feedstuff after calving for average 8 weeks. (b): Median (min-max) (c): Sum of non-ortho and mono-ortho PCBs with an assigned TEF value. (d): Upper bound (UB) concentrations. (e): Mean (range). (f): Only non-ortho PCBs. (g): Sheep grazed on PCP contaminated sawmill soil.

Sample n Location, Year WHO-TEF Dioxins DL-PCBs Sum Dioxins and DL-PCBs Reference

Sheep liver 5 n.r. 4.92-20.7 n.a. - Schulz et al., 2005 Sheep muscle 5 n.r. WHO-TEF1998 0.47-1.58 n.a -

Cow liver (a) 3 n.r. 2.27±1.3 n.a. - Lamb liver (d) (e) 11 Ireland n.r. WHO-TEF1998

3.43 (0.50-6.76) 0.80 (0.15-1.58) 4.23 (0.75-8.34) FSAI, 2005 Porcine liver (d) 1 Ireland, n.r. 1.44 0.22 1.67 Sheep liver (g) n.r. n.r., 2003

WHO-TEF1998 49 13(f) 61

Lindström et al., 2005 Sheep meat (g) n.r. n.r., 2003 3.2 3.9(f) 7.1 Sheep liver 5 Denmark, n.r.

WHO-TEF1998 6.8-16.7 1.0-3.0 n.r.

Lund et al., 2008 Sheep flank/shank 5 Denmark, n.r. 0.26-1.9 0.18-0.48 n.r. Sheep liver (b) 77 Germany WHO-TEF1998 18.3 (4.8-161.2) 14.66 (2.00-133.3) 33.89 (7.22-203.7) Bruns-Weller et al., 2010 Lamb liver 22 UK, 2005

n.r. 0.24-25 0.12-3.2 n.r.

Rose et al., 2010 Venison liver 10 UK, 2005 13-109 3.7-50 n.r. Lamb liver (d) 19 UK, 2005

WHO-TEF1998 6.97 1.39(c) 8.36

Fernandes et al., 2010 Deer liver (d) 10 UK, 2005 52 16.04(c) 68.2 Sheep liver (b) 43 Germany, 2009

WHO-TEF1998 11.72 (4.48-68.0) 8.56 (3.99-42.7) 20.28 (8.47-110.7)

CVUA-MEL, 2010 Sheep meat (b) 43 Germany, 2009 0.64 (0.24-3.9) 0.99 (0.14-4.44) 1.62 (0.38-8.34) Sheep liver 2 UK, n.r.

WHO-TEF1998

n.r. n.r. 8.21-19.55

Fernandes et al., 2011 Sheep meat 16 UK, n.r. n.r. n.r. 0.51-3.21 Pig liver 12 UK, n.r. n.r. n.r. 8.9-20.65 Pig meat 2 UK, n.r. n.r. n.r. 0.56-2.1


EFSA Journal 2011;9(7):2297 24

5.2. Current occurrence of dioxins and DL-PCBs in liver and meat from sheep and deer

5.2.1. Summary of data collected

EFSA collected and evaluated the analytical results from 524 samples originally reported by eight European countries for levels of dioxins and PCBs in liver and meat from sheep and deer. The data were either submitted directly to EFSA by Member States or sent to the European Commission and then forwarded to EFSA. In the latter case, as the format of the data itself was not compliant with the Standard Sample Description format normally adopted by EFSA, a standardisation was performed. Analytical results identified during the data cleaning steps with incomplete or incorrect description of any of the required variables (e.g. parameter type, food classification, results value or results LOD-LOQ) were amended when possible according to the respective data provider (after check). Finally, 516 samples were included in the analysis as eight samples were reported on a whole weight basis and no information on fat content was given. The final set includes the following sample types:

• 412 samples with both full set of 29 dioxins and DL-PCBs and 6 indicator PCBs • 104 samples with full set of 29 dioxins and DL-PCBs congeners (either individual or reported

as a calculated TEQ) only.

Data included in the dataset were provided by eight European countries, covering the period 2003 to 2010 (Figure 3 and 4). No data were provided for the year 2004. It should be stated that samples reported by a country do not necessarily originate from the respective country. Throughout this opinion the WHO-TEFs1998 are used since for a considerable number of samples only the calculated TEQ values were reported. Thus, a conversion with the WHO-TEF2005 was not possible. While dioxin results are expressed as pg WHO-TEQ/g fat, all NDL-PCB results are expressed as ng/g fat.

Figure 3: Distribution of samples reported across European countries.

278

68

2115 12 10 8

50

4

50

0

50

100

150

200

250

300

350

Germany

UK

France

Ireland

Denmark

Greece

Sweden

Italy

N. sam

ples

Sampling country

PCDD/Fs, DL‐PCBs

PCDD/Fs, DL‐PCBs, NDL‐PCBs


EFSA Journal 2011;9(7):2297 25

132

10 144

336

1514

67

23

0

50

100

150

200

250

300

350

400

450

2003 2005 2006 2007 2008 2009 2010

N. sam

ples

Year of sampling

PCDD/Fs, DL‐PCBs


Figure 4: Distribution of samples reported across sampling years.

5.2.2. Distribution of samples reported for sheep liver, sheep meat and deer liver

The distribution of the 516 samples reported for sheep liver, sheep meat and deer liver is shown in Figure 5. In the current summary the results are given on a fat weight basis.

Figure 5: Distribution of results for dioxins and PCBs as submitted by the eight European countries.

9

257

146

75

29

0 50 100 150 200 250 300 350

DEER_LIVER

SHEEP_LIVER

SHEEP_MEAT

N. samples

Food type


PCDD/Fs, DL‐PCBs


EFSA Journal 2011;9(7):2297 26

5.2.3. Contribution of dioxins and DL-PCBs to total TEQ in sheep liver and meat

To study the importance of the different compound classes, the mean contribution of PCDDs, PCDFs, non-ortho PCBs and mono-ortho PCBs to the total-WHO-TEQ1998 levels were calculated for sheep liver and sheep meat (Figure 6 and 7). As can be seen, when comparing the relative contribution of dioxins and DL-PCBs to total TEQ in sheep liver and meat respectively, there is a significant shift. The predominant compound class in liver is represented by PCDFs (49.7 %, Figure 6) while in meat non-ortho PCBs are the major contributors (43 %, Figure 7). The contribution of the mono-ortho PCBs are quite low and would be even lower if the WHO-TEF2005 were applied.

Figure 6: Mean contribution (%) of PCDDs, PCDFs, non-ortho PCBs and mono-ortho PCBs to total TEQ in sheep liver.

13.0

49.7

35.3

2.0

PCDDs

PCDFs

Non‐ortho‐PCBs

Mono‐ortho‐PCBs


EFSA Journal 2011;9(7):2297 27

Figure 7: Mean contribution (%) of PCDDs, PCDFs, non-ortho PCBs and mono-ortho PCBs to total-TEQ in sheep meat.

A similar approach has been used for investigating the average individual contribution of each of the 29 dioxin and DL-PCB congeners to total TEQ in both liver and meat samples from sheep. In this case, the contribution (% of total TEQ) is related to the subset of samples for which the information about the levels in the two different tissues was given (148 samples). What is described above for the different profiles of dioxins and DL-PCBs in sheep meat and liver is represented at a congener level in Figure 8, 9 and 10, strengthened by the fact that the samples here are taken from the same animals, therefore exposed to the same source of dioxins and DL-PCBs. It can be seen that in almost all cases the individual PCDF congeners considerably exceed the relative contribution to total TEQ in liver compared to meat. This is specially true for 2,3,4,7,8-PeCDF. In contrast, the relative contribution of the individual PCDD congeners is higher in meat that in liver. The major PCDD contributors to total TEQ in meat are 1,2,3,7,8-PeCDD and TCDD. Figure 10 shows PCB-126 as the major contributor to the total TEQ value in both sheep liver and meat not only amongst the 12 DL-PCBs but also amongst all 29 dioxin and DL-PCB congeners. Noteworthy is also the higher average impact of both PCB-156 and -118 on total TEQ in sheep meat than in the corresponding liver samples. Overall, the relative contribution of DL-PCBs to total TEQ is generally considerably higher in meat samples than in liver.

The same analysis was performed for NDL-PCBs for samples where the information about the levels in the two different tissues was given (132 samples). Figure 11 shows that there are two congeners (PCB-138 and -153) contributing to the sum of the six NDL-PCBs more in the liver than in meat, but the overall profile of the NDL-PCBs is very similar.

22.1

17.043.0

18.0

PCDDs

PCDFs

Non‐ortho‐PCBs

Mono‐ortho‐PCBs


EFSA Journal 2011;9(7):2297 28

Figure 8: Relative contribution of the 10 toxic PCDF congeners to total TEQ.

Figure 9: Relative contribution of the 7 toxic PCDD congeners to total TEQ.

0

5

10

15

20

25

30

35

40

2,3,7,8‐TCDF

1,2,3,7,8‐Pe

CDF

2,3,4,7,8‐Pe

CDF

1,2,3,4,7,8‐HxCDF

1,2,3,6,7,8‐HxCDF

1,2,3,7,8,9‐HxCDF

2,3,4,6,7,8‐HxCDF

1,2,3,4,6,7,8‐HpC

DF

1,2,3,4,7,8,9‐HpC

DF

OCD

F

% of total TEQ

liver

meat

0

2

4

6

8

10

12

14

2,3,7,8‐TCDD

1,2,3,7,8‐Pe

CDD

1,2,3,4,7,8‐HxCDD

1,2,3,6,7,8‐HxCDD

1,2,3,7,8,9‐HxCDD

1,2,3,4,6,7,8‐HpC

DD

OCD

D% of total TEQ

liver

meat


EFSA Journal 2011;9(7):2297 29

Figure 10: Relative contribution of the 12 DL-PCBs to total TEQ.

Figure 11: Relative contribution of individual NDL-PCBs to the sum of the six indicator PCBs.

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

PCB‐28 PCB‐52 PCB‐101 PCB‐138 PCB‐153 PCB‐180

% of sum

of 6

NDL‐PC

Bs

LIVER

MEAT

0

5

10

15

20

25

30

35

40

45

PCB‐77

PCB‐81

PCB‐105

PCB‐114

PCB‐118

PCB‐123

PCB‐126

PCB‐156

PCB‐157

PCB‐167

PCB‐169

PCB‐189

% of total TEQ

liver

meat


EFSA Journal 2011;9(7):2297 30

5.2.4. Occurrence data reported for dioxins and PCBs in sheep and deer

Tables 3 and 4 provide information on the statistical description for dioxins, DL-PCBs and NDL-PCBs in sheep liver, sheep meat and deer liver.

Due to the low number of samples with results below LOD and LOQ, the statistical data in both tables show almost no differences between lower bound (LB) and upper bound (UB) values.

For sheep liver, the mean UB concentrations for dioxins and the sum of dioxins and DL-PCBs amount to 14.9 pg WHO-TEQ/g fat (range: 0.27-116.3) and 26.1 pg WHO-TEQ/g fat (range: 0.47-279.1), respectively. The corresponding levels in sheep meat were considerably lower and calculated as 0.70 pg WHO-TEQ/g fat (range: 0.08-5.12) and 2.0 pg WHO-TEQ/g fat (range: 0.16-11.9), respectively. For deer liver the respective mean UB concentration are 47.0 pg WHO-TEQ/g fat (range: 12.9-109.4) and 62.4 pg WHO-TEQ/g fat (range: 16.6-159.3).

Regarding NDL-PCBs, the mean UB concentrations for the sum of the six NDL-PCBs (PCB-28, -52, -101, -138, -153 and -180) in sheep liver and sheep meat is 26.8 ng/g fat (range: 0.41-350.5) and 13.1 ng/g fat (range: 0.51-162.2), respectively. For deer liver the mean occurrence concentration is 29.1 ng/g fat (range: 7.7-76.9).


EFSA Journal 2011;9(7):2297 31

Table 3: Statistical description of concentrations for dioxins and DL-PCBs calculated on 516 samples of sheep liver, sheep meat and deer liver. Occurrence levels (mean, min, max, P5, P50, P90, P95, P99 concentration) are reported on fat weight (pg WHO-TEQ1998/g fat) for dioxins, DL-PCBs, and the sum of the two with both the lower bound (LB) and upper bound (UB) estimates.

n: number of samples; DL*PCBs: dioxin-like PCBs.

Dioxins DL-PCBs Sum of Dioxins + DL-PCBs

Food type n Estimation MIN P5 P50 MEAN MAX P90 P95 P99 MIN P5 P50 MEAN MAX P90 P95 P99 MIN P5 P50 MEAN MAX P90 P95 P99

Sheep liver 332 LB 0.27 0.94 7.6 14.84 116.3 36.14 55.53 92.55 0.02 0.18 5.8 11.21 198.24 21.93 41.64 110.37 0.43 1.33 14.26 26.05 279.14 61.12 98.06 167.5

332 UB 0.27 0.98 7.8 14.9 116.3 36.14 55.53 92.55 0.1 0.18 5.8 11.22 198.24 21.93 41.64 110.37 0.47 1.36 14.26 26.12 279.19 61.14 98.06 167.5

Sheep meat 175 LB 0 0.1 0.42 0.67 5.09 1.46 2.31 3.89 0.08 0.15 0.92 1.29 11.29 2.53 3.33 9.67 0.08 0.25 1.41 1.96 11.87 3.52 5.51 10.61

175 UB 0.08 0.15 0.45 0.70 5.12 1.46 2.31 3.9 0.08 0.18 0.92 1.29 11.29 2.53 3.33 9.67 0.16 0.32 1.42 2.00 11.87 3.54 5.51 10.62

Deer liver 9 LB 12.87 12.87 31.49 47.01 109.4 109.4 109.4 109.4 3.73 3.73 13.98 15.36 49.96 49.96 49.96 49.96 16.6 16.6 47.87 62.37 159.37 159.37 159.37 159.37

9 UB 12.87 12.87 31.49 47.01 109.4 109.4 109.4 109.4 3.75 3.75 14.02 15.42 49.98 49.98 49.98 49.98 16.63 16.63 47.98 62.42 159.38 159.38 159.38 159.38


EFSA Journal 2011;9(7):2297 32

Table 4: Statistical description of concentrations for the sum of the six NDL-PCB congeners calculated on 412 samples of sheep liver, sheep meat and deer liver. Occurrence levels (mean, min, max, P5, P50, P90, P95, P99 concentration) are reported on fat weight basis (ng/g fat).

Food type n TYPE MIN P5 P50 MEAN MAX P90 P95 P99

Sheep liver 257 LB 0.00 0.50 13.88 25.66 350.45 51.58 92.00 217.50

257 UB 0.41 1.38 14.55 26.78 350.45 52.69 93.50 230.00

Sheep meat 146 LB 0.00 0.69 7.25 11.84 162.15 22.00 35.14 121.89

146 UB 0.51 1.09 8.46 13.14 162.15 24.50 37.00 121.89

Deer liver

9 LB 7.65 7.65 24.08 28.80 76.87 76.87 76.87 76.87

9 UB 7.67 7.67 24.33 29.08 76.87 76.87 76.87 76.87 n: number of samples; NDL-PCBs: non dioxin-like PCBs.


EFSA Journal 2011;9(7):2297 33

6. Food consumption

In 2010 EFSA established the “Comprehensive European Food Consumption Database” (Comprehensive Database). This is built on existing information for adults and children at a detailed level, where all food consumption data were codified according to the FoodEx classification system (developed by the Dietary & Chemical Monitoring (former DATEX) Unit in 2009). It is important to stress that the database still includes methodological differences making these data not fully suitable for country-to-country comparisons.

Consumption data on sheep liver are scarce in Europe. Data extracted from the EFSA’s Comprehensive Database indicates that only a very small fraction of the European population consumed such products within the length of the survey period. Only 0.2 % of the individuals included in the Comprehensive Database indicated that they had consumed sheep liver at least once during the period of recording. At a country level, Ireland had the highest percentage of consumers (3 %). The methodology used to collect the food consumption data differ between surveys affecting comparability. In three cases (France, Ireland and UK) the surveys reported data taken on a 7-day food record basis, while the remaining three are either reported as 24 hour recall (Bulgaria and Germany) or as a 3-day food record (Italy), with consumption data reported either “as raw”, “as consumed” or “as cooked”. Consequently, calculation of average consumption of sheep liver expressed on a weekly basis for the very small number of sheep liver consumers in the total adult population would be affected by a high degree of uncertainty.

A viable alternative is to use the portion size distribution assuming that an arbitrary frequency of one eating occasion in a week can be taken as a conservative estimate.

In Table 5 average portion sizes for each country are reported for all liver consumers and for sheep liver consumers. The results point to an average sheep liver portion size14 of 106.3 g (or 1.5 g/kg b.w.) across six countries with the largest portion size in the UK of 141.0 g (or 1.9 g/kg b.w.). Comparing the portion sizes for the overall food class of liver with sheep liver, there are many more consumers for the former, but the average portion size of sheep liver is slightly larger than the average of 86.8 g for all liver. This might indicate that it is more common to eat sheep liver as such, while general liver captures its use as an ingredient in a dish. No average portion size for all liver exceeds the average sheep liver consumption in the UK.

As a conservative approach, the average sheep liver portion size recorded in the UK will be used for the exposure assessment with the assumption that it represents a weekly amount. It should be noted that although 10 individuals out of the 117 adults consumed sheep liver twice in the survey period as recorded in the 7-day record for individuals in the UK, the assumption is that this would not happen on a regular basis every week.

14 Portion size on body weight basis was calculated dividing the individual consumption values for each eating occasion by individual body weight values from the Comprehensive Database, then averaged at Country level.


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Table 5: Portion size data available for all liver and sheep liver consumption for adults across European countries: number of eating occasions (N), average portion size (g) and average portion size per kg body weight (b.w.) are given.

All liver Sheep liver

Country N Portion size (g) Portion size (g/kg b.w.) N Portion size (g) Portion size (g/kg b.w.)(a)

Austria 21 47.7 0.7 Belgium 14 95.1 1.3 Bulgaria 47 73.7 1.1 26 36.5 0.5 Czech Republic 91 45.4 0.6 Denmark 300 62.8 0.8 Estonia 38 132.5 1.9 Finland 85 48.0 0.6 France 227 99.6 1.5 14 101.1 1.5 Germany 109 138.4 1.8 4 132.8 1.6 Hungary 239 70.2 1.0 Ireland 103 55.5 0.8 38 114.2 1.6 Italy 50 112.3 1.6 2 100.0 1.5 Latvia 50 113.0 1.5 Netherlands 7 71.5 1.1 Slovakia 31 105.9 1.5 Slovenia 4 87.0 0.9 Spain 23 98.0 1.5 Sweden 1 125.0 2.3 UK 78 127.9 1.7 43 141.0 1.9

Total 1,561 86.8 1.2 127(b) 106.3 1.5 (a): Portion size on body weight basis was calculated dividing the individual consumption values for each eating occasion by individual body weight values from the Comprehensive Database,

then averaged at Country level; (b): on a total of 117 individuals, 107 reported 1 eating occasion for sheep liver in a week, while the remaining 10 reported a second eating occasion in the same period, for a total amount of 127 eating occasions.


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Consumption data for children

Due to the scarce availability of consumption data related to ovine liver consumption for children, an estimation of the consumption was performed using any liver consumption as a “sheep liver proxy” (Table 6). The data extracted from the Comprehensive Database refer to a total of 303 individuals covering the age range from 0 to 18 years old with the following frequencies: 13 infants (0-1 years old), 21 toddlers (1-3 years old), 172 other children (3-10 years old) and 97 adolescents (10-18 years old).

The results show an average weekly amount of 1.5 g/kg b.w. This might be a slight underestimation of the actual sheep liver consumption based on adult consumption patterns for general liver consumption. However, the highest average weekly amount of 2.8 g/kg b.w. from Bulgaria was selected as a conservative estimate for the exposure assessment to compensate for this potential bias.

Table 6: Portion size data available for all liver consumption for children across European countries: number of eating occasions (N), average portion size (g) and average portion size per kg body weight (b.w.) are given.

All liver

Country N Portion size (g) Portion size (g/kg b.w.)

Belgium 1 50 2.4 Bulgaria 41 43.4 2.8 Cyprus 4 111.2 2.1 Czech Republic 56 26.4 0.9

Denmark 24 34.6 1.3 Finland 80 8.8 0.5 France 56 79.4 2.0 Germany 4 12.5 0.8 Italy 9 84.3 2.7 Latvia 14 93.2 2.1 Spain 14 95.3 2.1

Total 303 43.5 1.5

7. Human exposure assessment

7.1. Human exposure to dioxins and PCBs via consumption of sheep liver

With less than 3 % of the European population consuming sheep liver during the length of the survey period, the CONTAM Panel decided to use “consumers only” of this food for the exposure assessment for adults. The assessment only includes the consumption of sheep liver as such, although a small amount might also be consumed through processed foods. This is considered small in relation to the amount identified here. On the other hand, sheep liver might also be included as a component in composite dishes not identified here. However, such inclusion would reduce the weekly amount for consumers only. It can thus be concluded that the amount selected in the consumption chapter reflects a conservative estimate.


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Based on the occurrence data reported by the eight European countries for dioxins and PCBs in sheep liver and the consumption data for adults (consumers only) from the UK and children from Bulgaria, the human exposure to dioxins and PCBs through consumption of sheep liver was calculated.

Dioxins and DL-PCBs

Table 7 shows the results of the dietary exposure calculation for adults (consumers only) and children with respect to dioxins and DL-PCBs through consumption of sheep liver. Two different concentrations for the sum of dioxins and DL-PCBs in sheep liver were used in this evaluation. These are the mean levels calculated from the results reported by the eight submitting European countries and the maximum level for the sum of dioxins and DL-PCBs laid down in Regulation (EC) No 1881/2006. The dietary intake was estimated using UB concentrations as UB and LB were coinciding when rounded to the first decimal. The mean fat content of 5.1 % of the sheep liver samples reported by the European countries was applied as a common value for the assessment.

Table 7: Dietary exposure (pg WHO-TEQ/kg b.w. per week) to the sum of dioxins and DL-PCBs through sheep liver consumption.

Average consumption (sheep liver)

Concentration in sheep liver

Average dietary Exposure through consumption of

sheep liver(e) g/kg b.w. per week pg WHO-TEQ/g fat pg WHO-TEQ/kg b.w. per week

Adults 1.9 (a) 26.1 (c) 2.53

12.0 (d) 1.16

Children 2.8 (b) 26.1 (c) 3.73

12.0 (d) 1.71 (a): Data extracted from EFSA’s Comprehensive European Food Consumption Database, sheep liver consumed by adults

(consumers only) (the portion size from the UK survey is taken as a conservative approach, see Table 5). (b): Data extracted from EFSA’s Comprehensive European Food Consumption Database, all liver consumed by children.

(the portion size from the Bulgarian survey is taken as a conservative approach, see Table 6). (c): Mean value for sum of dioxins and DL-PCBs in sheep liver samples submitted by European countries (see Table 3). (d): Maximum level for sum of dioxins and DL-PCBs laid down in Regulation (EC) No 1881/2006. (e): Fat content of sheep liver 5.1 %.

As can be seen from Table 7, the average weekly exposure in adults (consumers only) to dioxins and DL-PCBs based on the mean concentration in sheep liver calculated from the occurrence data submitted by the eight European countries is 2.53 pg WHO-TEQ/kg b.w. If liver were consumed at the maximum level laid down in Regulation (EC) No 1881/2006, the weekly exposure for adults (consumers only) is 1.16 pg WHO-TEQ/kg b.w. For children the average weekly exposures were calculated to be 3.73 pg WHO-TEQ/kg b.w. and 1.71 pg WHO-TEQ/kg b.w., respectively.

NDL-PCBs

A similar evaluation was performed for the dietary exposure to NDL-PCBs (Table 8). The fat content of sheep liver is the same as for the exposure assessment concerning dioxins and DL-PCBs. The exposure estimations were performed with the mean concentration for the sum of the six indicator NDL-PCBs (PCB-28, -52, -101, -138, -153 and -180) calculated from the occurrence data submitted by the eight European countries.


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Table 8: Dietary exposure (ng/kg b.w. per week) to NDL-PCBs through sheep liver consumption.

Average consumption (sheep liver)

Concentration in sheep liver

Average dietary Exposure through

consumption of sheep liver(d)

g/kg b.w. per week ng/g fat ng/kg b.w. per week

Adults 1.9 (a) 26.8(c) 2.60

Children 2.8 (b) 26.8(c) 3.83

(a): Data extracted from EFSA’s Comprehensive European Food Consumption Database, sheep liver consumed by adults (consumers only) (the portion size from the UK survey is taken as a conservative approach, see Table 5).

(b): Data extracted from EFSA’s Comprehensive European Food Consumption Database, all liver consumed by children (the portion size from the Bulgarian survey is taken as a conservative approach, see Table 6).

(c): Mean value for sum of NDL-PCBs in sheep liver samples submitted by European countries (see Table 4). (d): Fat content of sheep liver 5.1 %. Considering the mean level for the sum of the six indicator NDL-PCBs, the average weekly dietary exposure for adults (consumers only) is 2.60 ng/kg b.w. For children, the average weekly exposure to NDL-PCBs based on the consumption value derived by the “generic liver” data is 3.83 ng/kg b.w.

7.2. Previously reported literature data on dietary intake of dioxins and DL-PCBs

A number of studies have reported the dietary intake of dioxins and DL-PCBs in several European countries (Table 9). Comparison between studies should be done carefully due to the different methodologies used (sampling methods and food consumption data), year of sampling, food categories covered, choice of WHO-TEF model, number of DL- and NDL-PCBs measured and approach to express the concentrations (lower (LB), medium (MB) or upper bound (UB)).

In Belgium, Windal et al. (2010) carried out in 2008 a total diet study (TDS) where dioxins and DL-PCBs were analysed in 43 composite samples mainly divided into three groups, i.e. meat and meat products, fish and fish products and dairy and dairy products. The average estimated intake for the sum of dioxins and DL-PCBs in the adult Belgian population was 0.83 pg WHO-TEQ1998/kg b.w. per day (UB). For PCBs, Voorspoels et al. (2008) estimated the PCB intake based on a food market-basket study for the general Belgian population in 2005. A total of 23 PCB congeners, including the DL-PCB congeners -105, -118 and -156 were analysed. The average intake was estimated at 535 ng per day (UB).

In Germany, Fromme et al. (2009) estimated the dietary intake of DL-PCBs for the adult population living in Munich or nearby in the southern region of Germany. Duplicate diet samples were collected for seven consecutive days in 2005. The estimated intake was 0.19 pg WHO-TEQ1998/kg b.w. per day (MB).

In Spain, Fernández et al. (2004) estimated the dietary intake of dioxins and DL-PCBs based on the analysis of 258 samples randomly acquired from Spanish supermarkets in 2000-03. The estimated intake (± standard deviation) was 3.22 ± 0.75 pg WHO-TEQ1998/kg b.w. per day (UB). In a later study, Llobet et al. (2008) estimated the dietary exposure to dioxins and PCBs of the population of Catalonia. Samples were randomly acquired in supermarkets in this Spanish region in 2006. The dietary intake was estimated at 1.12 pg WHO-TEQ2005/kg b.w. per day (MB). In 2011, Marin et al. (2011) estimated the average daily intake of dioxins and DL-PCBs to be 2.86 pg WHO-TEQ1998/kg b.w. per day (UB) from food marketed in the Spanish region of Valencia based on the analysis of 150 randomly collected individual food samples.


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In Finland, Kiviranta et al. (2004) measured the concentrations of dioxins and DL-PCBs in ten market baskets, covering almost 4,000 individual food samples, and also in the total diet basket. The estimated average intake was 1.53 pg WHO-TEQ1998/kg b.w. per day (UB).

In France, Tard et al. (2007) estimated the dietary intake based on the analysis of almost 800 individual food samples from national monitoring programs carried out during 2001-04. The mean intake of dioxins and DL-PCBs was estimated to be 1.8 pg WHO-TEQ1998/kg b.w. per day (LB) and 2.8 pg WHO-TEQ1998/kg b.w. per day for adults and children, respectively.

In Italy, the dietary intake of dioxins and DL-PCBs (Fattore et al., 2006) and NDL-PCBs (Fattore et al., 2008) were estimated based on mean occurrence data obtained from an original database of the European Commission and on domestic food consumption data collected in a 1994-1996 survey covering nearly 2,000 subjects. Intakes were first estimated at the individual level and then analyzed altogether. The mean dietary intake estimate for dioxins and DL-PCBs was 2.28 pg WHO-TEQ1998/kg b.w. per day (UB). For NDL-PCBs (six indicator PCBs, i.e. PCB-28 -52, -101, -138, -153 and -180) the estimated mean intake was 11.0 ng/kg b.w. per day.

In the Netherlands, De Mul et al. (2008) estimated the dietary exposure to dioxins and DL-PCBs using representative occurrence data from food composite samples. The median dietary intake was estimated at 0.9 pg WHO-TEQ2005/kg b.w. per day (MB).

In Norway, Kvalem et al. (2009) estimated the dietary intake of dioxins, DL-PCBs and NDL-PCBs (i.e. six indicator PCBs) based on food frequency questionnaires and occurrence data in Norwegian foods from 2000-06. The mean dietary intake for dioxins and DL-PCBs was estimated to be 0.78 pg WHO-TEQ2005/kg b.w. per day, while for NDL-PCBs the intake was 4.26 ng/kg b.w. per day, in both cases for the representative consumer.

In Sweden, Tornkvist et al. (2011) estimated the dietary intake in a Swedish market basket from 2005. The estimated dietary intake was 0.86 pg WHO-TEQ1998/kg b.w. per day (UB) and 5.3 ng/kg b.w. per day (UB) for the sum of dioxins and DL-PCBs and NDL-PCBs, respectively.

In the UK, the Food Standards Agency (FSA) analysed food samples from the 2001 UK total diet study for the levels of dioxins and DL-PCBs (UK FSA, 2003). The estimated average dietary intake of adults was 0.9 pg WHO-TEQ1998/kg b.w. per day (UB).

Whilst these published studies on dioxins and DL-PCBs intake are e.g. based on different years(s) of food collection, different selection of food items and different calculation methods, the dietary intake of the sum of dioxins and DL-PCBs for the different European countries ranges between 0.5 and 3.2 pg WHO-TEQ1998/kg b.w. per day (Table 9). For NDL-PCBs, the dietary intake ranges from 4.3 to 11.0 ng/kg b.w. per day. However, in interpreting this range it has to be considered that the number of PCB congeners that were analysed differs in the various surveys.

Based on a number of studies dealing with dietary exposure assessment of adults and children to dioxins and DL-PCBs in the general population of European countries (see Appendix A), it may be concluded that children aged 0-14 years (breastfeeding excluded) experience a greater exposure than adults by an average factor of 2, likely due to a higher food consumption relative to body weight. The ratio of exposure in children relative to adults decreases from 3.0 to 1.2 as children get older. A similar evaluation for NDL-PCBs could not be carried out for lack of sufficient data.


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Table 9: Dietary exposure to dioxins and DL-PCBs reported in the literature for different European countries. n.a.: not analysed. n.r.: not reported. LB: lower bound (nd=0), MB: medium bound (nd=1/2 LOD), UB: upper bound (nd=LOD or LOQ).

(a): 70 kg b.w.; (b): 76 kg b.w.; (c): PCB-81 not included in the analysis; (d): Only DL-PCBs; (e): Median dietary intake for the sum of six indicator PCBs; (f): Assuming a 60 kg b.w. (Original data provided in ng per day.); (g): Sum of PCB-28, -52, -74, -95, -99, -101, -105, -110, -118, -128, -138/163, -149, -153, -156, -170, -180, -183, -187, -194, -196/203 and -199; (h): Assuming a 73.7 kg b.w. (Original data provided in pg TEQ per day.); (i): Assuming a 68.5 kg b.w.

Country Year TEFs Estimation Dietary intake

Reference PCDD/Fs + DL-PCBs (pg WHO-TEQ/kg b.w. per day)

NDL-PCBs (ng/kg b.w. per day)

BE 2008 WHO-TEF1998 LB MB UB

0.61 0.72 0.83

n.a. Windal et al., 2010

BE 2005 - LB MB UB

n.a. 6.6(f)(g) 7.8(f)(g) 9(f)(g)

Voorspoels et al., 2008

DE - WHO-TEF1998 MB 0.19(d) Fromme et al., 2009

ES 2003 WHO-TEF1998 LB UB

2.78 ± 0.70 3.22 ± 0.75 n.a. Fernández et al., 2004(a)

ES (Catalonia) 2006 WHO-TEF2005 MB 1.12 n.a. Llobet et al., 2008

ES (Valencia) 2006-08 WHO-TEF1998 UB LB

2.86 2.09 n.r. Marin et al., 2011(i)

FI 1997-99 WHO-TEF1998 LB UB

1.50 1.53 n.a. Kiviranta et al., 2004(b)(c)

FR 2001-04 WHO-TEF1998 LB 1.8 n.a. Tard et al., 2007

IT - WHO-TEF1998 UB 2.28 n.a. Fattore et al., 2006

IT - - UB n.a. 11.0(e) Fattore et al., 2008

NL 2004 WHO-TEF2005 LB MB

0.8 0.9 n.a. De Mul et al., 2008

NO 2003 WHO-TEF2005 LB 0.78 4.26(e) Kvalem et al., 2009

SE 2005 WHO-TEF1998 UB LB

0.86(h) 0.51

5.3(h) 4.5 Törnkvist et al., 2011

UK 2001 WHO-TEF1998 UB 0.9 n.a. UK FSA, 2003


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8. Hazard identification and characterization

8.1. Toxicokinetics in ruminants

8.1.1. Absorption

In ruminants as in other mammalian species the absorption of dioxins and PCBs depends on their physicochemical properties such as lipophilicity, and also on feed composition, especially lipid content (EFSA, 2005). Higher chlorinated PCBs (8-10 chlorines), are absorbed less easily than the lower chlorinated congeners (Thomas et al., 1999). No quantitative data specifically describing the absorption of dioxins and DL-PCBs in sheep following oral exposure were identified. However, the presence of dioxins or PCBs in tissues or milk of animals exposed through diet (Hansen et al., 1977; Jan et al., 1999; Schulz et al., 2005; Berg et al., 2010; Bruns-Weller et al., 2010) gives indirect evidence that gastro-intestinal absorption occurred to a significant extent. The toxicokinetics of [14C]-TCDD following a single oral administration (approximately 0.5 mg per animal) was investigated in lactating goats by Grova et al. (2002). Assuming that radioactivity excreted in faeces corresponded to unabsorbed TCDD, apparent absorption was estimated to be about 80 % in this small ruminant species. In contrast, faecal elimination accounted for 81.6 % of the administered dose (1.2 mg/kg b.w.) in a similar experiment performed in bull calf (Hakk et al., 2001).

8.1.2. Distribution

The dioxin concentration in muscle, liver, and adipose tissue of sheep grazing on flooding areas was reported by Schulz et al. (2005). At the end of the experiment (16 weeks), levels in muscle, liver and adipose tissue were 0.6, 16.2 and 0.83 pg WHO-TEQ/g fat, respectively.

The tissue distribution of PCB congeners in sheep fed a diet contaminated with the technical PCB mixtures Aroclor 1242 or 1254 at 20 mg/kg for 105 days was investigated by Hansen et al. (1977). In all tissues examined, total PCB concentrations from feeding Aroclor 1254 were higher than those from feeding Aroclor 1242. The tissue concentrations (total PCBs), on a fresh weight basis, were as follows: adipose tissue >> liver > muscle > kidney > blood.

The distribution pattern of PCBs was determined in 50-55 kg lactating sheep two months after a single intramuscular injection of the following mixture dissolved in olive oil: PCB-54: 1.3 mg, PCB-80: 0.5 mg, PCB-155: 1.4 mg and PCB-169: 0.7 mg (Jan et al., 1999). Two months after administration, the levels (ng/g fat) of PCB-54 and -80 were as follows: blood > liver > adipose tissue, whereas for PCB-155 and -169, levels were in the following order: liver > adipose tissue > blood.

8.1.3. Metabolism

8.1.3.1. Biotransformation pathways

No study was identified regarding the biotransformation of dioxins in sheep. Recently, Berg et al. (2010) identified 4-OH-PCB-107 and 4-OH-PCB-146 in plasma of ewes dosed by gavage PCB-118 and -153, respectively, suggesting a para-oxidation of these congeners without subsequent dechlorination. These data show that hydroxylation of PCBs occur in sheep, as previously described in the cow (Safe et al., 1975).


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8.1.3.2. Comparative expression and activity of CYP1 enzymes in sheep and other ruminant species

In mammalian species, CYP1 enzymes are involved in the kinetics of polycyclic aromatic compounds including dioxins and dioxin-like related compounds (Hakk et al., 2009). CYP1 is a relatively small and well conserved family comprising two subfamilies, 1A and 1B. The former, in turn, consists of two members, CYP1A1 and CYP1A2. CYP1A1 (Olson et al., 1994) and, to a lesser extent, CYP1B1 (Santostefano et al., 1997) are believed to be involved in the oxidative biotransformation of TCDD and related compounds generating -OH derivatives which can be metabolised by UDP glucuronosyltransferase (UGT) to glucuronides that are excreted via the biliary and urinary routes. In rodents and in humans only CYP1A2 is constitutively expressed to a notable extent in the liver, while CYP1A1 and CYP1B1 are predominantly expressed in extra-hepatic tissues (Ioannides, 2006).

TCDD and dioxin-like compounds are able to bind with different affinities to the Ah receptors (AhR) and hence to increase the transcription of a number of genes (the so called “Ah gene battery”) encompassing, among others, all the CYP1 family members and selected UGTs, i.e. the same enzymes involved in the biotransformation of dioxin-like chemicals (Mandal, 2005). A concentration-related increase in CYP1A1 and CYP1B1 transcripts has been recently reported in primary cultures of calf hepatocytes incubated with TCDD or a number of polybrominated dioxins and furans (Guruge et al., 2009).

In addition to the AhR, some dioxins bind to an inducible hepatic protein, which has been identified as the CYP1A2 (Diliberto et al., 1997), resulting in a dose-dependent liver sequestration of such chemicals which prevents any further CYP-mediated metabolism (Staskal et al., 2005). Recent experiments performed with both CYP1A2-knockout mice and parental (wild type) mice strongly suggest that CYP1A2 does not actively participate in the metabolism of TCDD and possibly other dioxins but rather governs their toxicokinetics by making them unavailable for hepatic CYP1A1 through sequestration and attenuating extrahepatic disposition (Hakk et al., 2009). However, the role of CYP1A2 in the sequestration of dioxin and dioxin-like compounds in the liver of ruminants has not been investigated.

There is scant information concerning the basal expression and the inducibility of the CYP1 family in ruminants. Cattle liver microsomes are able to O-dealkylate 7-ethoxyresorufin and 7-methoxyresorufin, two fluorescent substrates that in the rat are reported to be relatively specific for CYP1A1 and CYP1A2, respectively, and 7-ethoxyresorufin was found a reliable marker for hepatic CYP1A-mediated activity in the bovine species (Sivapathasundaram et al., 2001; Pegolo et al., 2010). The ability displayed by bovine preparations in dealkylating 7-ethoxyresorufin (EROD) was several fold greater than that measured in the rat or in other food producing species (Nebbia et al., 2003) including sheep (Watkins and Klaassen, 1986) and goats (Szotáková et al., 2004). Early comparative metabolic studies on the rate of biotransformation of model substrates in liver microsomes show that there may be important differences in metabolic rates of alkoxyresorufins between sheep and cattle. Smith et al. (1984) found that hepatic EROD in cattle (n=5) was 14 fold higher than in sheep (n=5). This difference was confirmed in a study carried out in cultured hepatocytes isolated from sheep (n=3) and cattle (n=4) showing a 3-fold greater EROD activity in cattle cells (Van t' Klooster et al., 1993). Whereas CYP1A1 is the only enzyme responsible for EROD in bovine liver (Sivapathasundaram et al., 2001; Pegolo et al., 2010), this specificity has not been established in sheep. As found in rats, some other CYPs could catalyse the O-deethylation of ethoxyresorufin (Nakajima et al., 1990), but their activity is limited compared to CYP1A1. These data suggest that in sheep the CYP isoform(s) displaying a high catalytic activity towards ethoxyresorufin, a substrate known to be biotransformed by the CYP P450 isoforms involved in the oxidative metabolism of dioxins and DL-PCBs, are likely to be less expressed and/or active than in cattle.

In contrast, no significant differences in the 7-methoxyresorufin O-demethylation (MROD) rate could be measured in bovine vs. ovine or goat liver microsomes (Szotáková et al., 2004). Machala and coworkers (2003) studied CYP expression in liver preparations from different breeds of deer,


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including red deer (Cervus elaphus). All breeds displayed EROD activity and, to a lesser extent, MROD activity; as for cattle, EROD activity was several fold higher than that detected in rat preparations.

No clear conclusions can be drawn from the immunochemical studies aimed at detecting CYP proteins in ruminant hepatic microsomes using antibodies raised to rat or human CYP counterparts. In this respect, one single band was detected when probing cattle preparations with a rat anti-CYP1A1/2 antibodies (Nebbia et al., 2003). In other studies (Sivapathasundaram et al., 2001; Grasso et al., 2005), two bands (probably related to CYP 1A1 and 1A2) were instead revealed using an anti-rat CYP1A1 antibody whilst the bovine protein(s) did not react with anti CYP1A2 antibodies raised in humans (Sivapathasundaram et al., 2001). Likewise, in deer microsomes, no strong immunoreaction was detected with human CYP1A2 antibodies, but at least one immunoreacting protein was apparent with rat anti-CYP1A.

Scant information is similarly available on the inducibility of CYP1 family in ruminants. No detectable bands could be observed in female goat, sheep, or cattle hepatocytes probed with anti-rat CYP1A1/2 but one immunoreacting protein was clearly visible upon the incubation with β-naphthoflavone (β-NAF), a CYP1A prototype inducer; as detected by eye, the band intensity was in the order sheep > goat > cattle (van’t Klooster et al., 1993). Despite that, EROD activity was higher in cattle than in sheep uninduced hepatocytes and it increased to a similar extent (40-60 fold) in both species as the result of β-NAF treatment.

Only quite recently studies performed at the molecular level have demonstrated for the first time in primary cultures of calf hepatocytes the expression and the strong inducibility by TCDD (1,000 fold with 5nM) and polybrominated dioxins of both CYP1A1 and CYP1B1 genes (Guruge et al., 2009). In addition, Bos taurus CYP1A2 mRNA has been cloned and sequenced.15 By contrast, the only information concerning the CYP1 family in other ruminant species is related to the ovine CYP1A1 mRNA sequence (Hazinski et al., 1995).

Finally, an in vivo study conducted in Suffolk ewes and Hereford heifers (Danielson and Golsteyn, 1996) showed that the hepatic capacity to clear the CYP1A substrate caffeine from the systemic circulation is similar between the two species. However, the preferred routes of biotransformation differ, paraxanthin (mainly due to CYP1A2) being the major plasma metabolite in cattle, whereas theophylline was predominant in sheep, suggesting that sheep may express CYP1A2 to a lesser extent than cattle.

In mammalian species NDL-PCBs are mainly metabolized by the CYP enzyme system to form hydroxylated metabolites and arene oxides including CYP2B in rodents (hydroxylation) and CYP2B, 2C or 3A in rodents and humans (arene oxide formation) (EFSA, 2005). Although the corresponding enzymes have not been identified in ruminants, the presence of hydroxylated metabolites has been reported in plasma of sheep dosed with either a DL-PCB (PCB-118) or a NDL-PCB (PCB-153) (Berg et al., 2010). As for dioxin-like compounds, the expression of CYPs involved in NDL-PCBs biotransformation may be increased under conditions of prolonged exposure with mechanisms similar to those of phenobarbitone and thus independent from the binding to AhR (Connor et al., 1995). Under field conditions, however, living organisms are exposed to mixtures of NDL-PCBs and DL-compounds, so that a wide spectrum of CYPs and other xenobiotic metabolizing enzymes may be induced. Accordingly, a sharp increase in the rate of the in vitro metabolism of typical CYP1A substrates along with that of CYP2B, CYP3A, and CYP2C substrates was observed in liver microsomes from PCB-contaminated bulls (Machala et al., 1998) (no details on the PCB composition are given in the cited reference). All the CYPs believed to be involved in NDL-PCB biotransformation are present in ruminants (Ioannides, 2006), but limited information exist concerning possible species differences in their expression. From both immunochemical and functional assays using model

15 http://www.ncbi.nlm.nih.gov/nuccore


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substrates (Szotáková et al., 2004; Dacasto et al., 2005), it would appear that CYP3A is expressed at a higher level in ovine than in bovine liver.

In conclusion, there is a molecular proof of the expression of CYP1A1, CYP1A2, and CYP1B1 in cattle and of CYP1A1 in sheep, while in deer only immunochemical and catalytic evidence of CYP1A expression is available. In vitro and in vivo comparative studies concerning (i) the ability in metabolizing CYP1A model substrates by liver preparations, including primary cultures, and (ii) the metabolic profile of caffeine, a CYP1A2-related substrate, point to a lower activity of CYP1A enzymes in sheep than in cattle. It remains to be established to which extent such differences could be relevant to explain the marked species-related differences in the liver storage of dioxins and related compounds, also in the light of the lack of ad hoc studies on the dioxin-related induction of CYP1A and other xenobiotic metabolizing enzymes in target species. However, it cannot be excluded that other mechanisms such as the sequestration of dioxins and dioxin-like compounds by hepatic CYP1A2 or their biotransformation by other enzymes, which have been observed in rodents, may partly explain these differences. Finally, the poor database does not allow concluding about the occurrence of species related differences in the expression of CYPs that are likely involved in NDL-PCBs biotransformation in ruminant species.

8.1.4. Elimination

Although no quantitative data have been reported concerning the relative importance of urinary, and faecal excretion routes of dioxins in sheep, it can be assumed, based on published data on goat (Grova et al., 2002) and calf (Hakk et al., 2001) that faeces should be the major route of elimination. As shown by Fries et al. (2002), recovery in faeces generally increases with the number of chlorine atoms. Milk represents an important route of excretion for dioxin and DL-PCBs in ruminants. Costera et al. (2006) found that in goat, the transfer of ingested DL-PCBs at a steady state situation to milk may vary from approximately 10 to 25 % for PCB-77, -81 and -123, and to more than 80 % for PCB-105, -118 and -157. The values reported by these authors for tetra- to hexachlorodibenzo-p-dioxins were from 14 % for 1,2,3,7,8,9-HxCDD to 39 % for 2,3,7,8-TCDD. For PCDFs, the transfer values were found to be below 28 %. The data reported by Ingelido et al. (2009) for sheep milk samples collected in farms located in the vicinity of incineration plants and for samples of feedstuffs used in the investigated farms, are in agreement with these transfer values.

Olling et al. (1992) investigated the elimination of dioxins in sheep purchased from farms contaminated with dioxin. At the beginning of the experiment animals received for eight days a mixture of dioxins (130 ng International-TEQ16/animal per day). Then animals were fed concentrate and hay obtained from a non-contaminated area. Dioxins were measured in fat biopsies sampled during a 32 week post-dosing period. The half-life of dioxins in non-lactating sheep expressed as International-TEQ was about 160 days. For individual congeners, the half-life ranged from 95 days for 1,2,3,7,8,9-HxCDD to 226 days for 1,2,3,4,7,8-HxCDF. In lactating sheep, the half-life ranged from 51 days for 1,2,3,7,8,9-HxCDD to 109 days for 1,2,3,4,7,8-HxCDF, and was about half of that in non-lactating sheep (ranging from 95 days for 1,2,3,7,8,9-HxCDD to 226 days for 1,2,3,4,7,8-HxCDF). The authors reported that the half-lives found in non-lactating sheep are somewhat shorter than in non-lactating cows and those in lactating sheep are twice as long as in lactating cows (Olling et al., 1991) which may be explained by assuming a relatively large fat compartment and a relatively low milk fat production rate in sheep as compared to cows. This lower elimination rate of dioxins in lactating sheep, together with the fact that the lactation period of sheep is much shorter than that of cows, so

16 The international toxicity equivalency factors (I-TEFs) were developed in the second half of 1980s, within the framework of activities carried out by the Committee on the Challenges of Modern Society of the North Atlantic Treaty Organization (NATO/CCMS, 1988a,b). I-TEQ estimates obtained by converting the analytical values of dioxin congeners by the I-TEFs would in general diverge only moderately from the WHO-TEQ estimates computed by applying the WHO-TEFs1998 to the same sets of data. I-TEFs, available for dioxins only, were a widely used conversion system to TEQs for approximately a decade.


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that accumulation of dioxins can take place over a longer period of time, may result in higher concentrations in ewe liver as compared with cow.

8.1.5. Transfer and accumulation ratios

In a study performed on sheep fed a diet containing 20 mg Aroclor 1242 or Aroclor 1254 per kg feed for 15 weeks, concentrations were measured in various tissues including muscle, liver and adipose tissue (Hansen et al., 1977). The CONTAM Panel noted that PCB concentrations in this study were determined by GC-ECD and were not based on the quantification of selected individual congeners but on the sum of all peaks present in the chromatogram The accumulation ratios (concentration in tissues relative to the concentration in the diet) for Aroclor 1242 were as follows: 0.78 (adipose tissue), 0.08 (liver) and 0.008 (muscle), whereas for the same tissues the values for Aroclor 1254 were 1.02, 0.08 and 0.01, respectively. No similar work was identified in cows.

Based on the dioxin concentrations (measured as WHO-TEQ) in tissues of sheep related to feed contamination reported by Schulz et al. (2005), and assuming that liver contains 5 % lipids, an accumulation ratio of approximately 2 can be calculated for the liver.

Recently, Fernandes et al. (2011) calculated the biotransfer factor (BTF, defined as the quotient of the contaminant in foodstuff [ng/kg fat] by the daily contaminant intake rate [ng/day]) for different dioxins and PCBs in sheep. A total of 16 sheep (8 pregnant lowland and highland sheep carrying twins) were investigated and BTFs corresponding to selected dioxins and PCBs were calculated for meat, liver and kidney samples from lambs slaughtered at 4 or 5 months. Average BTF values across all 17 PCDD/Fs and 22 PCBs measured were approximately 4 times higher in liver than in meat or kidney. In lowland sheep, BTF values for 2,3,7,8-TCDD, 1,2,3,7,8-PeCDD, 2,3,4,7,8-PeCDF, PCB-126, PCB-153 and PCB-169 in liver were 5.2, 23.5, 175.7, 117.2, 53.1 and 38.5, respectively, whereas in meat these values were 2.6, 5.5, 5.2, 12.6, 33.7 and 37.4, respectively. BTFs in highland sheep were reported to be appreciably higher than those corresponding to lowland animals.

8.2. Effects of dioxins and PCBs on ruminants, particularly in sheep

The comparative toxicity of the dioxins and the related compounds in laboratory and farm animals has been reviewed (McConnell, 1985). For ruminants, the most common toxic syndromes are the result of a subacute-chronic exposure. According to both field cases (Davies et al., 1985) and experimental studies (McConnell et al., 1980) the main features in cattle were unthriftiness, poor fertility and late abortion, impairment of the immune system with thymic atrophy, epithelial hyperplasia of the extrahepatic bile ducts and gallbladder, and skin lesions associated with alopecia and hyperkeratosis. As evidenced by body weights, feed consumption and observations by a licensed veterinarian, no adverse effects were observed in beef cattle administered a diet containing TCDD (24 ng/kg feed) for 4 weeks and kept under observation for a further 46 weeks (Jensen et al., 1981). More recently, a statistically significant increase in both chromosome fragility, expressed as increase in the percentage of chromosome aberrations and sister chromatid exchange, was noticed in circulating lymphocytes from dairy cows reared in a contaminated area from Northern Italy showing bulk milk TEQ values higher than those legally permitted (Di Meo et al., 2010).

Comparatively fewer reports can be found in the scientific literature addressing the adverse effects of dioxins and DL-PCBs in sheep. Reproduction and fertility are affected in this species as well. Sewage sludges contain a mixture of environmental pollutants, including heavy metals and a wide array of polycyclic aromatic hydrocarbons, and many organohalogen compounds (dioxins and DL-PCBs) (Umlauf et al., 2011). Both the male (Paul et al., 2005) and female offspring (Fowler et al., 2008) of pregnant ewes allowed to graze pastures fertilized twice annually with sewage sludge exhibited signs of cellular and hormone disruption of testicular and ovarian foetal development, respectively. In addition, biomechanical and peripheral Quantitative Computed Tomography measurements of femurs from sheep of either sex exposed as above revealed changes suggestive of a perturbation in bone


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homeostasis (Lind et al., 2009). In all the above studies, however, neither a thorough assessment of the carry over of dioxin-like compounds has been performed, nor the role of each pollutant could have been assessed.

A contamination of dairy products with high levels of dioxins occurred in 2002 in the provinces of Naples and Caserta (southern Italy) (Diletti et al., 2003a, 2003b, 2004, 2008; Borrello et al., 2008), which resulted in the compulsory slaughtering of about 12,000 head of cattle, river buffaloes, and sheep by the end of 2003. A careful check performed by Veterinary Services in an exposed farm over a three-year period (2002-2004) revealed an unusually high mortality rate (a total of 925 sheep and lambs) as compared with no deaths recorded in a control farm. Moreover, animals reared in the contaminated farm exhibited a higher percentage of abortions (12.2 vs. 1.2 %, mostly at the fourth month of gestation) and abnormal foetuses (4 % vs. none), as well as a huge rise (up to 17-fold) in both chromosome aberrations and sister chromatid exchange (Perucatti et al., 2006) compared with unexposed animals. The CONTAM Panel noted that co-contamination with genotoxic pollutants cannot be excluded.

In the study by Machala et al. (1998) mentioned before, an increase in the overall CYP-mediated liver microsomal catabolism of testosterone and a decrease in the activity of testicular mitochondrial CYP11A, the rate limiting enzyme in testosterone biosynthesis, were measured in bulls accidentally contaminated with PCBs from coat paints. Testosterone levels in bulls were not reported.

A chronic wasting disease was diagnosed in a sheep flock of 80 individuals showing a severe drop in milk production and emaciation, increased levels of serum aspartate aminotransferase and bilirubin, and severe liver atrophy at necropsy. Muscle and liver tissues were found to contain variable amounts of PCB-138, -153 and -180, but the source of contamination could not be assessed (Spengler, 1993).

8.3. Toxicological end-points for dioxins and DL-PCBs

A large number of scientific publications are available related to the adverse effects of TCDD and other dioxin-like compounds. No other group of organic chemicals has been investigated more extensively. In this chapter, a short summary of the current knowledge on a number of toxic endpoints is given, being by no means comprehensive. Due to the enormous number of publications (and reviews) on this issue, no references are provided in the text. For more detailed and comprehensive information, a number of risk assessments and scientific reviews are available (e.g. IARC, 1997; ATSDR, 1997; WHO, 1998; SCF, 2001; JECFA, 2001). The current risk assessment approach for dioxins (SCF, 2001) is mainly based on developmental effects of TCDD in rat offspring after exposure of the dams.

It is assumed that DL-PCBs have a large number of adverse effects in common with potent PCDDs and PCDFs, and that the activation of the AhR is a major common denominator of this group of chemicals. In contrast, the NDL-PCBs do not activate the AhR to a relevant extent, and have therefore been subject to separate risk assessment (EFSA, 2005).

8.3.1. Laboratory animals

8.3.1.1. Acute and sub-acute toxicity

Dioxins exert a broad range of toxic effects in laboratory animals. The effects themselves as well as their severity vary among different species, strains within a species, individual organs and tissues, as well as the age and gender of the animals.

The toxic potencies of different dioxin congeners correlate with their ability to bind to and activate the AhR. The same holds true for single congeners in different species. Although it is rather clear that


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dioxin toxicity depends on AhR-binding, this and especially the effects on target gene transcription can, at present, not fully explain the severe toxicities of these substances.

TCDD is the most toxic dioxin congener, i.e., most animal studies trying to elucidate the molecular mechanisms of dioxin toxicity have been conducted with TCDD. Oral lethal dose (LD50) values for TCDD vary between species, e.g. between 0.5 for Guinea pigs and 1,000 µg/kg b.w. for hamsters. Furthermore, among different strains within one species, LD50 values can vary by a factor of up to 100. A characteristic effect of single high-dose administration of TCDD in laboratory animals is a severe loss of body weight (wasting syndrome). This is accompanied by a decrease in food intake and a reduction of body fat. TCDD in sub-lethal doses leads to hepatic alterations such as lipid accumulation, accompanied by damage to liver cells in most animal species. It also causes hepatomegaly, an abnormal liver growth resulting from hypertrophy and hyperplasia of parenchymal liver cells, as well as a rapid decrease in liver retinol levels in laboratory animals.

Furthermore, TCDD causes atrophy of lymphatic organs such as thymus, spleen, and lymph nodes in all species tested. In rhesus monkeys and nude mice TCDD causes dermal symptoms resembling human chloracne (see below). Besides that, toxic effects on organs of the reproduction system including hormone producing organs (testes, prostate, uterus and thyroid) were observed in different species.

8.3.1.2. Sub-chronic and chronic toxicity

When administered sub-chronically or chronically, dioxins are hepatotoxic and cause dermal effects. In all species assessed TCDD causes hepatomegaly. Chronic exposure to TCDD also results in significantly decreased weight gain.

Estimated no-observed-adverse-effect levels (NOAELs) for sub-chronic toxicity of TCDD are in the range of 0.6 ng/kg b.w. per day in Guinea pigs, 10 ng/kg b.w. per day in rats, and 100 ng/kg b.w. per day in mice. There are several animal studies on the chronic effects of TCDD, the most conclusive is a two-year study in rats with doses ranging from 1 to 100 ng TCDD per kg b.w. per day. The most consistent effects were observed in the liver. TCDD caused porphyria and an elevation of serum amino-transferase activities. The livers of the animals showed multiple, degenerative, inflammatory and necrotic lesions. Furthermore, hyperplasia of the liver parenchymal tissue and canaliculi was observed. The NOAEL in this study was 1 ng TCDD/kg b.w. per day.

8.3.1.3. Effects on endocrine and reproductive functions

In laboratory animals, TCDD can directly affect reproductive organs in both males and females, on the other hand TCDD interferes with steroid hormone homeostasis. In particular, TCDD acts on estradiol homeostasis. This effect is thought to contribute to the anti-estrogenic actions of dioxins. Exposure of adult male rodents to TCDD decreases serum androgen concentrations and affects male fertility.

In adult rats TCDD also causes a decrease in T4 and concomitant increase in thyroid stimulating hormone (TSH).

8.3.1.4. Developmental toxicity

TCDD also exerts developmental toxicity and acts as a teratogen. Prenatal exposure to TCDD leads to decreased viability in virtually in all animal species tested, both in utero and postnatally. These effects might be caused mainly by the toxicity of TCDD to the dams rather than by direct embryotoxicity. In some experiments it was shown, however, that certain effects are induced in the offspring without pronounced toxicity to the dams. Likewise, the offspring of exposed animals showed impaired reproduction, and structural and functional abnormalities. Prenatal exposure to TCDD led to an


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increase in the incidence of cleft palate, hydronephrosis and thymic atrophy in mice. TCDD treatment of rat dams affected learning abilities and locomotor activity and delayed brain growth in the fetus/offspring. Maternal exposure to TCDD resulted in reduced testis weight in the male offspring.

Gestation and lactational exposure of rats to TCDD leads to a decrease in serum T4.

8.3.1.5. Immunotoxicity

A number of animal studies have identified the immune system as a target for dioxin toxicity. Dioxins can cause atrophy of lymphatic organs and inhibit both innate and adaptive immune responses. Because of the variety of immunotoxic effects observed with dioxins it seems plausible that these pollutants affect more than one single cell type. Furthermore, toxic effects in other than lymphatic organs seem to contribute to the immunotoxicity of dioxins. The AhR is thought to mediate the immunosuppressive effects of dioxins since the relative potency of immuno-suppression of different congeners correlates with their TEF values.

The human immune system appears to be affected by dioxins too, although there are only very limited data on this issue.

8.3.1.6. Carcinogenicity

1. Dioxins and among those especially TCDD, are so called multiple-site, multiple-species carcinogens. This means that they cause cancer in different animal species in various organs. Dioxins are not DNA-reactive, i.e., they do not bind covalently to nucleic acids. That is why other mechanisms have been proposed by which these substances cause tumours. A large number of studies have been conducted, most of them with the model compound TCDD.

2. Life-long exposure to TCDD resulted in a pronounced increase in the incidence of hepatocellular carcinoma in female rats, whereas the carcinogenic potency of TCDD in the liver was much less pronounced in males. Furthermore, TCDD caused an increase in tumour incidence in a number of different tissues in both sexes, e.g., tongue, nasal turbinates, hard palate and lung. In addition, male rats developed thyroid tumours. Interestingly, TCDD caused a decrease in the occurrence of some oestrogen-dependent tumours, e.g. in the uterus and mammary gland. It was discussed that an anti-estrogenic effect of TCDD might explain this finding.

TCDD also causes liver tumours in mice. In contrast to rats, no sex-difference could be observed. As with rats, TCDD caused an increase in the number of tumours in other tissues besides the liver, too.

In initiation-promotion studies in the liver of female rats, TCDD by itself did not initiate tumour growth. Following initiation of the animals with genotoxic carcinogens, however, TCDD is the most potent tumour promoter in rodent liver. TCDD is also a potent tumour promoter in mouse skin when applied topically.

There are only very limited studies assessing the carcinogenicity of other dioxins besides TCDD, but it was proposed that all dioxin congeners might be rodent carcinogens and act as liver tumour promoters. Since their relative potencies correlate with their respective TEF values, and animals bearing a low-affinity AhR lose their sensitivity towards the carcinogenicity of TCDD, the AhR seems to be involved in the carcinogenic effects of dioxins.

8.3.2. Adverse effects in humans

In humans, high TCDD exposure resulted mainly in chloracne and hepatic alterations. Chloracne is characterized by follicular hyperkeratosis, a thickening of the stratum corneum. The period of time from dioxin exposure to the occurrence of chloracne depends on dosage and can range from days


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following very high doses to weeks or months with lower doses. The causal mechanism of chloracne is unknown to date. It has been linked to growth factors affecting the proliferation and differentiation of epidermal cells. A general inflammatory response of the skin might also be involved.

Mixed exposure to dioxins and PCBs also seem to exert developmental and reproductive toxicities in humans. Two incidents in Asia point towards this assumption: the Yusho incident in Japan and the Yu-Cheng incident in Taiwan. Mothers exposed to PCBs and PCDFs gave birth to hyperpigmented children with increased perinatal mortality. Exposure also caused delayed development, both pre- and postnatally, and affected the central nervous system. Most of the mothers suffered from chloracne. Long-term exposure to TCDD in men led to a decrease in testosterone levels as observed in US Vietnam war veterans.

In the Seveso cohort, effects resulting from exposure to high levels of TCDD (828 to 56,000 pg/g blood serum lipid in ten children living in the most contaminated zone of the Seveso accident (Zone A) were mainly chloracne and hepatic alterations. However, nine adults from the same area, with TCDD concentrations between 1,770 and 10,400 pg/g serum lipids, were not diagnosed chloracne.

Paternal exposure to TCDD was linked to lowered male/female ratio in their offspring. The effect was reported to start at concentrations below 20 ng/kg b.w., with fathers exposed when they were young siring significantly more girls than boys. In another Seveso study, it was concluded that in utero and lactational exposure of children to relatively low TCDD doses can permanently reduce sperm quality.

Based on observations over the 1976-2001 period, the Seveso cohort did not show a mortality increase from all cancers (Consonni et al., 2008). However, the results of this later study confirmed previous findings of an excess of lymphatic and hematopoietic neoplasms in the areas with high-to-intermediate TCDD contamination levels (Zones A and B, respectively). In the whole area under detectable TCDD impact, including the aforesaid zones and Zone R, contaminated to a lower level, increased mortality from circulatory diseases, chronic obstructive pulmonary disease, and diabetes mellitus (female subjects only) was observed. According to the authors, the outcome of the study provides evidence of a toxic and carcinogenic risk to humans after high TCDD exposures

Exposure at low levels, being in the order of magnitude of background exposure in the general population, has been suggested to be involved in a variety of adverse effects in human subpopulations. These include effects on hormone homeostasis, endometriosis, delayed male pubertal onset, reproduction, learning performance etc. No convincing evidence has been provided so far to prove these hypotheses since in other studies, no correlation of adverse outcomes with dioxin levels and/or exposure was found. Furthermore, other (environmental, lifestyle etc.) factors are likely to influence the endpoints analyzed, and are very difficult if not impossible to rule out in a mixed exposure scenario with relatively low dioxin exposure, in particular since a ‘true control’ group without detectable dioxin exposure cannot be included.

For most of the toxic effects of dioxins and DL-PCBs, background exposure is well below those associated with overt toxicities. Variability in dioxin kinetics between human individuals may play an important role for individual risk. Human variation in half-life of TCDD is about 6-fold (EFSA, 2004) and the differences in age and body fat mass contribute to this variability. In developing a toxicokinetics model of human life time body burden regarding TCDD, Kreuzer et al. (1997) determined half-life in infants about 5 months, increasing to 10 years between 40 and 60 years of age.

9. Risk characterization

In order to characterize the risk of chronic consumption of sheep liver, calculations were made to compare how much the consumption of sheep liver would add to the total human exposure and how the chronic intake of dioxins and DL-PCBs compares with the tolerable weekly intake (TWI). As the


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TWI the CONTAM Panel used the value of 14 pg TEQ/kg b.w. per week established by the SCF in 2001 (SCF, 2001).

Consumption data for sheep liver are scarce in Europe. Based on limited consumption data for sheep liver from the German National Nutrition Survey II which shows portion sizes of 80-250 g (mean value: 160 g) sheep liver consumed 1-2 times per month (mean 1.2 times per month) and on food frequency questionnaires of Italian, Greek and Turkish female migrants in Germany which indicated that female migrants, especially from Turkey, consume offal of all animals more often in comparison to the other groups that participated in the survey, the German Federal Institute for Risk Assessment (BfR) in its risk assessment used a weekly consumption of 250 g sheep liver for high consumers.

The CONTAM Panel considered this value an overestimation for chronic sheep liver consumption.

Data extracted from the EFSA’s Comprehensive Database indicates that only a very small fraction of the European population consumed such products within the length of the survey period. Only 0.2 % of the individuals included in the Comprehensive Database indicated that they had consumed sheep liver at least once during the period of recording. Thus, the CONTAM Panel concluded to use consumers only for the exposure assessment. The average sheep liver portion size recorded in the UK (about 140 g or 1.9 g/kg b.w.) is used for the risk characterization for adults (consumers only) with the assumption that it represents a weekly intake. Assuming that sheep liver consumption in children is similar to consumption of “all liver”, the average weekly amount of 2.8 g/kg b.w. from a Bulgarian survey was selected as a conservative estimate for the exposure assessment for children (see Chapter 6). In this case the data extracted from the Comprehensive Database refer to a total of 303 individuals covering the age range from 0 to 18 years old with the following frequencies: 13 infants (0-1 years old), 21 toddlers (1-3 years old), 172 other children (3-10 years old) and 97 adolescents (10-18 years old).

Dioxins and DL-PCBs

As a starting point, a human background daily intake of dioxins and DL-PCBs was derived from the literature (see Table 9 in Chapter 7.2.). The median dietary intake of dioxins and DL-PCBs across European countries for which data were reported as WHO1998-TEQs (see Table 2) is 1.53 pg TEQ/kg b.w. per day (range 0.5-3.2 pg TEQ/kg b.w. per day) or, converted to a weekly basis, around 11 pg TEQ/kg b.w. (range 3.6-23 pg WHO-TEQ/kg). As mentioned earlier, the CONTAM Panel used the data based on the WHO-TEFs1998 because a considerable number of occurrence data were only reported as TEQs calculated with these TEFs without giving the raw data which made a conversion with the most recent WHO-TEFs2005 impossible. Applying the latter TEFs may lead to 10-15 % lower values.

Based on the occurrence data reported by the eight European countries and the maximum levels laid down in Regulation (EC) No 1881/2006 the exposure to dioxins and DL-PCBs for adults (consumers only) of sheep liver can be estimated. Table 10 shows the calculated weekly intakes through consumption of sheep liver and the resulting median total weekly dietary intake for dioxins and DL-PCBs.

The data in Table 10 indicate that for adults, consumption of about 140 g (or 1.9 g/kg b.w.) sheep liver with the concentration at the maximum level laid down in Regulation (EC) No 1881/2006 would result in a weekly intake of 1.2 pg WHO-TEQ/kg b.w. for consumers only, and in a total weekly intake of 12.2 pg WHO-TEQ/kg b.w., taking a median background exposure of 11.0 pg WHO-TEQ/kg b.w. into account.

For adults, consumption of about 140 g (or 1.9 g/kg b.w.) sheep liver with the mean value of 26.1 pg WHO-TEQ/g fat would result in a weekly intake of 2.5 pg WHO-TEQ/kg b.w. and in a total weekly intake of 13.5 pg WHO-TEQ/kg b.w., taking the same before mentioned median background exposure of 11.0 pg WHO-TEQ/kg b.w. into account. This is 96 % of the TWI.


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Due to lack of data, the consumption of sheep liver for children was based on an age range up to 18 years old. As respective current data on background exposure to dioxins and DL-PCBs for children are sparse, the CONTAM Panel decided not to estimate a median dietary background exposure for children. The assessment indicates that the exposure of children to dioxins and DL-PCBs through consumption of sheep liver is approximately 50 % higher compared to adults because of the higher food consumption relative to body weight (Table 10). This exposure would add up to a dietary exposure greater by a factor of 1.2-3 (average, 2) than estimated for adults (see Appendix A).

NDL-PCBs

A similar evaluation as for dioxins and DL-PCBs was performed for the dietary exposure to NDL-PCBs (PCB-28, -52, -101, -138, -153 and -180) (Table 11). The fat content of sheep liver and body weight of the adults are the same as for the exposure assessment concerning dioxins and DL-PCBs. The exposure estimation was performed with the mean concentration for the sum of the six indicator NDL-PCBs calculated from the occurrence data submitted by the eight European countries. Harmonized maximum levels for the sum of these six PCB congeners in various food categories are foreseen to be set soon.

As a starting point the CONTAM Panel used the background exposure to NDL-PCBs as derived in its risk assessment from 2005 (EFSA, 2005). There, an average daily intake of total PCBs of 10-45 ng/kg b.w. was estimated for adults, corresponding to 5-23 ng/kg b.w. per day or 35-161 ng/kg b.w. per week for the sum of the six indicator NDL-PCBs. The respective daily intake for young children, up to 6 years of age, was estimated as 27-50 ng/kg b.w. for total PCBs, corresponding to 14-25 ng/kg b.w. per day (or 98-175 ng/kg b.w per week) for the sum of the six indicator NDL-PCBs. The CONTAM Panel considered it unlikely that children up to six years of age consume sheep liver regularly and thus used the whole range of 35-175 ng/kg b.w. per week for the sum of the six NDL-PCBs as background exposure for children up to 18 years.

Considering the mean level for the sum of the six indicator NDL-PCBs, the average weekly dietary exposures for adults (consumers only) is 2.6 ng/kg b.w. Assuming a weekly background exposure of 35-161 ng/kg b.w., the additional contribution from sheep liver amounts to 1.6-7.4 %. The respective evaluation for children would result in an average weekly intake of 3.8 ng/kg b.w which is an additional weekly intake between 2.2 and 11 %.

Conclusions

In conclusion, regular consumption of sheep liver would result on average in an approximate 20 % increase of the background exposure to dioxins and DL-PCBs. On individual occasions, consumption of sheep liver could result in high intakes exceeding the TWI. The CONTAM Panel concluded that the frequent consumption of sheep liver, particularly by women of child-bearing age and children, may be a potential health concern.

Additional intake of NDL-PCBs from consumption of sheep liver does not add substantially to the total dietary intake.


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Table 10: Additional and total weekly intake of dioxins and DL-PCBs (pg WHO1998-TEQ/kg b.w.) for adults (consumers only) and additional weekly intake for children up to 18 years old through consumption of sheep liver. A median weekly background exposure for adults of 11 pg WHO-TEQ/kg b.w. was taken into account.

(a): Calculated with the mean value for the sum of dioxins and DL-PCBs in sheep liver samples submitted by eight European countries (see Table 3). (b): Calculated with the maximum level for the sum of dioxins and DL-PCBs laid down in Regulation (EC) No 1881/2006.

Table 11: Additional and total weekly intake of the sum of six indicator NDL-PCBs (ng/kg b.w.) for adults (consumers only) and children through consumption of sheep liver assuming a weekly background exposure of 35-161 ng/kg b.w. for adults and 35-175 ng/kg b.w. for children up to 18 years old.

(a): Calculated with the mean value for the sum of six NDL-PCBs in sheep liver samples submitted by eight European countries (see Table 4).

Population

Weekly consumption of

sheep liver (g/kg b.w.)

Concentration of dioxins and DL-PCBs in sheep liver (pg WHO-TEQ/g fat)

Weekly intake of dioxins and DL-PCBs from sheep liver (pg WHO-TEQ/kg b.w.)

Total median weekly intake of dioxins and DL-PCBs (pg WHO-TEQ/kg b.w.)

Adults 1.9 26.1 (a)

12.0 (b) 2.5 1.2

13.5 12.2

Children 2.8 26.1 (a)

12.0 (b) 3.7 1.7 -

Population

Weekly consumption of

sheep liver (g/kg b.w.)

Concentration in sheep liver (ng/g fat)

Weekly intake of NDL-PCBs from

sheep liver (ng/kg b.w.)

Total additional weekly intake of NDL-PCBs (ng/kg b.w.) (% of background

exposure)

Adults 1.9 26.8 2.6 1.6-7.4

Children 2.8 26.8 3.8 2.2-11


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For deer liver only nine results were reported on concentrations for dioxins, DL-PCBs and NDL-PCBs. This number of samples is too small to perform a meaningful risk assessment. However, as the reported concentrations for dioxins and DL-PCBs were generally higher than the concentrations for sheep liver (with an almost 2.5-fold higher mean value), the CONTAM Panel concluded that frequent consumption of deer liver, especially for high consumers may be of health concern.

10. Uncertainty

The evaluation of the inherent uncertainties in the assessment of exposure to dioxins and PCBs through consumption of sheep liver has been performed following the guidance of the Opinion of the Scientific Committee related to Uncertainties in Dietary Exposure Assessment (EFSA, 2007). In addition, the report on “Characterizing and Communicating Uncertainty in Exposure Assessment” has been considered (WHO/IPCS, 2008). According to the guidance provided by the EFSA opinion (EFSA, 2007) the following sources of uncertainties have been considered: assessment objectives, exposure scenario, exposure model, and model input (parameters).

10.1. Assessment objectives

The objectives of the assessment were clearly specified in the terms of reference. The CONTAM Panel assessed the new occurrence data on dioxins and PCBs in liver for human consumption from sheep and deer that were submitted by eight European countries to EFSA, and carried out an exposure assessment for adults (consumers only) and children. Due to the very low number of results for deer liver which were submitted by the reporting countries, an exposure assessment for deer did not seem meaningful and thus the exposure assessment was only performed for sheep liver. In its assessment, the CONTAM Panel evaluated the additional weekly exposure to dioxins and PCBs through consumption of sheep liver. As no data on processed food that may contain sheep liver as an ingredient were available, the exposure estimation is limited to sheep liver as such. The limited information on representative European consumption data for different age classes may have an impact on the accuracy of exposure estimates, especially in children, for which sheep liver consumption data are missing, adding to the overall uncertainty.

10.2. Exposure scenarios/Exposure model

EFSA collected data from 516 samples of sheep liver, sheep meat and deer liver for human consumption submitted by eight European countries (Germany, UK, Ireland, Sweden, Denmark, France, Greece and Italy). More than 60 % of the data were from Germany. Thus, there is uncertainty in possible regional differences in the contamination of sheep liver with dioxins and PCBs, and the CONTAM Panel recognised that the data set is not representative of liver on the EU market.

Data on consumption of sheep liver and liver from other terrestrial animals for the general population and high consumers in Europe are scarce. As the few consumption data are derived from studies from different countries which differ in study design and do not generally indicate frequency of sheep liver consumption, a derivation of a representative European consumption is hampered.

The overall uncertainty in the model estimation is considered to be high.

10.3. Model input (parameters)

There are no prescribed fixed official methods for the analysis of dioxins and PCBs in food and laboratories can use any method of analysis, provided it can be demonstrated in a traceable manner that they strictly fulfil the requirements according to Regulation (EC) No 1883/2006. This may have added to the uncertainty in the analytical results. However, recent inter-laboratory and proficiency tests have shown that the analytical results provided are satisfactory.


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10.4. Summary of uncertainties

In Table 12 a summary of the uncertainty evaluation is presented, highlighting the main sources of uncertainty and indicating an estimate of whether the respective source of uncertainty might have led to an over- or underestimation of the exposure or the resulting risk.

Table 12: Summary of qualitative evaluation of the impact of uncertainties on the risk assessment of the dietary exposure to dioxins and PCBs through consumption of sheep liver.

Sources of uncertainty Direction Extrapolation of occurrence data from a few countries to whole Europe +/- Very limited data on frequency of sheep liver consumption +/- Limited data on portion size + Lack of information on processed food containing sheep liver - Lack of information on the impact of food processing +/- Limited representative European background data on dietary exposure +/- Lack of sheep liver consumption data for children +/- Use of WHO-TEF1998 rather than WHO-TEF2005 + (a): + = uncertainty with potential to cause over-estimation of exposure/risk; - = uncertainty with potential to cause under-

estimation of exposure/risk.

The CONTAM Panel considered that the impact of the uncertainties on the risk assessment of exposure to dioxins and PCBs via consumption of sheep liver is considerable and concluded that its assessment of the risk is likely to be conservative, i.e. more likely to overestimate than to underestimate the risk.

CONCLUSIONS AND RECOMMENDATIONS

CONCLUSIONS

General

• Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) are two groups of tricyclic planar compounds that often are together referred to as “dioxins”. Dependent on the number of chlorine atoms and their positions in the rings 75 PCDDs and 135 PCDFs, termed “congeners”, can occur.

• Dioxins are generated in a number of thermal and industrial processes as unwanted and often unavoidable impurities or by-products. Important emission sources are inter alia metal production and processing, open-air waste incineration and on-site fires (“backyard emissions”).

• Polychlorinated biphenyls (PCBs) are a group of persistent organochlorine compounds that are synthesised by catalysed chlorination of biphenyl. Depending on the number of chlorine atoms (1-10) and their position in the two rings, 209 different compounds, also termed “congeners”, are possible.

• In contrast to dioxins, PCBs have had widespread use in numerous industrial applications, generally in the form of complex technical mixtures. They were massively produced for over four decades, from 1929 until they were banned, with an estimated total world production of


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1.2-1.5 million tons. As a result of their widespread use, leakages, and improper disposal practices PCBs have a global distribution in the environment.

• In fires and other thermal events, PCBs can be converted to PCDFs and other products. As PCBs are often mixed with polychlorobenzenes, their thermal degradation may also be associated with the production of PCDDs.

• Based on structural characteristics and toxicological effects, PCBs can be divided into two groups. One group consists of 12 congeners that easily can adopt a coplanar structure and show toxicological properties similar to dioxins. This group is therefore called “dioxin-like PCBs” (DL-PCBs). The other PCBs do not show dioxin-like toxicity and have a different toxicological profile. This group is called “non dioxin-like PCBs” (NDL-PCBs).

Occurrence

• Soil and sediments are natural sinks/reservoirs of dioxins and PCBs. Soil-to-plant transfer of dioxins and PCBs via the root apparatus is generally of minor importance.

• In the past few years a number of sheep liver samples from various European countries were found to contain high concentrations of dioxins and PCBs although not being associated with specific contamination sources.

• For sheep, grazing activity is a primary factor for exposure. When grazing, intake of soil can occur through particles deposited on vegetables or directly when feeding on pasture herbage close to ground surface. Soil intake is remarkably variable and strongly seasonal: a median soil intake has been reported in the order of 8 % of dry matter intake. On the whole, soil intake might contribute substantially to sheep’s exposure to dioxins and PCBs.

• Limited data are available concerning the transfer of dioxins and/or PCBs from feed to sheep liver. Depending on the PCDD, PCDF or PCB congeners considered, reported transfer ratios varied from 5 to 175 and were approximately 4 times higher for liver than for meat or kidney.

• EFSA evaluated the dioxin and PCB results from 332 sheep liver and 175 sheep meat samples submitted by eight European countries. For sheep liver the mean upper bound concentrations for dioxins and the sum of dioxins and DL-PCBs amounted to 14.9 pg WHO-TEQ/g fat (range: 0.27-116) and 26.1 pg WHO-TEQ/g fat (range: 0.5-279), respectively. The corresponding levels in sheep meat were considerably lower and calculated as 0.70 pg WHO-TEQ/g fat (range: 0.08-5.1) and 2.0 pg WHO-TEQ/g fat (range: 0.16-11.9), respectively.

• Occurrence data for NDL-PCBs were submitted by eight European countries for 257 sheep liver and 146 sheep meat samples. For sheep liver and sheep meat the mean upper bound concentrations for the sum of the six indicator NDL-PCBs (PCB-28, -52, -101, -138, -153 and -180) amounted to 26.8 ng/g fat (range: 0.41-350) and 13.1 ng/g fat (range: 0.51-162), respectively.

• The CONTAM Panel evaluated the fat content of sheep liver reported in literature and the data submitted by the European countries. A range of 3 to 8 % fat with a mean content of 5.1 % was identified. Comparable fat contents are found for liver samples of other terrestrial animals, such as bovine, pigs and chicken. These ranges of fat content are considerably narrower than for a number of other food categories regulated in Regulation (EC) No 1881/2006.

• In general, the expression of results on a product basis would be preferable from a dietary exposure point of view as this would better reflect the exposure to the consumed products.


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However, a change of the expression of maximum levels seems only meaningful if all food categories would be considered. Therefore, the CONTAM Panel sees no justification to change the basis in future Regulation from fat basis to fresh weight basis solely for liver of terrestrial animals.

• Even if there would be a possible hepatic sequestration and the dioxins and PCBs would not be totally associated with the fat fraction of the liver, this would have no influence on the result, whether expressed on lipid fresh weight basis, as all dioxins and PCBs are extracted during the analytical procedure irrespective of the liver compartment where they are present.

• For deer liver only nine results were reported on concentrations for dioxins, DL-PCBs and NDL-PCBs. The mean value for the sum of dioxins and DL-PCBs was almost 2.5-fold the mean value in sheep liver. For NDL-PCBs the mean values for sheep and deer liver were comparable.

Consumption

• Consumption data on sheep liver are scarce in Europe. Data extracted from the EFSA’s “Comprehensive European Food Consumption Database” show that only a very small fraction of the European population consumed sheep liver during the time of the survey. The available data from six countries show that less than 3 % are consumers of “mutton and lamb liver”.

• As calculation of average consumption of sheep liver expressed on a weekly basis for the small number of sheep liver consumers in the total adult population is subject to a high degree of uncertainty, the alternative chosen is to use the portion size distribution assuming that an arbitrary frequency of one eating occasion in a week can be taken as a conservative estimate.

• The results show an average sheep liver portion size of 106 g (or 1.5 g/kg b.w.) across six countries with the highest average value being in the UK of 141 g (or 1.9 g/kg b.w.). The average sheep liver portion size recorded in the UK was used for the exposure assessment.

• For children, due to the scarce availability of consumption data related to ovine liver consumption, any liver consumption for children covering the age range from 0 to 18 years old was used as a proxy. The highest average weekly amount of 43.4 g (or 2.8 g/kg b.w.) from Bulgaria was selected as a conservative estimate for the exposure assessment.

Human exposure

• Due to the low fraction of the European population that consumes sheep liver the CONTAM Panel decided to perform the risk assessment for consumers only, based on the consumption data reported and the mean value calculated from the reported occurrence data by the European countries and the maximum level for the sum of dioxins and DL-PCBs in the European Legislation.

• The average weekly exposure to dioxins and DL-PCBs from sheep liver based on the concentration at the maximum level laid down in Regulation (EC) No 1881/2006 is 1.2 pg WHO-TEQ/kg b.w. for adults (consumers only). This value would increase to 2.5 pg WHO-TEQ/kg b.w. when considering the mean value calculated from the occurrence data submitted by the European countries. For children, the respective values are 1.7 and 3.7 pg WHO-TEQ/kg b.w.

• Considering the above consumption data and the mean level for the sum of the six indicator NDL-PCBs calculated from the occurrence data submitted by the eight European countries,


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the average dietary exposure for adults (consumers only) of sheep liver is 2.6 ng/kg b.w., and 3.8 ng/kg for children.

• No exposure estimation was made for deer liver, as insufficient occurrence data were available.

Hazard identification and characterization

• A limited number of disposition studies carried out in sheep indicate that the liver is an important storage organ of dioxins and PCBs in this species.

• Sheep are able to metabolize PCBs to hydroxy-PCB, very likely through cytochrome CYP1A enzyme(s). Studies in vitro and in vivo with prototype substrates for rat, human and cattle P450 enzymes indicate lower CYP1A activity in sheep than in cattle, suggesting that differences in metabolism could partly explain the relatively high liver storage of dioxins and related compounds in sheep compared to cattle.

Risk characterization

• In order to characterize the risk of consumption of sheep liver, as this concerns a chronic intake, calculations were made to compare how much the consumption of sheep liver would add to the total human exposure and how this compares with the tolerable weekly intake (TWI) of dioxins and DL-PCBs. As TWI the CONTAM Panel used the value of 14 pg TEQ/kg b.w. per week established by the SCF in 2001.

• As a starting point, a human background daily intake for adults was derived from the literature. The median dietary intake of dioxins and DL-PCBs across European countries for which data were reported as WHO1998-TEQs is 1.53 (range 0.51-3.2) pg/kg b.w. per day or on a weekly basis around 11 (range 3.6-23) pg WHO-TEQ/kg b.w. The CONTAM Panel noted that the available data on dietary exposure was only available from a limited number of European countries and might not reflect the most recent exposure.

• The data based on the WHO-TEFs1998 were used because a considerable number of occurrence data were only reported as TEQs calculated with these TEFs without giving the raw data which made a conversion with the most recent WHO-TEFs2005 impossible. Applying the latter TEFs may lead to 10-15 % lower values.

• For adults (consumers only), consumption of 141 g sheep liver with the mean value of 26.1 pg WHO-TEQ/g fat would result in an additional intake of 2.5 pg WHO-TEQ/kg b.w. and in a total weekly intake of 13.5 pg WHO-TEQ/kg b.w., based on a median background exposure of 11.0 pg WHO-TEQ/kg b.w. This is close to the TWI.

• As a number of sheep liver samples showed considerably higher concentrations for dioxins and DL-PCBs, it can be assumed that consumers may be exposed on individual occasions to much higher values.

• Assuming that sheep liver consumption in children is similar to consumption of “all liver”, their exposure through consumption of sheep liver is calculated to be approximately 50 % higher as compared to adults because of the higher food consumption relative to body weight.

• Considering the mean level for the sum of the six indicator NDL-PCBs, the average weekly dietary exposures for adults (consumers only) is 2.6 ng/kg b.w. Assuming a weekly background exposure of 35-161 ng/kg b.w., the additional contribution from sheep liver amounts to around 2-7 %.


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• For children the respective estimation would result in an average weekly dietary exposure of 3.8 ng/kg b.w. The additional contribution from sheep liver is in a comparable range as for adults.

• Regular consumption of sheep liver would result on average in an approximate 20 % increase of the background exposure to dioxins and DL-PCBs. On individual occasions, consumption of sheep liver could result in high intakes exceeding the TWI. The CONTAM Panel concluded that the frequent consumption of sheep liver, particularly by women of child-bearing age and children, may be a potential health concern. Additional intake of NDL-PCBs from consumption of sheep liver does not add substantially to the total dietary intake.

• For deer liver only nine results were reported on concentrations for dioxins, DL-PCBs and NDL-PCBs. This number of samples is too small to perform a meaningful risk assessment. However, as the reported concentrations for dioxins and DL-PCBs were generally higher than the concentrations for sheep liver (with an almost 2.5-fold higher mean value), the CONTAM Panel concluded that frequent consumption of deer liver, especially for high consumers, may be of health concern.

RECOMMENDATIONS

• As information on consumption of sheep and deer liver is scarce, respective data should be collected. In addition, analyses of deer liver for dioxins and PCBs should be performed in order to evaluate whether regular consumption might pose a potential health risk.

• Studies on the potential role of hepatic CYP enzymes to the reported high levels of dioxins and DL-PCBs in sheep liver are needed.

• Dioxin and PCB transfer from feed, feed components, and soil to sheep liver should be investigated further.

• An updated dietary intake assessment should be performed based on the most recent consumption and occurrence data across European countries for dioxins and PCBs.

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APPENDIX A. AVERAGE DIETARY INTAKE OF DIOXINS AND DL-PCBS FOR CHILDREN REPORTED IN THE LITERATURE FOR DIFFERENT EU COUNTRIES AND ITS RELATIONSHIP TO ADULT AVERAGE DIETARY EXPOSURE

(a): Assessment based on the second nation-wide food consumption survey INN-CA 1994–1996 and on European representative mean dioxin and dioxin-like PCB occurrence data. Breastfeeding excluded.

(b): Undistinguishable from median.

Country Year TEFs Estimation

Dioxin + DL-PCB dietary intake (pg WHO-TEQ/kg b.w. per day) Children’s

age range (years)

C / A Reference Children (C) Adults (A)

FR 2001-2004 WHO-TEF1998 LB = UB 2.8 (mean)

2.4 (median) 1.8 (mean) 1.5 (median) 3-14 1.6

1.6 Tard et al., 2007

UK 2001 WHO-TEF1998

UB UB LB LB

1.95 (average) 1.35 (average) 1.7 (average) 1.1 (average)

0.9 (average) 0.9 (average) 0.7 (average) 0.7 (average)

1.5-4.5 4-14

1.5-4.5 4-14

2.2 1.5 2.4 1.6

UK FSA, 2003

ES (Valencia) 2006-2008 WHO-TEF1998

UB LB

4.58 (average) 3.83 (average)

2.86 (average) 2.09 (average) 7-12 1.6

1.8 Marin et al., 2011

ES (Catalonia) 2006 WHO-TEF2005 MB ~2.75 (mean) 1.12 (mean) 4-9 2.5 Llobet et al., 2008

Sweden 1998-2004 WHO-TEF1998 MB

4.15 (mean) 3.7 (median) 4.05 (mean) 3.2 (median) 2.75 (mean) 2.9 (median) 1.95 (mean) 1.55 (median)

1.5 (mean) 1.25 (median)

1-3 1-3 4-6 4-6

7-10 7-10

11-14 11-14

2.8 3.0 2.7 2.6 1.8 2.3 1.3 1.2

Bergkvist et al., 2008

NL WHO-TEF1998 LB 2.8 (median) 1.5 (median) 1.1 (median) 2

10 2.5 1.4 Baars et al., 2004

IT 1994-1996(a) WHO-TEF1998 UB 5.34 (b) (mean)

3.37 (b (mean) 2.28 (b) (mean) 0-6 7-12

2.3 1.5 Fattore et al., 2006


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ABBREVIATIONS

2,4-D 2,4-Dichlorophenoxyacetic acid β-NAF β-naphthophflavone AhR Aryl hydrocarbon receptor BfR Bundesinstitut für Risikobewertung BTF Biotransfer factor b.w. Body weight CONTAM Panel EFSA Panel on Contaminants in the Food Chain COR Carry-over ratio/rate CRM Certified reference material DL-PCB Dioxin-like PCB DM Dry matter EFSA European Food Safety Authority EROD 7-ethoxyresorufin EU/RL European Reference Laboratory FSA United Kingdom’s Food Safety Authority FSAI The Food Safety Authority of Ireland GC-ECD Gas chromatography with electron capture detection GC-HRMS Gas chromatography/high resolution mass spectrometry GC-LRMS Gas chromatography-low resolution mass spectrometry GS-MS Gas chromatography – mass spectrometry GC-MS/MS Gas chromatography/ tandem mass spectrometry HHRAP Human Health Risk Assessment Protocol for Hazardous Waste Combustion

Facilities IARC International Agency for Research on Cancer JECFA Joint FAO/WHO Expert Committee on Food Additives Kow n-octanol-water partition coefficient LB Lower bound concentration LD50 Lethal dose LOD Limit of detection LOQ Limit of quantification ML Maximum level MROD 7-methoxyresorufin N Number of subjects NDL-PCB Non dioxin-like PCB NOAEL No-observed-adverse-effect-level NRL National Reference Laboratory OFL Official National Laboratory PCB Polychlorinated biphenyl PCDDs Polychlorinated dibenzo-p-dioxins PCDFs Polychlorinated dibenzofurans PCP Pentachlorophenol POP Persistent organic pollutant PTMI Provisional tolerable monthly intake RSDR Relative standard deviation calculated from results generated under

reproducibility conditions SCF Scientific Committee on Food TCDD 1,2,3,4-tetrachlorodibenzo-p-dioxin TDS Total diet study TEF Toxicity equivalency factor TEQ Toxicity equivalent TSH Thyroid stimulating hormone


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TWI Tolerable weekly intake UB Upper bound concentration UGT UDP glucuronosyltransferase WHO World Health Organization

scientific opinion on the risk to public health related to the presence of high levels of dioxins...

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