Toxic effects of mineral oil saturated hydrocarbons (MOSH) and relation to accumulation in rat liver.

Humans are daily exposed to mineral oil saturated hydrocarbons (MOSH) from the diet. We exposed female Fischer 344 rats to a broad mixture and sub-fractions of MOSH. Chemical characterization of the MOSH mixture used and material accumulated in rat tissues were previously reported (Barp et al. 2017a, 2017b). Rats were exposed to feed containing 0-4000 mg/kg broad MOSH mixture for 30, 60, 90 and 120 days; and for 120 days to feed containing different MOSH fractions: i) mainly molecular masses < C25 (S-C25), ii) dewaxed, mainly molecular masses > C25 (L-C25) and iii) the L-C25 fraction mixed with wax largely consisting of n-alkanes > C25 (L-C25W). Treatments related effects were increased liver and spleen weight, as well as vacuolization and granuloma formation with lymphoid cell clusters in the liver, but effects varied strongly between the MOSH fractions tested. We conclude that increased liver and spleen weights were related to accumulated n-alkanes (wax) above C25, presumably not relevant for humans, but also to MOSH from S-C25, mainly consisting of iso-alkanes and substituted cycloalkanes below C25 with a small proportion of n-alkanes. Induction of liver granuloma appeared to be related to n-alkanes > C25 and not to the accumulated amount of MOSH. Immune responses to an injected antigen were not affected. Iso-alkanes and substituted cycloalkanes accumulating in rat liver and spleen were similar to those accumulating in humans.

How to make the use of recycled paperboard fit for food contact? A contribution to the discussion.

Recycled paperboard contains hundreds of non-evaluated or even unidentified substances that could endanger human health if they turn out to be highly toxic. It seems as unrealistic to evaluate each of them as it is to phase out the use of the problematic ones or sort out the papers and boards introducing them into the recyclate. Therefore, measures should be taken that generally reduce migration into food, such as functional barriers or functional sorbents. A general approach is used for the recycling of plastics, particularly poly(ethylene terephthalate), PET: as not every potential contaminant can be regulated, a pragmatic approach is applied, for PET mainly on the required decontamination efficiency. Criteria are required on the required efficacy of the measures to be taken. Recycled paperboard is used for various types of food contact: mostly contact is through the gas phase (evaporation and recondensation), often indirect through other layers (e.g. internal bags or for transport boxes), seldom in wetting contact. Numerous factors have to be considered. For typical folding boxes and at least strongly dominating gas phase contact, it was proposed that no more than 1% of each contaminant in the recycled paperboard should enter the food. The efficiency of the measures required to comply with this criterion depends on the application. The three main measures are reviewed with regard to this criterion: (i) internal bags with an incorporated functional barrier (successfully used for some time), (ii) a barrier layer on the internal wall of the box (for which the design of the closures might be most critical) and (iii) functional sorbents added to the paperboard (for which the sorbent capacity is critical). For transport boxes, commonly of corrugated board (quantitatively the most important use of recycled paperboard in food contact), an adjusted or different criterion is needed.

Epoxidation for the analysis of the mineral oil aromatic hydrocarbons in food. An update.

On-line coupled high performance liquid chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID) used for determining mineral oil aromatic hydrocarbons (MOAH) in foods, particularly in certain oils and fats, may be disturbed by interfering olefins present as natural food components or resulting from raffination of the oils and fats. While some interference can be coped with by disregarding their peaks, others overload GC to the extent of obscuring the MOAH or form humps which need to be distinguished from the hump formed by the MOAH. In the latter cases, it is necessary to remove these interferences prior to HPLC-GC analysis. So far, epoxidation of the olefins to increase their retention time beyond that of the MOAH in HPLC is the best method available, though imperfect by causing some loss of MOAH and sometimes incomplete removal of the interference. Two methods are re-evaluated; preference is given to a slightly modified version of that proposed by Nestola and Schmidt. The performances are comparable: the losses of MOAH are similar and with both methods not all interfering olefins may be removed from refined edible oils. However, the Nestola/Schmidt method has practical advantages, the main ones being that no cooling is necessary and no solvent needs to be evaporated, which facilitates automation. Potential residual interferences must be recognized and subtracted, which can be by the characteristics of the hump they form in HPLC-GC-FID, by GCxGC-FID or by GCxGC-MS using characteristic mass fragments.

The role of the European Food Safety Authority (EFSA) in a better European regulation of food contact materials - some proposals.

For most substances migrating from food contact materials (FCMs) to food, the producers have to perform the safety assessment as self-control, since only a minor part has been officially evaluated and listed. This implies that the main official control of the safety assessment is by the national control authorities. The present EU legislation requires this, but in its structure, it still reflects the concept of providing specific and detailed regulation for all FCMs. Over the last about 40 years, only a few types of FCMs were covered by specific regulation and even these only partially; the reaction products and impurities (usually the majority of the migrants) were hardly addressed at all. Taking this into account, the focus of the regulation should shift towards implementing and supporting self-assessment by the producers and harmonised control by the control authorities. This also affects the role of the European Food Safety Authority (EFSA): presently its main mandate is to evaluate new monomers and additives for plastics to support their authorisation (roughly 10 substances per year) - a niche job when considering the field in its entirety. Since risk assessment and the specification of the requirements to ensure safety are in the remit of EFSA, EFSA should play a key role also in the control of the FCMs on the market. It should be mandated to provide guidelines on safety assessment with requirements that can realistically be implemented (eased where possible), support control authorities in the evaluation of difficult cases and arbitration in cases of disagreement. If private bodies are engaged to approve materials on request of producers in order to be listed, expected to be the most powerful trigger for implementing compliance work, these would have to be closely guided.

Advantages of comprehensive two-dimensional gas chromatography for comprehensive analysis of potential migrants from food contact materials.

All substances migrating from food contact materials (FCMs), such as packagings, into food must be safe. This presupposes comprehensive analysis of all constituents potentially reaching a concentration in food that may be of toxicological concern. There is no single technique meeting this task and usually several need to be combined. In many cases, comprehensive two-dimensional gas chromatography (GCxGC) is the best technique available to start with. It provides high resolution and an overview in well-structured plots, grouping similar substances in a manner facilitating identifications. Further, flame ionization detection (FID) enables approximate quantitation without standards, and electron impact (EI) fragmentation in mass spectrometry (MS) provides access to large libraries for identification. GC is limited in amenable molecular mass, but the characterization of the lower mass constituents is usually helpful also for the identification of higher mass ones by techniques like HPLC-MS. The scope of this paper is to advocate the use of GCxGC for comprehensive migrate analysis, based on advantages illustrated by examples.

Conclusions from a Swiss official control of the safety assessment for food contact polyolefins through the compliance documentation of the producers.

Swiss control authorities checked the safety assessment of nine major producers of polyolefin granulates for making food contact materials. It was a pilot project to gain experience on the procedure of collecting and evaluating compliance documentation, but also to obtain insight into the quality of compliance work performed by the main plastic producers. It revealed that there are fundamental problems in performing such control. These are reported with proposals for improvement. For most products, the safety assessment made available did not correspond to the requirements, as confirmed by a group of internationally recognised experts, who were asked for their opinion on whether the safety of the migrates was assessed in accordance to 'internationally accepted scientific methods on risk assessment', as required by Art. 19 of Regulation (EU) 10/2011 and specified by EFSA.

Toxic effects of mineral oil saturated hydrocarbons (MOSH) and relation to accumulation in rat liver.

Humans are daily exposed to mineral oil saturated hydrocarbons (MOSH) from the diet. We exposed female Fischer 344 rats to a broad mixture and sub-fractions of MOSH. Chemical characterization of the MOSH mixture used and material accumulated in rat tissues were previously reported. Rats were exposed to feed containing 0-4000 mg/kg broad MOSH mixture for 30, 60, 90 and 120 days; and for 120 days to feed containing different MOSH fractions: i) mainly molecular masses < C25 (S-C25), ii) dewaxed, mainly molecular masses > C25 (L-C25) and iii) the L-C25 fraction mixed with wax largely consisting of n-alkanes > C25 (L-C25W). Treatments related effects were increased liver and spleen weight, as well as vacuolization and granuloma formation with lymphoid cell clusters in the liver, but effects varied strongly between the MOSH fractions tested. We conclude that increased liver and spleen weights were mainly related to accumulated iso-alkanes and substituted cycloalkanes, but also wax n-alkanes. Induction of liver granuloma appeared to be related to n-alkanes > C25 and not to the accumulated amount of MOSH. Immune responses to an injected antigen were not affected. MOSH fractions associated with increased liver and spleen weights were similar to those accumulating in humans.

Activated carbon added to recycled paperboard to prevent migration into food: approach for determining efficacy, and first results.

Catcherboard MB12® from Smurfit Kappa is a recycled paperboard incorporating activated carbon to reduce the release of contaminants into food. An approach is proposed to determine the efficacy of the activated carbon. Sorption into activated carbon increases the concentration ratio paperboard/food (distribution coefficient) and reduces the migration rate, i.e. prolongs the time for equilibration. Using silicone paper as food simulant, the concentration ratio was increased by a factor of at least 1000 compared to recycled paperboard without activated carbon, which is sufficient to meet the 1% criterion proposed for barriers. Sorbents have limited capacity. A load with in total 4000 mg surrogate substances/kg paperboard (in addition to the material from the paperboard) exceeded the capacity: concentration ratios were reduced and the release of paperboard constituents increased. Capacity is consumed by constituents from the printing inks and the packed food. Total amounts of substances in dry foods of sufficient volatility to potentially migrate into the paperboard through the gas phase at ambient temperature were determined by GC-FID. For the large majority of the dry foods, these amounts were clearly below the capacity limit even under the exaggerated assumption of total transfer. Migration of mineral oil hydrocarbons into cat food over up to 1 year was not detectable at 1 mg/kg. It is concluded that recycled paperboard with activated carbon is promising for respecting the 1% criterion stipulated for functional barriers to avoid food contamination from recycled paperboard.

Toxicological Assessment of Mineral Hydrocarbons in Foods: State of Present Discussions.

The evaluation of mineral oils by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) provided high acceptable daily intakes for classes largely falling into the mass range strongly accumulated by humans. Because they are roughly 2 orders of magnitude above the present exposure, they authorize strongly increased exposure. An approach based on accumulation seems more adequate. Increased organ weights might be more critical than granulomas. Aromatic hydrocarbons with 1-2 aromatic rings should be distinguished from those with at least 3 aromatic rings. If mineral oil saturated hydrocarbon limits were low, no limit might be needed for the 1-2 ring aromatics. It should be considered to phase out substantial use of mineral oils in food application.

Mineral oil hydrocarbons in food: a review.

Work on mineral oil hydrocarbons (MOH) contaminating food is reviewed up to about 2010, when the subject received broad publicity. It covers the period of the main discoveries and elimination or reduction of the dominant sources: release agents used in industrial bakeries, spraying of rice, additions to animal feed, contamination of edible oils from various sources and migration from paperboard packaging. In most cases, highly refined ('white') oils were involved, but also technical oils, e.g. from the environment, and more or less crude oil fractions from jute and sisal bags. There were numerous unexpected sources, and there might still be more of those. The exposure of the consumers to MOH must have been markedly reduced in the meantime. Environmental influx may have become dominant, particularly when taking into account that these MOH go through several degradation processes which might enrich the species resisting metabolic elimination. Major gaps are in the systematic investigation of sources and the largely unavoidable levels from environmental contamination, but also in the toxicological evaluation of the various types of hydrocarbons. A regulation is overdue that avoids the present discrepancy between the low tolerance to MOH perceived as contaminants and the very high legal limits for some applications - the MOH are largely the same.

Recycled paperboard with a barrier layer for food contact: set-off during stacking or reeling. Analytical method and preliminary results.

The use of recycled paperboard for packaging dry foods is in the interest of sustainability of resources, but in most applications, the food must be protected against contamination, such as by a functional barrier on the internal surface of the paperboard box. After application, the paperboard is usually stacked or reeled before making boxes. During this period, the food-contact surface of the barrier layer is in contact with the outer side of the paperboard, which may result in set-off and subsequent contamination of food. A method is described for the determination of this path of migration, based on the taped format also used for the measurement of the barrier efficiency. Recycled paperboard containing the three surrogate substances n-heptadecane, 4-methyl benzophenone and dipropyl phthalate was taped to the food-contact side of the barrier layer. Pressure onto the test packs did not seem to be a relevant parameter. After periods of interest, a piece of the paperboard with the barrier layer was extracted and analysed for the surrogate substances. Another piece may be brought into contact with silicone paper to simulate the transfer to food. After 2 weeks at 60°C (simulating about 1 year at 25°C), set-off and the transfer to the silicone paper exceeded 1% for all barrier materials tested, but after 6 weeks at 40°C (around half a year at 25°C), set-off remained below 1% for all barrier layers except a multilayer with polyethylene on the food-contact surface. The preliminary conclusion is that set-off should be taken seriously, but may be kept low enough to provide sufficient protection of the packed food.

Update of on-line coupled liquid chromatography - gas chromatography for the analysis of mineral oil hydrocarbons in foods and cosmetics.

On-line coupled high performance liquid chromatography-gas chromatography-flame ionization detection (HPLC-GC-FID) is the most widely used method for the analysis of mineral oil hydrocarbons in food, food contact materials, tissues and cosmetics. With comprehensive two-dimensional gas chromatography (GCxGC), a tool became available for better establishing the elution sequence of the various types of hydrocarbons from the HPLC column used for isolating the mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). The performance of a heavily used HPLC column with reduced retention for MOAH was investigated to improve the robustness of the method. Updates are recommended that render the MOSH/MOAH separation less dependent of the state of the HPLC column and more correct in cases of highly refined mineral oil products of high molecular mass. Cyclohexyl cyclohexane (Cycy), used as internal standard, turned out to be eluted slightly after cholestane (Cho); apparently the size exclusion effect predominates the extra retention by ring number on the 60Å pore size silica gel. Hence, Cycy can be used to determine the end of the MOSH fraction. Long chain alkyl benzenes were eluted earlier than tri-tert. butyl benzene (Tbb). It is proposed to start the MOAH transfer immediately after the MOSH fraction and use a gradient causing breakthrough of dichloromethane (visible in the UV chromatogram) at a time suitable to elute perylene (Per) at the end of the fraction. In this way, a decrease in retention power of the HPLC column can be tolerated without adjustment of the MOAH fraction until some MOAH start being eluted into the MOSH fraction. This critical point can be checked either with di(2-ethylhexyl) benzene (DEHB) as a marker or the HPLC-UV chromatogram. Finally, based on new findings in rats and human tissues, it is recommended to integrate the MOSH and MOAH up to the retention time of the n-alkane C40.

Effect of dietary pristane and other saturated mineral oils (MOSH) on autoimmune arthritis in rats.

Pristane and other adjuvants based on mineral oil saturated hydrocarbons (MOSH) may induce autoimmunity in rodents after intradermal injection; however there is a lack of information on immune effects after oral MOSH exposure. The aim of our study was to determine the impact of dietary exposure to pristane and other MOSH on the development of autoimmune arthritis. Dark Agouti (DA) rats were given feed containing 4000 mg/kg pristane or a broad MOSH mixture in various concentrations (0-4000 mg/kg) for 90 days, or a single intradermal injection of 200 µl pristane (positive control). Arthritis scores, and serum and splenocyte markers previously associated with arthritis development, were determined. All rats injected with pristane displayed arthritis symptoms and higher levels of certain serum markers. None of the rats fed pristane or MOSH developed arthritis symptoms or demonstrated clear changes in any measured arthritis-associated biological markers in serum or splenocytes. The absence of clinical arthritis symptoms or any increase in common arthritis-associated biological markers in sera and spleen following dietary exposure to pristane or a broad MOSH mixture in a sub-chronic rat model of arthritis suggest that dietary MOSH have low capacity to promote development of autoimmunity.

The European system for the control of the safety of food-contact materials needs restructuring: a review and outlook for discussion.

The present European system to assure the safety of migrates from food-contact materials (FCMs) needs improvement. It is proposed to implement better the self-control by the producers through improved official control and more attractive listing of approved substances and materials (the latter being subject of another discussion paper). The initial concept of a positive list for the substances used, a limit for the overall migration and regulation of compliance testing was recognised as insufficient long ago, as it does not properly cover reaction products (including oligomers) and impurities. It also turned out to be unrealistic to cover all 17 types of FCMs owing to lack of resources by the authorities. Therefore, European Union legislation shifted the focus to the compliance work carried out by the business operators (in-house documentation and declaration of compliance). However, this approach has not been properly implemented. This is partly due to lack or unsuccessful control by authorities. A suitable structure of this control still needs to be built. It is proposed that specialised document-collection centres working with dedicated tools harmonised throughout Europe be created. Further, since most migrating substances are not listed, the toxicological evaluation reported by industry must be checked by risk-assessment authorities. Finally, effective and harmonised measures are needed to react in case of non-compliance. The currently large gap between the legal requirements and reality must be bridged by introducing flexibility: authorised work plans by industry are proposed. It is also proposed to encourage certified private institutes to approve compliance work, driven by the attractive listing of approved materials and exploiting market forces. In the long run, the focus of the authorities might change from evaluating the substances used and regulating migration testing to the evaluation of the compliance work performed by industry, which means moving from legislation supporting industry towards checking self-control.

Mineral oil saturated hydrocarbons (MOSH) in female Fischer 344 rats; accumulation of wax components; implications for risk assessment.

Female Fischer 344 rats were exposed to three MOSH mixtures: oils largely below and above C25 (S-C25 and L-C25) and a 1:1 mixture of L-C25 with a wax; doses of 400, 1000 and 4000mg/kg feed were administered during 120days. MOSH were determined by on-line HPLC-GC-FID in liver, spleen, adipose tissue and the carcass. The composition of the hydrocarbons accumulated in the tissues was further analyzed by comprehensive two-dimensional GC (GC×GC). MOSH in the mass range of C26-30 were more strongly accumulated than those between C20-25, which does not support the present classification of MOSH differentiating at n-C25 for risk assessment. Compared to the total of the MOSH, n-alkanes and n-alkyl monocyclic naphthenes were generally enriched in adipose tissue. In liver and spleen, n-alkanes up to C25 were eliminated, but strongly accumulated at around C30. Based on this profile, poor solubility and the melting points, it is hypothesized that crystallization protects these wax components against metabolism and elimination. In the liver, relative retention of n-alkanes decreased again beyond C30, accentuated at high exposure, suggesting reduced absorption. Compared to the animal data, accumulation of n-alkanes from food sources, such as apples, into human tissues seems low, perhaps because of low absorption due to their presence in crystalline form. A series of dominant isoalkanes, accumulated in all tissues analyzed, was characterized, though without proposing a structure. Implications on present regulation of white mineral oil products are discussed.

Accumulation of mineral oil saturated hydrocarbons (MOSH) in female Fischer 344 rats: Comparison with human data and consequences for risk assessment.

Female Fischer 344 rats were orally exposed to a mixture of mineral oil saturated hydrocarbons (MOSH) of broad molecular mass range at doses of 40, 400 and 4000mg/kg feed. Amounts and compositions of the MOSH were analyzed in liver, spleen, adipose tissue and the carcass after exposure during 30, 60, 90 and 120d as well as after 90d exposure followed by 30d depuration. At 40mg/kg in the feed, after 30d of exposure, 10.9% of the ingested MOSH were recovered from the animal body; after 90d plus 30d depuration it was 3.9%. In liver and spleen, the maximum retention in terms of molecular mass (simulated distillation) was at n-C29; in adipose tissue and carcass it was at n-C15/16. The differentiation between MOSH below and above n-C25 (Class I versus Class II and III oils), used for present regulation, is not supported by the present data on accumulation; structural characteristics seem more pertinent than molecular mass. Concentrations in the tissues increased far less than proportionally with the dose, rendering linear extrapolation to low doses questionable. No steady state was reached after 120d. In fact, comparing with the concentrations in human tissues at the estimated exposure, extrapolation from animal experiments seems to grossly underestimate human internal exposure. Comprehensive two-dimensional gas chromatography (GCxGC) was used to characterize the MOSH residues in the tissues with the aim of identifying the most strongly accumulated types. In the liver and spleen, the highly branched hydrocarbons dominated, whereas in the adipose tissue it was the n-alkanes and species with main n-alkyl moieties. Strong MOSH accumulation is not of concern per se, but the safety at the high concentrations in human tissues needs to be re-evaluated, possibly taking into account also end points other than granuloma formation.

Required barrier efficiency of internal bags against the migration from recycled paperboard packaging into food: a benchmark.

The use of recycled paperboard and corrugated board for food packaging is in the interest of the sustainability of resources, but in most applications the food must be protected against contamination from these materials, such as by an internal bag with a functional barrier. Producers of packaging need a specification to find the most suitable and economical barrier for a given application, and the customer needs the confidence that a solution offered to him is adequate. An accurate determination of the barrier efficiency is not possible due to the large number of migrants, most of which have not been evaluated or not even identified. Hence the specification must be based on assumptions and verifiable by a simple test. The proposed benchmark presumes that the migration of all non-evaluated or even unknown substances in recycled paperboard will remain below 0.01 mg kg(-1) food, the conventional detection limit, if their transfer does not exceed 1% of the content in the paperboard. Some substances, such as mineral oil or fatty acids, will exceed the 0.01 mg kg(-1) limit, but they are known, evaluated and of no concern at the reduced migration. Since the critical substances must be assumed to be unknown, the criterion of the 1% migration is tested with three surrogate substances of similar volatility and covering a broad range of polarity. The cornerstones of the method are specified.

Analysis of saturated and aromatic hydrocarbons migrating from a polyolefin-based hot-melt adhesive into food.

Hot-melt adhesives are widely utilised to glue cardboard boxes used as food packaging material. They have to comply with the requirements of Article 3 of the European Framework Regulation for food contact materials (1935/2004). The hot melt raw materials analysed mainly consisted of paraffinic waxes, hydrocarbon resins and polyolefins. The hydrocarbon resins, functioning as tackifiers, were the predominant source of hydrocarbons of sufficient volatility to migrate into dry foods: the 18 hydrocarbon resins analysed contained 8.2-118 g kg(-1) saturated and up to 59 g kg(-1) aromatic hydrocarbons eluted from GC between n-C16 and n-C24, substantially more than the paraffinic waxes and the polyolefins. These tackfier resins, especially the oligomers ≤ C24, have been characterised structurally by GC×GC-MS and (1)H-NMR spectroscopy. Migration into food was estimated using a simulating system with polenta as food simulant, which was verified by the analysis of a commercial risotto rice sample packed in a virgin fibre folding box sealed with a hot melt. About 0.5-1.5% of the potentially migrating substances (between n-C16 and n-C24) of a hot melt were found to be transferred into food under storage conditions, which can result in a food contamination in the order of 1 mg kg(-1) food (depending on the amount of potentially migrating substances from the hot melt, the hot melt surface, amount of food, contact time etc.). Migrates from hot melts are easily mistaken for mineral oil hydrocarbons from recycled cardboard.

Argentation high performance liquid chromatography on-line coupled to gas chromatography for the analysis of monounsaturated polyolefin oligomers in packaging materials and foods.

Multidimensional chromatography based on two-dimensional high performance liquid chromatography on-line coupled to gas chromatography (on-line HPLC-HPLC-GC) enables the separate analysis of saturated, monounsaturated and aromatic hydrocarbons in packaging materials like polyolefins or paperboard and their migrates into foods. Since normal-phase HPLC on silica gel did not preseparate saturated from monounsaturated hydrocarbons, a separation step on a normal-phase HPLC column treated in the laboratory with an optimized amount of silver nitrate was added. The preparation of this HPLC column and the instrumental set-up are described, followed by examples showing the potential of the method. In a preliminary investigation of 11 polyolefin granulates for food contact up to 40% monounsaturated hydrocarbons among the oligomers C16-35 were determined.

Mineral oil in human tissues, part II: characterization of the accumulated hydrocarbons by comprehensive two-dimensional gas chromatography.

Mineral oil hydrocarbons are by far the largest contaminant in the human body. Their composition differs from that in the mineral oils humans are exposed to, and varies also between different tissues of the same individual. Using the presently best technique for characterizing the composition of mineral oil hydrocarbons, comprehensive two-dimensional gas chromatography (GC×GC), the hydrocarbons in human tissues were compared to those of various mineral oils. This provided information about the strongly accumulated species and might give hints on the flow path through the human body. The selectivity of accumulation is probably also of interest for the risk assessment of synthetic hydrocarbons (polyolefins). GC×GC grouped the MOSH into classes of n-alkanes, paraffins with a low degree of branching, multibranched paraffins and naphthenes (alkylated cyclic hydrocarbons) with 1-4 rings. Metabolic elimination was observed for constituents of all these classes, but was selective within each class. The MOSH in the subcutaneous abdominal fat tissues and the mesenteric lymph nodes (MLN) had almost the same composition and included the distinct signals observed in mineral oil, though in reduced amounts relative to the cloud of unresolved hydrocarbons. The MOSH in the liver and the spleen were different from those in the MLN and fat tissue, but again with largely identical composition for a given individual. Virtually all constituents forming distinct signals were eliminated, leaving an unresolved residue of highly isomerized hydrocarbons.