Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions
Introduction
Mineral oil hydrocarbons (MOH) are complex mixtures commonly divided into mineral oil saturated hydrocarbons (MOSH), the main part in mineral oil which includes n-alkanes, iso-alkanes and cycloalkanes, and mineral oil aromatic hydrocarbons (MOAH), which are almost exclusively alkylated.
In 2012, the European Food Safety Authority (EFSA) published an opinion on MOH in food (EFSA, 2012). The pivotal toxicological end point of the MOAH was considered to be genotoxic carcinogenicity of some of its constituents, whereas MOSH may be accumulated in human tissue and form microgranuloma. No values for tolerable daily intakes (TDIs) were specified, for the MOAH since no safe dose can be defined for genotoxic compounds and for the MOSH because of insufficient data, particularly with regard to accumulation. It was concluded on potential toxicological concern for the MOSH as well as for the MOAH. Estimated MOSH exposure ranged from 0.03 to 0.3 mg/kg body weight (bw) per day, with higher exposure of children. This means that an adult of 60 kg bw may be exposed to 18 mg MOSH per day, summing up to 6.6 g per year.
In 2002, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) specified a temporary acceptable daily intake (ADI) of 0.01 mg/kg bw for Class II and III mineral oils free of MOAH (JECFA, 2002), from which a limit of 0.6 mg/kg MOSH in food had been derived (Biedermann and Grob, 2010). MOSH corresponding to Class II and III oils are those most frequently detected in food. For Class I oils, of a higher molecular mass, an ADI of 10 mg/kg bw has been established. In 2012, JECFA withdrew the temporary ADI for Class II and III mineral oils (JECFA, 2012).
Albro and Fishbein (1970) found that after administration of a single dose to rats, the retention of the aliphatic hydrocarbons was inversely proportional to the number of carbon atoms and ranged from 60% for C14 to 5% for C28 compounds, independent of the dose and the hydrocarbon type (alkanes, alkenes or alkynes). Tulliez (1986) determined absorption rates in rats and pigs of 52% for MOSH with a high proportion of cycloalkanes when the average carbon number was 20, and of 20% if the average carbon number was 28, with lower absorption if the dose was reduced.
n-Alkanes are metabolized to fatty alcohols and then fatty acids in the small intestine and the liver through the cytochrome P450 system (Ichihara et al., 1981, Perdu-Durand and Tulliez, 1985). The metabolism of some branched and cyclic alkanes has been investigated (e.g. Le Bon et al., 1988, Halladay et al., 2002), but little is known about the rate of degradation for structurally differing hydrocarbons, in particular about structures which are virtually not degradable.
Baldwin et al. (1992) determined mean hepatic concentrations in male and female Fischer-344 rats (considered most sensitive) of about 2 mg/g and 10 mg/g, respectively, after feeding them a diet containing 20 g/kg mineral oil for 90 days. The concentrations in mesenteric lymph nodes (MLN) were approximately half of those found in liver. Firriolo et al. (1995) measured MOSH concentrations of 5.6 and 1.7 mg/g in the liver of Fischer 344 and Sprague Dawley rats, respectively, when 2 g/kg MOSH in the diet were fed for 90 days and of 8.2 and 4.1 mg/g with a ten times higher dose. The MOSH ranged from C18 to C30, with a mean molecular mass of 350 Da. Smith et al. (1996) found 0.6–4.3 mg/g MOSH in the liver and up to 3.3 mg/kg in the MLN of female rats fed a diet containing 20 g/kg MOSH during 90 days. Spleens and kidneys contained less than 0.1 mg/g MOSH. Scotter et al. (2003) analyzed liver, intestine, heart, kidney, cervical lymph nodes and MLN after administration of a diet containing 20 g/kg of various mineral waxes and white oils during 90 days to female Fischer-344 rats. MOSH, mainly ranging from C20 to C35, were detected in the small intestine, heart and kidney at concentrations between 0.1 and 7.5 mg/g. Le Bon et al. (1988) found that the radioactivity in tissues after oral administration of 3H-pristane to rats decreased from liver and adipose tissue to spleen, kidney, heart and lung.
Trimmer et al. (2004) daily exposed female Fischer 344 rats to 60–1200 mg/kg b.w. P70(H) and P100(H) white oils in the diet and measured MOSH in liver, kidneys, MLN and spleen during 2 years. MOSH were only detected in the liver for the highest dose (detection limit not indicated). After 3 months it was 900 and 1600 mg/kg for P100(H) and P70(H) oil, respectively, and reached about 1400 and 2300 mg/kg after 2 years. For a subgroup, exposure was stopped after 12 months. During the following year, concentrations in the liver fell to 10–15%. However, also the controls contained some 400 mg/kg MOSH in the liver, for which no explanation was given.
For oils specified as Classes II and III (JECFA, 2002), the formation of granulomas or microgranulomas, i.e. droplets containing MOSH surrounded by proteins, was observed at elevated doses (Smith et al., 1996, Fleming et al., 1998, Carlton et al., 2001). In the MLN of Fischer 344 rats these granulomas sometimes caused inflammations. It has been argued that such inflammations were not observed in other rat strains and other species (Firriolo et al., 1995, Griffis et al., 2010) and that granuloma formation was non-specific, adaptive and not progressing to more severe pathological effects (Carlton et al., 2001, EFSA, 2009). In analogous experiments, no granuloma formation or other effect was noted for Class I mineral oils.
Use of animal data for human safety assessment faces uncertainties. Among other differences, animal tests last for far less time than human lives and, therefore, accumulation is not properly reflected. In none of the animal tests a steady state concentration in tissue was reached for MOSH. It is also unknown whether granuloma formation is merely determined by the MOSH concentration in a tissue (a kind of oversaturation) or also by other factors.
Granulomas may be formed for many reasons (Lagana et al., 2010, Coash et al., 2012). Granulomas containing mineral oil in human tissues have been described in literature long ago (e.g. by Boitnott and Margolis, 1970, Nochomovitz et al., 1975, Blewitt et al., 1977, Dincsoy et al., 1982, Wanless and Geddie, 1985). They ranged from several μm to over 100 μm. Cruickshank (1984) pointed out the rapid and continuous increase in occurrence of what they called follicular lipidosis since the late 1940s, with incidences ranging from 10% to 50% of the spleens investigated in the 1950ies and 1960ies. Occurrence of granulomas was correlated with age and area of residence. In 24–76% of the spleens sampled in Canada in 1970/1971 granulomas were detected, depending on the provinces, with a higher incidence in males than females. The medicinal use of mineral oil was confirmed in a minority of subjects, indicating a low probability to represent the main cause. Occurrence was higher in North America and Australia compared to Europe. In a subsequent publication, Cruickshank and Thomas (1984) found a higher incidence in spleen (approximately 80%) compared to liver (approximately 40%). Wanless and Geddie (1985) found a correlation of the incidence and severity of granulomas with age for liver but not for spleen. No correlation was found with diseases.
Noti et al. (2003) determined mineral oil hydrocarbons in human milk. Related to the fat, the mean concentration was 95 mg/kg (n = 33), with a maximum at 1300 mg/kg. In abdominal fat from 144 women living in Austria collected during Caesarian section, Concin et al. (2008) measured MOH concentrations from 15 to 360 mg/kg fat (average of 60.7 mg/kg). Milk fat from days 4 and 20 contained up to 355 mg/kg MOSH, with averages of 45 and 22 mg/kg on days 4 and 20, respectively. The composition of the MOSH was largely identical in all fat tissue and milk samples, in gas chromatographic retention times ranging from n-C17 to n-C32 and centered at n-C23/C24. Virtually no mineral n-alkanes were detectable. Since the mineral oil products humans are exposed to range from much smaller to much higher molecular mass and may contain prominent n-alkanes, the MOSH in the fat tissue and milk must have been a residue from selective uptake, elimination by evaporation and metabolic degradation. For the evaluation of the exposure it should be taken into account that the MOSH correspond to those the mother could not get rid of and which are likely to be persistent also in the baby.
In order to identify the most relevant sources, MOSH concentrations in fat tissues obtained from Caesarian section were correlated with women’s personal data, nutrition habits and cosmetics use. The age resulted as the predominant predictor for accumulated MOSH, supporting the assumption that part of the MOSH are persistent for a long time, possibly accentuated by a wider use of mineral oils in the food industry up to the 1990ies. There was also some association with cosmetics, which suggests relevant dermal uptake (Concin et al., 2011).
In Part I of the present work, human tissues, namely spleen, liver, MLN, lung, fat tissues, brain, kidney and heart, were sampled during autopsy. They were analyzed for MOH (mainly MOSH) in terms of concentration and molecular mass distribution. In Part II (Biedermann et al., 2014), the accumulated MOSH are characterized in more detail, principally by comprehensive two-dimensional gas chromatography (GCxGC), and compared to the MOSH in mineral oil products.
Section snippets
Materials
HPLC-grade hexane, ethanol and dichloromethane were from J.T. Baker (Deventer, The Netherlands); hydrochloric acid 37% was from Scharlau Chemie S.A. (Sentmenat, Spain). Internal standard solutions were prepared as described by Biedermann and Grob (2012a) and contained 0.3 mg/mL n-C11, cyclohexyl cyclohexane (Cycy), n-pentyl benzene (5B), 1- and 2-methyl naphthalene (MN) and 1,3,5-tri-tert-butyl benzene (TBB), 0.6 mg/mL cholestane (Cho) and perylene (Per) as well as 0.15 mg/mL n-C13 in
Method validation
The yield of the extraction was determined for human tissues (liver, lung, spleen, lymph nodes and fat tissue) and their inherent MOSH content. After the 1 h equilibration with ethanol, samples were extracted with hexane for 1 h. The centrifuged residues were extracted another time overnight. These second extracts still contained 25% MOSH for the lung, but only 0.5–9% for the other tissues, indicating that 1 h is insufficient for a robust quantitative extraction. Hydrolysates of the residues from
MOSH concentrations
MOSH concentrations in subcutaneous abdominal fat tissues were higher than those previously determined in abdominal fat obtained during Caesarean sections (Concin et al., 2008; in both cases Austrian population). The reported data for the latter ranged from 15 to 360 mg/kg, with an average of 60.7 mg/kg and a median of 52.5 mg/kg. This data referred to the fat, while the values reported in this work refer to the tissue. As adipose fat tissue includes 5–30% of water, the concentrations reported by
Conclusions
The concentrations measured confirm that the MOSH are the by far largest contamination of the human body (Concin et al., 2008): The body of a quarter of the subjects probably contained more than 5 g MOSH, and the concentrations in active organs reached 1.4 g/kg (spleen and MLN). This is not necessarily indicative of a health problem, but calls for a careful toxicological evaluation.
For the toxicological evaluation of white mineral oils, the data in human tissues provide information about
Conflict of Interest
The authors declare that there are no conflicts of interest.
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References (48)
- et al.
Absorption of aliphatic hydrocarbons by rats
Biochim. Biophys. Acta
(1970) - et al.
On line coupled high performance liquid chromatography–gas chromatography (HPLC–GC) for the analysis of contamination by mineral oil; Part 1: Method of analysis. A review
J. Chromatogr. A
(2012) - et al.
On-line coupled high performance liquid chromatography – gas chromatography (HPLC–GC) for the analysis of mineral oil. Part 2: Migrated from paperboard into dry foods: interpretation of chromatograms. A review.
J. Chromatogr. A
(2012) - et al.
Is comprehensive analysis of potentially relevant migrants from recycled paperboard into foods feasible?
J. Chromatogr. A
(2013) - et al.
Granulomatous liver diseases: a review
J. Formosan Med. Assoc.
(2012) - et al.
Mineral oil paraffins in human body fat and milk
Food Chem. Toxicol.
(2008) Follicular (mineral oil) lipidosis: I. Epidemiologic studies of involvement of the spleen
Hum. Pathol.
(1984)- et al.
Mineral oil (follicular) lipidosis: II. Histologic studies of spleen, liver, lymph nodes, and bone marrow
Hum. Pathol.
(1984) - et al.
Granulomas in the livers of humans and Fischer rats associated with the ingestion of mineral hydrocarbons: a comparison
Regul. Toxicol. Pharm.
(1998) - et al.
Comparative 90-day dietary study of paraffin wax in Ficher-344 and Sprangue–Dawley rats
Food Chem. Toxicol.
(2010)