FCT 7945
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Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 5 6
Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions
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a
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Università di Udine, Dipartimento di Scienze degli Alimenti via Sondrio 2/A, I-33100 Udine, Italy Clinical Institute for Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Wien, Austria Official Food Control Authority of the Canton of Zurich, Fehrenstrasse 15, P.O. Box, CH-8032 Zurich, Switzerland d Department of Pathology, Danube Hospital, Langobardenstr. 122, A-1220 Vienna, Austria e Department of Obstetrics and Gynecology, Innsbruck Medical University, Anichstr. 35, A-6020-Innsbruck, Austria b
Q2
14 1 2 6 9 17 18 19 20
Laura Barp a, Christoph Kornauth b, Tanja Wüger b, Margaretha Rudas b, Maurus Biedermann c, Angelika Reiner d, Nicole Concin e, Koni Grob c,⇑ c
a r t i c l e Q3
i n f o
Article history: Received 15 February 2014 Accepted 12 April 2014 Available online xxxx Keywords: Mineral oil saturated hydrocarbons (MOSH) Fat tissue Liver Spleen Granuloma Accumulation
a b s t r a c t Of 37 subjects aged 25–91 y (mean 67 y), mineral oil hydrocarbons were measured in subcutaneous abdominal fat tissue, mesenteric lymph nodes (MLN), spleen, liver and lung, for some of them also in kidney, heart and brain. No mineral oil aromatic hydrocarbons (MOAH) were detected. The mean concentration of mineral oil saturated hydrocarbons (MOSH) in the mesenteric lymph nodes was 223 mg/kg, in liver 131 mg/kg, in fat tissue 130 mg/kg, in spleen 93 mg/kg and in lung 12 mg/kg. They were clearly lower in kidney, heart and brain. The maxima, found in MLN and spleen, were 1390 and 1400 mg/kg, respectively. For a quarter of the subjects a total amount of MOSH in the body above 5 g was calculated. The MOSH composition in the fat tissue and the MLN appeared virtually identical and varied little between the subjects. It was centered on the n-alkanes C23 –C24, ranged from C16 to C35 and included hydrocarbons of plant origin. The MOSH in spleen and liver had almost the same composition for a given subject, but varied somewhat between subjects. They were centered between C25 and C27, ranged from C18 to beyond C45 and were without hydrocarbons of plant origin. Part of the MOSH seem to be strongly accumulated, resulting in far higher concentrations in human tissues related to exposure than observed in shorter term animal experiments. The composition of the accumulated MOSH does not support that Class I mineral oils, sometimes termed ‘‘food grade’’, are less accumulated in the human body than Class II and III oils, which questions the present classification. Ó 2014 Elsevier Ltd. All rights reserved.
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49 50
1. Introduction
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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
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⇑ Corresponding author. Tel.: +41 432447131; fax: +41 432447101. E-mail address: [email protected] (K. Grob).
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 ran-
http://dx.doi.org/10.1016/j.fct.2014.04.029 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Barp, L., et al. Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.029
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81
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L. Barp et al. / Food and Chemical Toxicology xxx (2014) xxx–xxx
ged 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 lm to over 100 lm. 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 nC32 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.
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2. Materials and methods
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2.1. Materials
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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
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Please cite this article in press as: Barp, L., et al. Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.029
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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) and 0.15 mg/mL n-C13 in 1,1,2-trichloroethane. The dimethyl polysiloxane PS255 for coating the capillary gas chromatography (GC) column was from Fluka (Buchs, Switzerland). Raw fused silica tubing was from BGB Analytics, Boeckten, Switzerland.
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2.2. Sample collection
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Between February and August 2013, liver, spleen, MLN, lung and subcutaneous abdominal fat tissues (approximately 7 g each) were collected from 37 patients (11 females and 26 males) during autopsy at the Medical University of Vienna. From 14 of these subjects (5 females and 9 males) also brain, heart and kidney tissues were collected. Of each tissue, 5 g was stored at 20 °C until subjected to MOSH/MOAH analysis. Personal information, such as age, sex, weight, height, occupation, relevant diseases during life and cause of death, were available for each patient. The age varied between 25 and 91 years (Fig. 1). The study was approved by the Ethics Committee of the Medical University of Vienna (Project number 1785/2012).
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2.3. Sample preparation and extraction
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The thawed tissues were rinsed with 1% hydrochloric acid in order to avoid infection problems, dried with absorbent paper to remove water and blood, immersed in ethanol at a 1:1 (w/w) sample/ethanol ratio and homogenized using a PolytronÒ homogenizer (PT 10–35 GT). MOH were extracted as described by Biedermann and Grob (2012a). Briefly, to 2 g of tissue/ethanol homogenate weighted in a 10 mL centrifuge tube, 4 mL ethanol and 20 lL internal standard solution were added. This mixture was intensively shaken and left standing for at least 1 h to allow ethanol entering the particles. After centrifugation, ethanol was decanted into a 30 mL centrifuge tube. The tissue residue was homogenized with 5 mL hexane. After extraction overnight at ambient temperature, the tube was centrifuged and the supernatant hexane added to the previously collected ethanol. To separate the hexane phase from ethanol, approximately 10 mL of water was admixed.
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2.4. Chromatographic analysis
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MOSH and MOAH were analyzed by on-line normal phase high performance liquid chromatography (HPLC)-GC-flame ionization detection (FID) as described previously (Biedermann et al., 2009; Biedermann and Grob, 2012a). Briefly, 100 lL extract in hexane was injected into a 25 cm 2 mm i.d. HPLC silica gel column (LiChrospher Si 60, 5 lm) using hexane as eluent at 300 lL/min. The MOSH fraction was eluted between 1.45 and 2.45 min. The MOAH were eluted by a steep gradient with dichloromethane. The column was backflushed with 1 mL dichloromethane. HPLC-GC transfer occurred by the retention gap technique with partially concurrent eluent evaporation. A 7 m 0.53 mm i.d. uncoated, deactivated pre-column was connected to the solvent vapor exit and a 15 m 0.25 mm i.d. separation column coated in the laboratory with a 0.13 lm film of PS-255, a dimethyl polysiloxane. GC oven temperature was programmed from 65 °C (5 min) to 350 °C at 15 °C/min. The area corresponding to the MOSH was determined by the integration of the whole hump of largely unresolved material above the baseline transferred from a blank run (obtained by injecting hexane). Internal standards and endogenous hydrocarbons were subtracted from the hump as described previously (Biedermann et al., 2009; Biedermann and Grob, 2012b).
Fig. 1. Subjects sorted by age, with females in gray and males in black.
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3. Results
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3.1. Method validation
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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 the overnight extracts obtained by treatment with hydrochloric acid at 80 °C for 30 min (Concin et al., 2008) contained less than 1% MOSH compared to the previous extracts. The amount of water added to split the hexane/ethanol 1/1 (v/v) solution from the combined extracts was optimized using pyrene as marker, since this polyaromatic compound is better soluble in the ethanol/water than the MOSH. It was measured by HPLC-UV detection (245 nm) being part of the HPLC-GC analysis. Adding a volume of water equal to the ethanol, 82% of the pyrene was recovered in the hexane phase. Recovery increased to 97% when adding a doubled volume of water. It was previously established that the HPLC column used would safely retain 20 mg triglycerides, leaving enough packing for the MOSH/MOAH separation (Biedermann and Grob, 2012a). However, the capacity to retain the lipids from liver and brain was lower: injections of 50 lL of an extract of 1 g tissue in 1 mL hexane caused breakthrough and loaded GC with lipids such that the system needed being rinsed with solvent. For this reason the aliquot injected was reduced to 20 mg tissue. As sensitivity was abundant, this was also applied to the other tissues (see Section 2). However, there was still some breakthrough for brain extracts. These lipids were retained in a programmed temperature vaporizing (PTV) injector that was inserted between the transfer line and the uncoated GC precolumn, as described by Biedermann and Grob (2013), with an upper injector temperature of 250 °C. Repeatability was calculated by performing 5 replicate extractions from the same human liver homogenate containing 18 mg/kg MOSH. The relative standard deviation was 3%. The limit of detection (LOD) was determined performing 3 times the analysis of the extract from a liver tissue containing 32 mg/kg MOSH mixed at various ratios with an extract from a cow liver without detectable MOSH. At the lower calibration point (2.7 mg/kg for a range also including 5, 8, 20 and 30 mg/kg), the LOD was calculated as 3.3 times the standard deviation and plotting this value in the calibration line. The limit of quantification (LOQ) was obtained analogously multiplying the standard deviation by 10. The resulting LOD and LOQ were 0.7 and 2 mg/ kg, respectively.
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3.2. Concentrations
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Fig. 2 reports the MOSH concentrations (referring to fresh mass) in the liver, MLN, subcutaneous abdominal fat tissue, spleen and lung from 37 individuals sorted by increasing concentration. The highest concentrations, 1400 and 1390 mg/kg in the spleen and in the MLN, respectively, out of the scale shown, were from the same 87 y old male subject of 88 kg body weight. The highest value in liver, 900 mg/kg, was from a female subject of 65 y and 50 kg body weight. The plots for liver, MLN, fat tissue and spleen, all with the same scale, show MOSH concentrations in similar ranges, but values varied less for the MLN than the spleen. Many values in spleen were low, but also the highest concentration was found in spleen. Lung tissue contained far less MOSH than the other four.
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Please cite this article in press as: Barp, L., et al. Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.029
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Fig. 2. MOSH concentrations in 5 tissues sorted by increasing values.
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Ranges of concentrations, mean values and medians are listed in Table 1. Values are given separately for female and male subjects as well as for the complete set of samples. This table includes data for brain, heart and kidney which were analyzed only for 14 subjects. Mean and median concentrations were clearly highest for the MLN (223 and 166 mg/kg, respectively). Liver was next in
concentration, but only third in terms of the median, for which fat tissue was second. Spleens, for which most data on granulomas was reported in the past, contained 2.4 times less MOSH in term of mean values and 5.9 times less in terms of median than MLN. Brain, heart and kidney contained far less MOSH than the other tissues analyzed.
Table 1 Minimum/maximum MOSH concentrations (mg/kg fresh weight), mean values and medians listed for females, males and for the total of samples. Females (n = 11)
MLN Liver Fat tissue Spleen Lung
Males (n = 26) Average
Median
Min – max
Average
Median
Min – max
Average
Median
71–512 38–901 49–493 12–343
No. of Pages 10, Model 5G
12 May 2014 Food and Chemical Toxicology xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox 5 6
Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions
3 4 7
Q1
8 9 10 11 12 13
a
21 22 23 24 25 26 27 28
Università di Udine, Dipartimento di Scienze degli Alimenti via Sondrio 2/A, I-33100 Udine, Italy Clinical Institute for Pathology, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Wien, Austria Official Food Control Authority of the Canton of Zurich, Fehrenstrasse 15, P.O. Box, CH-8032 Zurich, Switzerland d Department of Pathology, Danube Hospital, Langobardenstr. 122, A-1220 Vienna, Austria e Department of Obstetrics and Gynecology, Innsbruck Medical University, Anichstr. 35, A-6020-Innsbruck, Austria b
Q2
14 1 2 6 9 17 18 19 20
Laura Barp a, Christoph Kornauth b, Tanja Wüger b, Margaretha Rudas b, Maurus Biedermann c, Angelika Reiner d, Nicole Concin e, Koni Grob c,⇑ c
a r t i c l e Q3
i n f o
Article history: Received 15 February 2014 Accepted 12 April 2014 Available online xxxx Keywords: Mineral oil saturated hydrocarbons (MOSH) Fat tissue Liver Spleen Granuloma Accumulation
a b s t r a c t Of 37 subjects aged 25–91 y (mean 67 y), mineral oil hydrocarbons were measured in subcutaneous abdominal fat tissue, mesenteric lymph nodes (MLN), spleen, liver and lung, for some of them also in kidney, heart and brain. No mineral oil aromatic hydrocarbons (MOAH) were detected. The mean concentration of mineral oil saturated hydrocarbons (MOSH) in the mesenteric lymph nodes was 223 mg/kg, in liver 131 mg/kg, in fat tissue 130 mg/kg, in spleen 93 mg/kg and in lung 12 mg/kg. They were clearly lower in kidney, heart and brain. The maxima, found in MLN and spleen, were 1390 and 1400 mg/kg, respectively. For a quarter of the subjects a total amount of MOSH in the body above 5 g was calculated. The MOSH composition in the fat tissue and the MLN appeared virtually identical and varied little between the subjects. It was centered on the n-alkanes C23 –C24, ranged from C16 to C35 and included hydrocarbons of plant origin. The MOSH in spleen and liver had almost the same composition for a given subject, but varied somewhat between subjects. They were centered between C25 and C27, ranged from C18 to beyond C45 and were without hydrocarbons of plant origin. Part of the MOSH seem to be strongly accumulated, resulting in far higher concentrations in human tissues related to exposure than observed in shorter term animal experiments. The composition of the accumulated MOSH does not support that Class I mineral oils, sometimes termed ‘‘food grade’’, are less accumulated in the human body than Class II and III oils, which questions the present classification. Ó 2014 Elsevier Ltd. All rights reserved.
30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
49 50
1. Introduction
51
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
52 53 54 55 56 57 58 59 60 61 62 63
⇑ Corresponding author. Tel.: +41 432447131; fax: +41 432447101. E-mail address: [email protected] (K. Grob).
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 ran-
http://dx.doi.org/10.1016/j.fct.2014.04.029 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Barp, L., et al. Mineral oil in human tissues, Part I: Concentrations and molecular mass distributions. Food Chem. Toxicol. (2014), http://dx.doi.org/10.1016/j.fct.2014.04.029
64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81
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ged 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 lm to over 100 lm. 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 nC32 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.
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2. Materials and methods
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2.1. Materials
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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
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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) and 0.15 mg/mL n-C13 in 1,1,2-trichloroethane. The dimethyl polysiloxane PS255 for coating the capillary gas chromatography (GC) column was from Fluka (Buchs, Switzerland). Raw fused silica tubing was from BGB Analytics, Boeckten, Switzerland.
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2.2. Sample collection
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Between February and August 2013, liver, spleen, MLN, lung and subcutaneous abdominal fat tissues (approximately 7 g each) were collected from 37 patients (11 females and 26 males) during autopsy at the Medical University of Vienna. From 14 of these subjects (5 females and 9 males) also brain, heart and kidney tissues were collected. Of each tissue, 5 g was stored at 20 °C until subjected to MOSH/MOAH analysis. Personal information, such as age, sex, weight, height, occupation, relevant diseases during life and cause of death, were available for each patient. The age varied between 25 and 91 years (Fig. 1). The study was approved by the Ethics Committee of the Medical University of Vienna (Project number 1785/2012).
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2.3. Sample preparation and extraction
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The thawed tissues were rinsed with 1% hydrochloric acid in order to avoid infection problems, dried with absorbent paper to remove water and blood, immersed in ethanol at a 1:1 (w/w) sample/ethanol ratio and homogenized using a PolytronÒ homogenizer (PT 10–35 GT). MOH were extracted as described by Biedermann and Grob (2012a). Briefly, to 2 g of tissue/ethanol homogenate weighted in a 10 mL centrifuge tube, 4 mL ethanol and 20 lL internal standard solution were added. This mixture was intensively shaken and left standing for at least 1 h to allow ethanol entering the particles. After centrifugation, ethanol was decanted into a 30 mL centrifuge tube. The tissue residue was homogenized with 5 mL hexane. After extraction overnight at ambient temperature, the tube was centrifuged and the supernatant hexane added to the previously collected ethanol. To separate the hexane phase from ethanol, approximately 10 mL of water was admixed.
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2.4. Chromatographic analysis
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MOSH and MOAH were analyzed by on-line normal phase high performance liquid chromatography (HPLC)-GC-flame ionization detection (FID) as described previously (Biedermann et al., 2009; Biedermann and Grob, 2012a). Briefly, 100 lL extract in hexane was injected into a 25 cm 2 mm i.d. HPLC silica gel column (LiChrospher Si 60, 5 lm) using hexane as eluent at 300 lL/min. The MOSH fraction was eluted between 1.45 and 2.45 min. The MOAH were eluted by a steep gradient with dichloromethane. The column was backflushed with 1 mL dichloromethane. HPLC-GC transfer occurred by the retention gap technique with partially concurrent eluent evaporation. A 7 m 0.53 mm i.d. uncoated, deactivated pre-column was connected to the solvent vapor exit and a 15 m 0.25 mm i.d. separation column coated in the laboratory with a 0.13 lm film of PS-255, a dimethyl polysiloxane. GC oven temperature was programmed from 65 °C (5 min) to 350 °C at 15 °C/min. The area corresponding to the MOSH was determined by the integration of the whole hump of largely unresolved material above the baseline transferred from a blank run (obtained by injecting hexane). Internal standards and endogenous hydrocarbons were subtracted from the hump as described previously (Biedermann et al., 2009; Biedermann and Grob, 2012b).
Fig. 1. Subjects sorted by age, with females in gray and males in black.
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3. Results
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3.1. Method validation
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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 the overnight extracts obtained by treatment with hydrochloric acid at 80 °C for 30 min (Concin et al., 2008) contained less than 1% MOSH compared to the previous extracts. The amount of water added to split the hexane/ethanol 1/1 (v/v) solution from the combined extracts was optimized using pyrene as marker, since this polyaromatic compound is better soluble in the ethanol/water than the MOSH. It was measured by HPLC-UV detection (245 nm) being part of the HPLC-GC analysis. Adding a volume of water equal to the ethanol, 82% of the pyrene was recovered in the hexane phase. Recovery increased to 97% when adding a doubled volume of water. It was previously established that the HPLC column used would safely retain 20 mg triglycerides, leaving enough packing for the MOSH/MOAH separation (Biedermann and Grob, 2012a). However, the capacity to retain the lipids from liver and brain was lower: injections of 50 lL of an extract of 1 g tissue in 1 mL hexane caused breakthrough and loaded GC with lipids such that the system needed being rinsed with solvent. For this reason the aliquot injected was reduced to 20 mg tissue. As sensitivity was abundant, this was also applied to the other tissues (see Section 2). However, there was still some breakthrough for brain extracts. These lipids were retained in a programmed temperature vaporizing (PTV) injector that was inserted between the transfer line and the uncoated GC precolumn, as described by Biedermann and Grob (2013), with an upper injector temperature of 250 °C. Repeatability was calculated by performing 5 replicate extractions from the same human liver homogenate containing 18 mg/kg MOSH. The relative standard deviation was 3%. The limit of detection (LOD) was determined performing 3 times the analysis of the extract from a liver tissue containing 32 mg/kg MOSH mixed at various ratios with an extract from a cow liver without detectable MOSH. At the lower calibration point (2.7 mg/kg for a range also including 5, 8, 20 and 30 mg/kg), the LOD was calculated as 3.3 times the standard deviation and plotting this value in the calibration line. The limit of quantification (LOQ) was obtained analogously multiplying the standard deviation by 10. The resulting LOD and LOQ were 0.7 and 2 mg/ kg, respectively.
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3.2. Concentrations
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Fig. 2 reports the MOSH concentrations (referring to fresh mass) in the liver, MLN, subcutaneous abdominal fat tissue, spleen and lung from 37 individuals sorted by increasing concentration. The highest concentrations, 1400 and 1390 mg/kg in the spleen and in the MLN, respectively, out of the scale shown, were from the same 87 y old male subject of 88 kg body weight. The highest value in liver, 900 mg/kg, was from a female subject of 65 y and 50 kg body weight. The plots for liver, MLN, fat tissue and spleen, all with the same scale, show MOSH concentrations in similar ranges, but values varied less for the MLN than the spleen. Many values in spleen were low, but also the highest concentration was found in spleen. Lung tissue contained far less MOSH than the other four.
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Fig. 2. MOSH concentrations in 5 tissues sorted by increasing values.
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Ranges of concentrations, mean values and medians are listed in Table 1. Values are given separately for female and male subjects as well as for the complete set of samples. This table includes data for brain, heart and kidney which were analyzed only for 14 subjects. Mean and median concentrations were clearly highest for the MLN (223 and 166 mg/kg, respectively). Liver was next in
concentration, but only third in terms of the median, for which fat tissue was second. Spleens, for which most data on granulomas was reported in the past, contained 2.4 times less MOSH in term of mean values and 5.9 times less in terms of median than MLN. Brain, heart and kidney contained far less MOSH than the other tissues analyzed.
Table 1 Minimum/maximum MOSH concentrations (mg/kg fresh weight), mean values and medians listed for females, males and for the total of samples. Females (n = 11)
MLN Liver Fat tissue Spleen Lung
Males (n = 26) Average
Median
Min – max
Average
Median
Min – max
Average
Median
71–512 38–901 49–493 12–343
