In vitro metabolic study on alkanes in hepatic microsomes from humans and rats 1

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1 Supporting Publications 2012:EN-263 EXTERNAL SCIENTIFIC REPORT humans and rats 1 ABSTRACT Drs. Jean-Pierre Cravedi and Elisabeth Perdu 2, 3 Institut National de la Recherche Agronomique INRA - France The purpose of this in vitro study was to measure the efficiency of rats and humans at linear, branched and naphthenic hydrocarbon metabolism. The biotransformation of three radiolabelled alkanes, namely 14 C-heptadecane (n-alkane, 17 carbon atoms), 3 H-pristane (branched-alkane, 19 carbon atoms) and 3 H-dodecylcyclohexane (cyclo-alkane, 18 carbon atoms) was investigated using liver microsomes from three rat strains (Wistar, Sprague-Dawley and Fischer 344) and from three different pools of human donors (at least 10 donors in each case). In each case, both males and females were studied and three concentrations (20, 40 and 60 µm) were tested. After incubation with microsomes, the hydrocarbon substrate and produced metabolites were extracted then separated and quantified by radio-hplc. The hydroxylation rate was evaluated from the sum of the different metabolites. On average, the hydroxylation rates of heptadecane incubated with male rat liver microsomes at the highest concentration tested (60 µm) varied from 78 ± 32 moles/hr/mg proteins in Sprague Dawley rats to 101 ± 20 pmoles/hr/mg proteins (total amount of metabolites formed) in Fischer 344 rats. For females, incubations carried out at the same concentration resulted in values varying from 76 ± 38 pmoles/hr/mg proteins (Fischer 344) to 148 ± 47 pmoles/hr/mg proteins (Sprague Dawley). In humans, the heptadecane hydroxylation rates determined for 60 µm incubations was 159 ± 46 pmoles/hr/mg proteins and 180 ± 10 pmoles/hr/mg proteins, for males and females, respectively. No metabolism was detected when pristane or dodecylcyclohexane were incubated with human or rat hepatic microsomes (irrespective of the rat strain investigated). Institut National de la Recherche Agronomique INRA - France KEY WORDS Alkanes, metabolism, in vitro biotransformation, liver microsomes, rat, human 1 Question No EFSA-Q Jean-Pierre Cravedi, Elisabeth Perdu. 3 Acknowledgement: Sandrine Bruel for the technical assistance. Any enquiries related to this output should be addressed to contam@efsa.europa.eu Suggested citation: Institut National de la Recherche Agronomique INRA - France; In vitro metabolic study on alkanes in hepatic microsomes from. Supporting Publications 2012:EN-263. [64 pp.]. Available online: European Food Safety Authority, 2012

2 TABLE OF CONTENTS Abstract... 1 Table of contents... 2 Background as provided by the European Food Safety Authority... 3 Terms of reference as provided by the European Food Safety Authority INTRODUCTION METHODOLOGY Chemicals Subcellular fraction preparation Incubation of radiolabelled alkanes with rat and human liver microsomes Radioactivity counting Analytical procedures Statistics RESULTS Radiopurity Characteristics of microsome preparations Incubations of alkanes with boiled microsomes Heptadecane biotransformation Pristane and dodecylcyclohexane biotransformation DISCUSSION CONCLUSIONS RECOMMENDATIONS References Appendix Abbreviations Supporting publications 2012:EN-263 2

3 BACKGROUND AS PROVIDED BY THE EUROPEAN FOOD SAFETY AUTHORITY The European Commission asked the European Food Safety Authority (EFSA) to issue a Scientific Opinion on the risk to human health arising from the presence of mineral hydrocarbons in food (EFSA-Q ). The request has been allocated to the Scientific Panel on Contaminants in the Food Chain (CONTAM Panel) of EFSA. Mineral oil hydrocarbons can be classified into mineral oil aromatic hydrocarbons and mineral oil saturated hydrocarbons. The latter group includes paraffins (consisting mainly of linear and branched alkanes) and naphthenes (consisting mainly of cycloalkanes). Highly refined mineral oil saturated hydrocarbons intended for food use were classified by FAO/WHO, based on viscosity, average molecular mass and carbon number at 5 % distillation point, into the following groups: microcrystalline wax, low melting point wax, mineral oil (high viscosity), mineral oil (medium and low viscosity) class I, mineral oil (medium and low viscosity) class II, and mineral oil (medium and low viscosity) class III (FAO/WHO, 1996, 2002). Subchronic exposure to low melting point waxes (LMPW) and medium- and low-viscosity mineral oils (class II and III) caused inflammatory responses in the liver (microgranulomas) and mesenteral lymph nodes (hystiocytosis) in Fischer rats, with higher severity in females. Such effects were not observed in Sprague Dawley rats, Long-Evans rats and beagle dogs when exposed to the same products at similar concentrations (Smith et al., 1996, Firriolo et al, 1995; Griffis et al., 2010). The greater sensitivity of Fischer rats to LMPW and medium- and low-viscosity mineral oils (class II and III) has been associated with a higher retention of the mineral oil saturated hydrocarbons in the tissues, possibly due to a reduced ability to metabolise absorbed hydrocarbons in this rat strain in comparison to Sprague Dawley rats (Miller et al., 1995; FAO/WHO, 2002; Halladay et al., 2002). However, no information is available on metabolism rates of the main components of these products, i.e. linear alkanes, branched alkanes and alkylated cycloalkanes, from which such a conclusion could be drawn. In addition, no information is available to assess which rat strain is most suitable to compare to the situation in humans. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN FOOD SAFETY AUTHORITY The objective of the assignment is to conduct an in vitro study to determine the rates of metabolism for three substances representative of the main components of mineral oil saturated hydrocarbons (linear alkanes, branched alkanes and alkylated cycloalkanes). The rates of metabolism shall be determined using hepatic microsomes obtained from male and female rats of three strains (Fischer F-344, Sprague Dawley and Wistar) and from both male and female human liver samples. This contract was awarded by EFSA to: Contractor: Institut National de la Recherche Agronomique INRA, France Contract title: Contract number: CT/EFSA/CONTAM/2011/02 Supporting publications 2012:EN-263 3

4 1. INTRODUCTION The sensitivity of Fischer 344 rats to mineral oil adverse effects, compared with other strains such as Sprague Dawley rats, has been reported in several studies (Miller et al., 1995). This difference could be due to a more efficient metabolism of n-, branched- and/or cyclo-alkanes, the major classes of saturated hydrocarbons present in mineral oil, in Sprague Dawley rats, resulting in a more effective clearance of these compounds in this strain compared to Fischer 344 rats. Accordingly, measuring the extent of biotransformation of these hydrocarbons in different rat strains and in humans could help to understand the discrepancies observed in rat toxicological studies and in identifying which strain is appropriate to predict the possible effects of mineral oil in humans. The objective of this study was to measure the efficiency of saturated hydrocarbons metabolism in three rat strains (Fischer 344, Sprague-Dawley and Wistar) as well as humans. Since oxidation is the first step in the biotransformation pathways of these compounds, this metabolic step was investigated using liver microsomes prepared from male and female rats of the three strains, but also from male and female human donors. The rate and extent of biotransformation of the selected compounds were measured in incubations supplemented with an excess of NADPH as a cofactor (directly or as part of a regenerating system) to allow cytochrome P450 system to operate. For such studies, radiolabelled substrates are very useful in measuring the metabolic rates, since they permit to follow the parent compound clearance, but also the formation of all radiolabelled metabolites, even when they are unknown compounds. In addition, radioactivity monitoring associated to chromatography techniques is appropriate for the quantitative determination of 14 C- or tritiated analytes. In this study, 14 C-heptadecane (17 carbon atoms), 3 H-pristane (19 carbon atoms) and 3 H- dodecylcyclohexane (18 carbon atoms) were investigated as selected substances for normal-, branched- and cyclo-alkanes, respectively. The selection criteria were based on availability as radiochemical, specific activity, and carbon number (relevant for alkanes present in food). Quantitative analyses of metabolites were carried out by radio-chromatography. 2. METHODOLOGY 2.1. Chemicals 1-14 C-n-Heptadecane (1069 MBq/mmol) was from Isotopchim (Ganagobie-Peyruis, France); uniformly labelled 3 H-pristane (35.5 GBq/mmol) was from the Commissariat à l Energie Atomique (CEA, Saclay, France) ; ring labelled 3 H-dodecylcyclohexane (1073 MBq/mmol) was prepared from radiolabelled dodecylbenzene (provided by the CEA) as indicated by Cravedi and Tulliez (1987). All these radiochemicals were already available in the Laboratory. The radiochemical purity of radiolabelled hydrocarbons was checked by reversed phase radio-hplc and was found to be higher than 96 % for all. Other chemicals, including unlabelled heptadecane, pristane and dodecylcyclohexane were from Sigma-Aldrich (Saint Quentin Fallavier, France), except Flo-Scint II and Ultima Gold liquid scintillation cocktails (from PerkinElmer Life and Analytical Sciences, Courtaboeuf, France) and chromatography-grade solvents (from Scharlau, Barcelona, Spain). Ultrapure water from Milli-Q system (Millipore, Saint Quentin en Yvelines, France) was used for incubations and for the preparation of HPLC mobile phases Subcellular fraction preparation Male and female Sprague Dawley, Wistar and Fischer 344 rats weighing g and fed standard commercial diets were used in this study. The animals were killed by asphyxiation in CO 2 gas, followed by decapitation. The livers were removed immediately after slaughtering and perfused with 0.9 % saline. For each strain, liver microsomes from 3 males and 3 females were prepared as described by Perdu-Durand and Tulliez (1985) and stored at 80 C in phosphate buffer (0.1 M, ph 7.4) containing 20 % glycerol. The protein concentration was determined by the method of Lowry et al. Supporting publications 2012:EN-263 4

5 (1951). Three different pools of male and female cryopreserved human liver microsomes prepared from donors were obtained from Xenotech, Tebu-bio (Le Perray en Yvelines, France), Gentest BD Biosciences (Woburn, MA USA ) and Celsis/ In vitro Technologies (Baltimore, MD USA) and used in this experiment Incubation of radiolabelled alkanes with rat and human liver microsomes Incubations were performed in vials containing 2 mg of rat or human microsomal proteins, 1mM NADPH, 1mM NADP, 1mM NAD, 3 mm glucose 6 phosphate, 2 IU of glucose 6 phosphate dehydrogenase in a volume of 0.5 ml phosphate buffer (0.1 M, ph 7.4) containing 5mM MgCl 2. After 5 min pre-incubation, the reaction was started by adding radiolabelled alkanes (heptadecane, dodecylcyclohexane or pristane) in 5µL ethanol. For rat and human samples, incubations were carried out at 37 C for 2 hr under agitation with 20, 40, 60 μm, of radiolabelled substrate, then stopped by adding 200 µl 5 % trichloracetic acid. Three separate incubations were performed for each concentration of heptadecane, dodecylcyclohexane and pristane investigated in the different rat strains (males and females) and in humans (males and females) Radioactivity counting Aliquots of incubation media were counted directly in a Packard scintillation counter (Model Tricarb 2200CA; Packard Instruments, Meriden, CT, USA) using Ultimagold as the scintillation cocktail. An external standard method was used for automatic quench correction Analytical procedures The hydrocarbon substrate and hydroxylation products were extracted twice with 2 ml ethyl acetate. The organic solvent was evaporated to dryness and the residue was dissolved in 0.5 ml methanol. The radioactivity in the extract was determined by counting a 5 µl aliquot in a scintillation counter. For heptadecane, pristane and dodecylcyclohexane metabolic profiling, the analyses were performed on a Spectra P1000 pump (Thermo Separation Products, Les Ulis, France) associated with a Zorbax SB-C 18 column (5 µm, 250 x 4.6 mm) (Agilent, Interchim, Montluçon, France) protected by a Kromasil C 18 guard precolumn and connected for radioactivity detection to a Packard Flo-One / β A500 detector (Packard Instruments Co., Meriden, CT) with Flow-scint II as scintillation cocktail. Metabolites were quantified by integrating the area under peaks monitored by radioactivity detection. Mobile phases used at a flow rate of 1 ml/min consisted of methanol and water, 35:65 (v/v) as solvent A and methanol as solvent B. The elution gradient was as follows: 0-5 min, 50:50 (v/v) A/B; 5-10 min, linear gradient leading to 100 % B; min, 100 % B; min, return to 50:50 (v/v) A/B Statistics Intergroup comparisons (alkane concentration*biological model or sex) were performed by a two-way ANOVA followed by Bonferroni and Tukey post tests. Differences between groups were considered significant when p< RESULTS 3.1. Radiopurity The radiopurity of 14 C-heptadecane, 3 H-dodecylcyclohexane and 3 H-pristane was found to be 97.3 %, 96.5 % and 96.1 %, respectively (Figure 1). Supporting publications 2012:EN-263 5

6 A B C Figure 1: Radio-HPLC analyses of radiochemicals used in this study. A = 14 C-heptadecane, B = 3 H- dodecylcyclohexane, C = 3 H-pristane. Supporting publications 2012:EN-263 6

7 3.2. Characteristics of microsome preparations Table 1: Protein, cytochrome b5 and cytochrome P450 contents in hepatic microsomes prepared from Wistar, Sprague Dawley and Fischer 344 rat strains. Hepatic microsomes prepared from different rat strains were characterized before use (Table I). Protein contents were determined by the Lowry method (1951). Cytochrome b5 and cytochrome P450 were measured as described by Omura and Sato (1964). Values are according to those usually described for rat microsomes and indicate similar cytochrome P450 contents for each strain. Biochemical characteristics of Human microsomes were provided by the suppliers (Table II). Supporting publications 2012:EN-263 7

8 Table 2: Protein, cytochrome b5 and cytochrome P450 contents of Human hepatic microsomes as provided by the suppliers. Pool 1 and pool 4 were from 10 different individuals each and were purchased from Xenotech-TEBU-bio, pool 2 and pool 5 were from 25 different individuals each and were from Gentest-BD Biosciences, pool 3 and 6 were from 25 different donors each and were obtained from Celsis- In vitro Technologies. ND = not determined. [Protein] (mg/ml) [b5] (nmol/mg protein) [P450] (nmol/mg prot) Humans (males) Pool Pool Pool 3 24 ND 0.35 Mean ± SD 21.3 ± ± 0.08 Hum. (females) Pool Pool Pool 6 20 ND 0.38 Mean ± SD 20.0 ± ± 0.05 SD: standard deviation Incubations of alkanes with boiled microsomes In order to check if a non-enzymatic degradation of alkanes could occur during the incubation, 14 C- heptadecane, 3 H-dodecylcyclohexane and 3 H-pristane were incubated during 2 h with boiled Wistar rat liver microsomes. Radio-HPLC analyses showed no trace of degradation compound (Figure 2), indicating the absence of chemical transformation of the substrates. Supporting publications 2012:EN-263 8

9 A B C Figure 2: Radio-HPLC analyses of 14 C-heptadecane (A), 3 H-dodecylcyclohexane (B), and 3 H- pristane (C) incubated with boiled rat hepatic microsomes Heptadecane biotransformation Incubation of 14 C-heptadecane with rat liver microsomes results in biotransformation levels varying between 0.7 % and 7.1 % of the dose, depending on the tested concentration, the strains, the gender and the individuals (see Tables A, B, C in annex). Radio-HPLC chromatograms (Figure 3) show the presence of several metabolites having retention times between 12 and 22 min. Supporting publications 2012:EN-263 9

10 A B C Figure 3: Typical radio-hplc metabolic profile of 14 C-heptadecane (20 µm) incubated with male (left side) and female (right side) rat liver microsomes. A = Wistar rats, B = Sprague Dawley, C = Fischer The peak highlighted in blue (R T = 17 min) corresponds to an impurity (see figure 1 radio-purity testing) and was not taking into account in the sum of produced metabolites for the calculation of heptadecane metabolic rates. Supporting publications 2012:EN

11 In Humans, these levels ranged from 1.4 to 3.8 % of the dose (Table D) and metabolic profiles were similar to those observed in rats (Figure 4). Figure 4: Typical radio-hplc metabolic profile of 14 C-heptadecane (20 µm) incubated with male (left side) and female (right side) Human liver microsomes. The peak highlighted in blue (R T = 17 min) corresponds to an impurity (see figure 1 radio-purity testing) and was not taking into account in the sum of formed metabolites for the calculation of heptadecane metabolic rates. The results, expressed as pmoles of metabolites formed per hour and per mg of microsomal proteins, are summarized in figure 5 (see also Tables A, B, C, D in annex). They indicate that in rats, saturation kinetics was attained at the lowest concentration tested (20 µm). The average hydroxylation rates (calculated as the total amount of metabolites formed) of heptadecane incubated with male rat liver microsomes at the highest concentration tested (60 µm) were 95 ± 28 pmoles/hr/mg proteins in Wistar strain, 78 ± 32 pmoles/hr/mg proteins in Sprague Dawley, and 101 ± 20 pmoles/hr/mg proteins in Fischer 344. For females, incubations carried out at the same concentration resulted in values corresponding to 134 ± 49 pmoles/hr/mg proteins (Wistar), 148 ± 47 pmoles/hr/mg proteins (Sprague Dawley), and 76 ± 38 pmoles/hr/mg proteins (Fischer 344). Statistical analyses based on two-way ANOVA, indicate no significant differences between males and females, irrespective of the strain considered, and no significant differences between strains regarding males. However, with respect to females, a significantly lower (p < 0.05) heptadecane hydroxylation activity was observed in Fischer 344 rats compared to Sprague Dawley. Supporting publications 2012:EN

12 pmol/hr/mg prot pmol/hr/mg prot pmol/hr/mg prot INRA France 250 WISTAR Males Females Sprague Daw ley M ales Females Fischer Males Females Figure 5: Production rates of heptadecane metabolites by liver microsomes prepared from rats (Wistar, Sprague Dawley and Fischer-344) and humans. Data are means ± SD from 3 animals (rats) or from 3 different pools (prepared each from at least 10 individuals) of human microsomes. Supporting publications 2012:EN

13 In humans, the heptadecane hydroxylation rates, determined for 60 µm incubations was 159 ± 46 pmoles/hr/mg proteins and 180 ± 10 pmoles/hr/mg proteins, for males and females, respectively (Figure 5, Table D in Annex). The comparison of the metabolic rates at 20, 40, and 60 µm suggests that the enzyme responsible for the hydroxylation of heptadecane is not saturated with the substrate, at the highest concentration tested (60 µm). The statistical analyses of the data show significant differences (p < 0.05) between 20 µm and 40 µm and between 20 µm and 60 µm, confirming that the saturation kinetics was not reached. These results suggest that no major differences, if any, exist between rat stains or between males and females regarding heptadecane hydroxylation. Nevertheless, they provide evidence that the biotransformation of heptadecane occurs at a significantly higher extent in Humans (females) as compared with Fisher 344 rats (females) (p < 0.05) Pristane and dodecylcyclohexane biotransformation Radio-HPLC analyses of incubates from human and rat liver microsomes with 3 H-pristane or 3 H-dodecylcyclohexane showed no trace of metabolites (see Annex, figures C,D,E,F), indicating that in our experimental conditions, the biotransformation of these alkanes did not occur. 4. DISCUSSION Several in vivo metabolic studies on n-, cyclo- and branched alkanes have been reported in rodents (McCarthy, 1964; Tulliez and Bories, 1978, 1979; Le Bon et al, 1988), showing that oxidation of these hydrocarbons occurred in mammals. In contrast, in vitro studies on the biotransformation of these compounds are scarce and are limited to n-alkanes. Perdu-Durand and Tulliez (1985) investigated the biotransformation of heptadecane using hepatic microsomes from chickens, rabbits, rats and trout. The higher hydroxylation rate was found for chicken: in this species the rate of heptadecane metabolism was approximately 10-fold greater than in rats or rabbits. In this study we investigated the biotransformation of 3 saturated hydrocarbons, namely n-heptadecane, pristane, and dodecylcyclohexane, as model compounds for linear-, branched- and cyclo-alkanes, respectively. Since hepatic microsomes have been found to be able to hydroxylate n-alkanes via a cytochrome P450 dependent oxidation reaction (Perdu-Durand and Tulliez, 1985), this in vitro system was selected to study the metabolism of alkanes in three rat strains (Wistar, Sprague Dawley, and Fischer 344). Using the same approach, the biotransformation of the 3 alkanes was also investigated in humans. In each case, the incubations were performed with microsomes prepared from both males and females. The results obtained with n-heptadecane indicate that both rats and humans are able to metabolize this saturated hydrocarbon; nevertheless there is no evidence that alkanes having a higher MW than n-heptadecane are biotransformed at similar rates. No strong differences were observed between rat strains, and between rats and humans, nevertheless for females, our data give evidence that biotransformation of heptadecane occurs at a higher extent in Humans compared to Fischer 344 rats. Our results also indicate that the activities of microsomal enzymes involved in the hydroxylation of heptadecane were saturated from 20 µm in rats whereas the highest concentration tested (60 µm) was likely below the saturating substrate concentration for humans. It can be speculated that the difference between would have been higher at saturation conditions. Unfortunately, due to analytical limitations, concentrations higher than 60 µm were not investigated. The objective of this study was not to identify the biotransformation products. Furthermore, the very low amount of hydroxylation compounds formed from heptadecane did not allow further characterisation. However, the co-elution of two metabolites with heptadecanoic acid and Supporting publications 2012:EN

14 heptadecan-1-ol standards is in accordance with the metabolic pattern of this hydrocarbon in rodents (Tulliez and Bories, 1978) and suggests that similar pathways occur in humans. In rats as in humans, assays for oxidation of pristane and dodecylcyclohexane were unsuccessful, irrespective of the rat strain, the gender or the concentration tested. These results suggest that branched- and cyclo-alkanes are more resistant to biotransformation than linear alkanes. In addition to the protocol described above, a pilot study was carried out with hepatic microsomes prepared from male rats induced by 3-methylcholanthrene (a CYP1A inducer) injected intraperitoneally (40 mg/kg bw) or dietary administered (4 mg/kg feed) clofibrate (a CYP4A inducer). At 40 µm, a 3-fold induction of 14 C-heptadecane hydroxylation activity was observed for both types of treatment, but no trace of metabolites were detected when 3 H-dodecylcyclohexane or 3 H-pristane were incubated with hepatic microsomes from induced rats (data not shown). Although this pilot study was not duplicated, it suggests that if the biotransformation of pristane and dodecylcyclohexane happens in rat microsomes, it would occur at much lower rates than heptadecane. CONCLUSIONS Both rat and human hepatic microsomes showed the ability to metabolise n-heptadecane. No strong differences were observed in the biotransformation of n-heptadecane between the three rat strains, and between rats and humans, with the exclusion of females, for which biotransformation of n-heptadecane occurs at a higher extent in humans compared to Fischer 344. For n-heptadecane microsomal enzyme involved in the hydroxylation were saturated from 20 µm in rats whereas the highest concentration tested (60 µm) was likely below the saturating substrate concentration for humans. There is no evidence that alkanes having a higher MW than n-heptadecane are biotransformed at similar rates. Heptadecanoic acid and heptadecan-1-ol were tentatively identified as biotransformation products of n-heptadecane. No biotransformation was observed for pristane and dodecylcyclohexane either in rat or in human samples, irrespective of the rat strain, the gender or the concentrations tested (from 20 to 60 µm). These results suggest that branched- and cyclo-alkanes are more resistant to biotransformation than linear alkanes. RECOMMENDATIONS Knowing that dodecylcyclohexane and pristane biotransformation exists in rat in vivo, it can be anticipated that a different methodology, such as cultured primary hepatocytes, allowing incubation times of several days instead of few hours, would result in a measurable metabolite production and could be used to compare the toxicokinetics of cyclo- and branched-alkanes between Sprague Dawley and Fischer 344 rats, and between rats and humans. Nevertheless, in that case, a preliminary experiment should be undertaken to make sure that hepatocytes are able to reveal the metabolism of these alkanes. Since the metabolic rates of mineral oil saturated hydrocarbons depend on the molecular weight of these compounds, a broader view of their fate would necessitate incubations with various model compounds representative of the different classes of alkanes. Supporting publications 2012:EN

15 REFERENCES Cravedi JP and Tulliez JE Urinary metabolites of dodecylcyclohexane in Salmo Gairdneri: evidence of aromatization and taurine conjugation in trout. Xenobiotica, 17, FAO/WHO (Food and Agriculture Organization/World Health Organization), Evaluation of certain food additives and contaminants: forty-fourth report of the Joint FAO/WHO Expert Committee on food Additives. WHO Technical Report Series 859. Available from FAO/WHO (Food and Agriculture Organization/World Health Organization), Evaluation of certain food additives: fifty-ninth report of the Joint FAO/WHO Expert Committee on food Additives. WHO Technical Report Series 913. Available from Firriolo JM, Morris CF, Trimmer GW, Twitty LD, Smith JH and Freeman JJ, Comparative 90- day feeding study with Low viscosity white mineral oil in Fischer-344 and Sprague Dawleyderived CRL:CD rats. Toxicologic Pathology, 23, Griffis LC, Twerdok LE, Francke-Carroll S, Biles RW, Schroeder RE, Bolte H, Faust H, Hall WC and Rojko J, Comparative 90-day dietary study of paraffin wax in Fischer-344 and Sprague Dawley rats. Food and Chemical Toxicology, 48, Halladay JS, Mackerer CR, Twerdok LE and Sipes IG, Comparative pharmacokinetic and disposition studies of [1-14C]1-eicosanylcyclohexane, a surrogate mineral hydrocarbon, in female Fischer-344 and Sprague-Dawley rats. Drug Metabolism and Disposition, 30, Le Bon AM, Cravedi JP and Tulliez JE, Disposition and metabolism of pristane in rat. Lipids, 23, Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, McCarthy RD, Mammalian metabolism of straight-chain saturated hydrocarbons. Biochimica et Biophysica Acta, 84, Miller MJ, Lonardo EC, Greer RD, Bevan C, Edwards DA, Smith JH and Freeman JJ, Variable response of species and strains to white mineral oils and paraffin waxes. Regulatory Toxicology and Pharmacology, 23, Omura T and Sato R The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. Journal of Biological Chemistry, 239, Perdu-Durand EF and Tulliez JE, Hydrocarbon hydroxylation system in liver microsomes from four animal species. Food and Chemical Toxicology, 23, Smith JH, Mallett AK, Priston RAJ, Brantom PG, Worrell NR, Sexsmith C, Simpson BJ, day feeding study in Fischer-344 rats of highly refined petroleum-derived food-grade white oils and waxes Toxicology Pathology 24: Tulliez JE and Bories GF, Metabolism of a n-paraffin, heptadecane, in rats. Lipids, 13, Tulliez JE and Bories GF, Metabolism of naphthenic hydrocarbons. Utilization of a monocyclic paraffin, dodecylcyclohexane, by rat. Lipids, 14, Supporting publications 2012:EN

16 APPENDIX Table A: Heptadecane hydroxylation activity in Wistar rat liver microsomes (M = males, F = females). WISTAR Heptadecane concentration (µm) Metabolites (% total radioactivity) Formed metabolites (pmol/hr/mg prot) Mean (pmol/hr/mg prot) Standard deviation M M M M M M M M M F F F6 20 2,54 64 F F F F F F Supporting publications 2012:EN

17 Table B: Heptadecane hydoxylation activity in Sprague Dawley rat liver microsomes (M = males, F = females). Sprague Dawley Metabolites (% total radioactivity) Formed metabolites (pmol/hr/mg prot) Mean (pmol/hr/mg prot) Heptadecane concentration M M M Standard deviation M M M M M M F F F F F F F F F Supporting publications 2012:EN

18 Table C: Heptadecane hydroxylation activity in Fischer 344 rat liver microsomes (M = males, F = females). Fischer 344 Heptadecane concentration (µm) Metabolites (% total radioactivity) Formed metabolites (pmol/hr/mg prot) Mean (pmol/hr/mg prot) Standard deviation M M M M M M M M M F F F F F F F F F Supporting publications 2012:EN

19 Table D: Heptadecane hydroxylation activity in Human liver microsomes (M = males, F = females). Heptadecane concentration (µm) Metabolites (% total radioactivity) Formed metabolites (pmol/hr/mg prot) Mean (pmol/hr/mg prot) Standard deviation Human Tebu : Pool M BD : Pool M Celsis : Pool M Tebu : Pool M BD : Pool M Celsis : Pool M Tebu : Pool M BD : Pool M Celsis : Pool M Tebu : Pool F BD : Pool F Celsis : Pool F Tebu : Pool F4 40 2, BD : Pool F5 40 2, Celsis : Pool F6 40 2, Tebu : F BD : Pool F Celsis : Pool F Supporting publications 2012:EN

20 Figure A1: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Wistar) hepatic microsomes (20 µm). Supporting publications 2012:EN

21 Figure A2: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Wistar) hepatic microsomes (40 µm). Supporting publications 2012:EN

22 Figure A3: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Wistar) hepatic microsomes (60 µm). Supporting publications 2012:EN

23 Figure A4: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Wistar) hepatic microsomes (20 µm). Supporting publications 2012:EN

24 Figure A5: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Wistar) hepatic microsomes (40 µm). Supporting publications 2012:EN

25 Figure A6: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Wistar) hepatic microsomes (60 µm). Supporting publications 2012:EN

26 Figure A7: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Sprague Dawley) hepatic microsomes (20 µm). Supporting publications 2012:EN

27 Figure A8: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Sprague Dawley) hepatic microsomes (40 µm). Supporting publications 2012:EN

28 Figure A9: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Sprague Dawley) hepatic microsomes (60 µm). Supporting publications 2012:EN

29 Figure A10: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Sprague Dawley) hepatic microsomes (20 µm). Supporting publications 2012:EN

30 Figure A11: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Sprague Dawley) hepatic microsomes (40 µm). Supporting publications 2012:EN

31 Figure A12: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Sprague Dawley) hepatic microsomes (60 µm). Supporting publications 2012:EN

32 Figure A13: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Fischer 344) hepatic microsomes (20 µm). Supporting publications 2012:EN

33 Figure A14: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Fischer 344) hepatic microsomes (40 µm). Supporting publications 2012:EN

34 Figure A15: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with male rat (Fischer 344) hepatic microsomes (60 µm). Supporting publications 2012:EN

35 Figure A16: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Fischer 344) hepatic microsomes (20 µm). Supporting publications 2012:EN

36 Figure A17: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Fischer 344) hepatic microsomes (40 µm). Supporting publications 2012:EN

37 Figure A18: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with female rat (Fischer 344) hepatic microsomes (60 µm). Supporting publications 2012:EN

38 Figure B1: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with Human (males) hepatic microsomes (20 µm). Supporting publications 2012:EN

39 Figure B2: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with Human (males) hepatic microsomes (40 µm). Supporting publications 2012:EN

40 Figure B3: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with Human (males) hepatic microsomes (60 µm). Supporting publications 2012:EN

41 Figure B4: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with Human (females) hepatic microsomes (20 µm). Supporting publications 2012:EN

42 Figure B5: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with Human (females) hepatic microsomes (40 µm). Supporting publications 2012:EN

43 Figure B6: Radio-HPLC metabolic profile of 14 C-heptadecane incubated with Human (females) hepatic microsomes (60 µm). Supporting publications 2012:EN

44 Figure C1: Radio-HPLC metabolic profile of 3 H-pristane incubated with male rat (Wistar) hepatic microsomes (20 µm). Supporting publications 2012:EN

45 Figure C2: Radio-HPLC metabolic profile of 3 H-pristane incubated with female rat (Wistar) hepatic microsomes (20 µm). Supporting publications 2012:EN

46 Figure C3: Radio-HPLC metabolic profile of 3 H-pristane incubated with male rat (Sprague Dawley) hepatic microsomes (20 µm). Supporting publications 2012:EN

47 Figure C4: Radio-HPLC metabolic profile of 3 H-pristane incubated with female rat (Sprague Dawley) hepatic microsomes (20 µm). Supporting publications 2012:EN

48 Figure C5: Radio-HPLC metabolic profile of 3 H-pristane incubated with male rat (Fischer 344) hepatic microsomes (20 µm). Supporting publications 2012:EN

49 Figure C6: Radio-HPLC metabolic profile of 3 H-pristane incubated with female rat (Fischer 344) hepatic microsomes (20 µm). Supporting publications 2012:EN

50 Figure D1: Radio-HPLC metabolic profile of 3 H-pristane incubated with Human (males) hepatic microsomes (20 µm). Supporting publications 2012:EN

51 Figure D2: Radio-HPLC metabolic profile of 3 H-pristane incubated with Human (females) hepatic microsomes (20 µm). Supporting publications 2012:EN

52 Figure D3: Radio-HPLC metabolic profile of 3 H-pristane incubated with Human (males) hepatic microsomes (60 µm). Supporting publications 2012:EN

53 Figure D4: Radio-HPLC metabolic profile of 3 H-pristane incubated with Human (females) hepatic microsomes (60 µm). Supporting publications 2012:EN

54 Figure E1: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with male rat (Wistar) hepatic microsomes (20 µm). Supporting publications 2012:EN

55 Figure E2: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with female rat (Wistar) hepatic microsomes (20 µm). Supporting publications 2012:EN

56 Figure E3: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with male rat (Sprague Dawley) hepatic microsomes (20 µm). Supporting publications 2012:EN

57 Figure E4: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with female rat (Sprague Dawley) hepatic microsomes (20 µm). Supporting publications 2012:EN

58 Figure E5: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with male rat (Fischer 344) hepatic microsomes (20 µm). Supporting publications 2012:EN

59 Figure E6: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with female rat (Fischer 344) hepatic microsomes (20 µm). Supporting publications 2012:EN

60 Figure F1: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with Human (males) hepatic microsomes (20 µm). Supporting publications 2012:EN

61 Figure F2: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with Human (females) hepatic microsomes (20 µm). Supporting publications 2012:EN

62 Figure F3: Radio-HPLC metabolic profile of 3 H-dodecylcyclohexane incubated with Human (males) hepatic microsomes (60 µm). Supporting publications 2012:EN

63 Figure F4: Radio-HPLC metabolic profile of (females) hepatic microsomes (60 µm). 3 H-dodecylcyclohexane incubated with Human Supporting publications 2012:EN

64 ABBREVIATIONS CEA ND EFSA INRA LMPW SD Commissariat à l Energie Atomique Not determined European Food Safety Authority Institut National de la Recherche Agronomique Low melting point waxes Standard deviation Supporting publications 2012:EN