MINERAL OILS (FOOD-GRADE), PARAFFIN WAXES AND MICROCRYSTALLINE WAXES First draft prepared by Ms F D Pollitt, Dr S Barlow and Ms S O'Hagan Health Aspects of the Environment and Food (Medical) Division Department of Health, London, United Kingdom Explanation Biological data Toxicological studies Short-term toxicity studies Observations in humans Comments Evaluation References 1. EXPLANATION Food grade mineral oils were last evaluated by the Committee at its thirty-seventh meeting (Annex 1, reference 94). At that meeting, it reconsidered two 90-day feeding studies in Fischer 344 (F344) rats given both oleum-treated and hydrogenated mineral oil (the "Shell" studies, Baldwin et al., 1992). In the first study, haematological changes and deposition of mineral oil in the liver, spleen and lymph nodes were reported; in the second, deposition in the liver, spleen and lymph nodes was again reported, but no haematological investigations were conducted. The Committee considered that both effects required further investigation and recommended that an adequate long-term feeding study should be performed using food-grade mineral oils representative of those in commercial use. The temporary ADI "not specified" was extended. Petroleum jelly was last evaluated at the thirty-third meeting (Annex 1, reference 83). At that meeting, the Committee reiterated its concern about whether the chemical composition of petroleum jelly in current use met the specifications for the materials tested in toxicological studies submitted to it. It concluded that, for newer formulations of petroleum jelly, new specifications were required and that adequate long-term, mutagenicity, and reproductive toxicity/teratogenicity studies should be completed. No ADI was allocated. Paraffin wax and microcrystalline wax were last evaluated by the Committee at its thirty-ninth meeting (Annex 1, reference 101). At that meeting, the Committee prepared separate specifications for these waxes and concluded that, because these specifications limit the number of waxes that can be used for food applications as compared with those tested in previous studies, previous long-term toxicity studies were suitable for evaluating the safety of hydrocarbon waxes in current use. The Committee reviewed the results of extraction and migration tests on waxes and wax-bearing products, information on the absorption and metabolism of hydrocarbon waxes, a long-term feeding study in Sprague-Dawley rats and a series of 180-day feeding studies in rats, and concluded that petroleum-derived paraffin and microcrystalline waxes were non-toxic and non-carcinogenic. A group ADI "not specified" was established for micro-crystalline wax and paraffin wax for the uses indicated in the specifications (chewing-gum base, protective coating, defoaming agent and surface-finishing agent). The Committee was informed that a 90-day study on hydrocarbon waxes made both by newer processes and by traditional methods was under way, and asked to be informed of the results when they became available. Since the previous evaluations, additional toxicological data have become available on a range of mineral oils, microcrystalline wax and paraffin wax representative of materials currently in use. These data are summarized and discussed in the following monograph addendum. 2. BIOLOGICAL DATA 2.1 Toxicological studies 2.1.1 Short-term toxicity studies 2.1.1.1 Rats A range of mineral oils and waxes were administered in the diet to groups of F344 rats (20/sex) at levels of 0.002%, 0.02%, 0.2% or 2% for 90 days (equivalent to 2, 20, 200 or 2000 mg/kg bw/day). The oils used in the study were N10(A), P15(H), N15(H), N70(H), N70(A) and P100(H)1. This range of oils was chosen to cover the variables previously identified as potential influences on the toxicity of mineral oils (viz. viscosity, method of refining and oil type). The 3 waxes tested were a hydrotreated, low-melting point, paraffinic wax (LMPW); a hydrotreated, high-melting point, microcrystalline wax (HMPW) and a clay treated, microcrystalline wax (high sulphur wax, HSW). Groups of rats (60/sex) were fed the control diet for the same period of time. A further group of rats (20/sex) were fed diets containing 2% coconut oil. These animals comprised the main study. Additional groups of 10 males and 10 females were fed diets containing 2% test article or 2% coconut oil for 90 days followed by a 28-day period on control diet. Groups of 30 males and 30 females were fed control diet for the same period of time. These animals comprised the reversal study. Further additional groups of 5 male and 5 female rats received control diet, diet containing 2% test article or 2% coconut oil for 90 days (tissue level study). Further groups of 5 male and 5 female rats received control diet, diet containing 2% test article or 2% coconut oil for 90 days followed by a 28-day period on control diet (tissue level reversal study). 1 Oils may be obtained from crude oil sources of naphthenic (N) or of paraffinic (P) origin and by either the conventional acid (oleum)-treatment process (A) or the hydrogenation or hydrotreatment process (H). Their viscosity ranges from 10-100 centistokes (cSt) (10-100 mm2/s). Thus a P100(H) oil refers to a paraffinic oil with a viscosity of 100 cSt produced by the hydrogenation process and a N10(A) oil to a naphthenic oil with a viscosity of 10 cSt produced by the acid-treatment process. All animals were monitored for body weight, food intake and clinical condition. Main study and reversal study animals were subject to a full necropsy. End of test haematology and clinical chemistry parameters were measured. All tissues from the high-dose and control groups were processed and examined by light microscopy, as were limited tissues from all intermediate-dose animals. A limited list of tissues was taken from the tissue level and tissue level reversal animals, weighed and the level of mineral hydrocarbon material present determined. All increases or decreases in parameters or incidence of lesions cited below refer to comparisons with the concurrent control group, unless otherwise stated. Body weights were largely unaffected by any of the treatments in this study. All treatments apart from the HSW caused statistically significant increases in food consumption in male rats in the main study at the 2% level. In females, increased food intake was observed in the 2% N15(H), N70(H) and N70(A) oil and HMPW groups only, although there were intermittent increases in food intake in females receiving coconut oil. In the reversal study (dosed at the 2% level only), food intake was increased in all male groups, including the HSW group, throughout the study. In the reversal period, intake returned to normal by day 105 in all groups with the exception of the N70(A) oil group. Among females, there were intermittent increases in food intake throughout the study and the reversal period in all groups including the coconut oil group. In general, the increases did not differ from controls by more than 10%. Increased organ weights were recorded in a number of organs, particularly the kidney, liver, spleen, lymph node and caecum. The weight changes were generally more marked in female rats than in males. Two main types of haematological changes were observed. The first was characterized by an increased WBC count, often accompanied by increases in the neutrophil, lymphocyte and monocyte population and a decrease in platelet level. Such changes were seen in both male and female rats receiving 2% N15(H), P15(H) and N10(A) oils, LMPW and HSW (females only). Similar changes were observed at the 0.2% level in both sexes receiving LMPW. The second type of change was characterized by decreased RBC and haemoglobin levels and was reported in most instances in females, especially those receiving 2% N15(H), N70(H), P15(H) and N10(A) oils and LMPW and 0.2% P15(H) and N10(A) oils. This latter change seemed to be readily reversible because, apart from the LMPW group, no effect was seen in the reversal study. In males receiving LMPW, decreased haemoglobin and mean corpuscular haemoglobin (MCH) levels were seen in both the main and reversal studies. There were no significant changes in haematological parameters in rats receiving coconut oil. Raised serum liver enzyme levels (ALT, ASAT and gamma-GT were seen with the N15(H), N70(A), P15(H), P100(H) and N10(A) oils in female rats and with LMPW in both female and male rats. There was a slight but statistically significant decrease in alkaline phosphatase levels in a number of treatment groups. A lower albumin:globulin ratio seen in some groups generally reflected decreased total protein and albumin levels. There was a slight but statistically significant higher glucose levels in all but the highest dose group of many of the female treatment groups. Males showed a much more limited range of effects than females. Reversal animals of both sexes given LMPW had statistically significantly increased levels of serum liver enzymes. Gamma-GT was also higher than control in the female N10(A) oil group after the reversal period. Some other clinical chemistry parameters remained altered in the reversal study, particularly in the LMPW, N15(H) and N10(A) oil groups. Serum vitamin E levels were measured in the tissue level and tissue level reversal studies (2% test article). All the oils significantly reduced the serum vitamin E concentration in both male and female rats (to approximately 30-75% of control). This may simply be due to a reduction in bioavailability of the vitamin resulting from its preferential solubility in the oils. Serum vitamin E was significantly increased in males and females receiving LMPW and in females receiving coconut oil (to approximately 130% of control). The only groups to show an effect of treatment after the 4-week reversal period were the male groups treated with N70(H) and N10(A) oils. Liver granulomas, occasionally accompanied by centrilobular vacuolation, were seen in female groups receiving N15(H), N70(H), N70(A), P15(H) and N10(A) oils at the highest dose level and in both sexes in the LMPW group at the 2% and 0.2% levels. The granulomas were focal and consisted of collections of macrophages, some with necrotic cells, surrounded by inflammatory cells and variable fibrosis. In general, one month after cessation of treatment, granulomas were still present in livers of animals from these groups although in some cases a reduction in the severity of the lesion was evident. In the mesenteric lymph nodes, lesions were seen which comprised focal collections of macrophages, often in the cortical region. The macrophages were vacuolated and some had a yellowish-brown pigmentation of varied intensity. The focal collections of macrophages were classified as histiocytosis. They were not homogeneously distributed but were often restricted to one node or even part of one node, with some areas severely affected and others relatively free of any effect. In view of the uneven distribution, the author commented that recording the presence of this lesion was subject to chance variation related to selection of tissue and sectioning and that this may have led to an under-reporting of the incidence of lesion. Histiocytosis was increased compared to controls in males given N 10(A), N70(A) and N70(H) oils at levels down to 0.2%, P15(H) oil and LMPW at levels down to 0.02%, and in those given N15(H) oil at levels down to 0.002%. It was seen in females given any of the N15(H), N70(A), N70(H), P15(H) or N10(A) oils down to 0.02% and in those given LMPW at all dose levels. In each case the severity increased with dose and the effect was more severe in females than in males. There were also significant levels of histiocytosis in the same treatment groups in the reversal study. HSW, HMPW and P100(H) oil were indistinguishable from controls at any dose level in either sex. Macrophage accumulation in Peyers patches was observed in both male and female animals receiving 0.2 and 2% LMPW. Among females, macrophage infiltration of the lamina propria was also observed at the 2% level. In the heart, focal inflammatory lesions were observed within the cusps of the mitral valve. The lesion was characterized by an increased cellularity of the valve with destruction of the fibrous core. The increased cellularity was composed of a mixed population of macrophages, plasma cells and lymphocytes. Pyknotic nuclei and cell debris were scattered throughout the lesions. In many lesions mitotic figures could be identified. The lesion was seen in 11/20 males and 11/20 females in the 2% LMPW groups, and in 5/20 females in the 0.2% LMPW group. The lesion was present in 1/60 male control animals and in occasional males from the high-dose treatment groups. Following a 28-day reversal period there was still a significantly increased incidence of this lesion in both male and female animals treated with LMPW, although the incidences were lower than at the end of the treatment period. Tissue levels of mineral hydrocarbons were determined in the kidney, liver, mesenteric lymph node and perirenal fat in the tissue level study. Increased levels were seen in both males and females in one or more of these tissues for all oils and waxes except the HSW and HMPW. A decrease in hepatic levels was seen following the 28-day reversal period (from 16% for the N70(H) oil to 76% for the N70(A) oil). Little reduction was seen in the mesenteric lymph nodes or in the kidneys. Livers of female rats in the control and 2% dose groups from the main and reversal studies (excluding the coconut oil group) were subsequently analyzed for content of hydrocarbons of different chain length. Four animals/group were used from the control, P15(H) oil and LMPW groups but only one animal/group from the other groups. Chromatograms of liver extracts of the control, reversal P100(H) oil group, main and reversal HMPW groups and main and reversal HSW groups showed no oil or wax profile. The authors concluded that, for the other test articles, the composition of the liver deposits was comparable but not identical to the original material. For the LMPW, the relative content of higher boiling hydrocarbon specimens in the liver deposit was found to have further increased after the 28-day reversal period. Also, the isoparaffin content of the residues was relatively higher compared to the original test material. The authors suggested that mobilization takes place and that the rate depends on molecular weight and structure of the hydrocarbon: the lower molecular weight, linear molecules being metabolized fastest. It was further concluded from these results that no hydrocarbons with carbon numbers over C35 passed the intestinal membranes. The NOELs in this study were as follows: N15(H) oil and LMPW: less than 0.002%, equivalent to < 2 mg/kg bw/day; N70(H), N70(A), P15(H) and N10(A) oils: 0.002%, equivalent to 2 mg/kg bw/day; P100(H) oil, HSW and HMPW: 2%, equivalent to 2000 mg/kg bw/day (Worrell, 1992). In a further 90-day study in F344 rats, a P70(H) oil was administered in the diet to groups of 20 male and 20 female rats at levels of 0.002%, 0.02%, 0.2% or 2% for 90 days. Groups of 20 males and 20 females were fed control diet for the same period. These animals comprised the main study. A further 5 male and 5 female rats received control diet or diet containing 2% test article for 90 days to provide tissue for measurement of mineral hydrocarbon levels (tissue level study). Study parameters monitored were as for Worrell, 1992, above, except that histopathological examination was limited to the liver and mesenteric lymph nodes of rats in the main study. There were no treatment-related effects on body weight. Food consumption was increased by approximately 10% in both sexes at the 2% level in the main study. Increased spleen weights were seen in male rats at the 0.2 and 2% levels in the main study only; in female rats, increased liver, spleen and kidney weights were seen at the 2% level in the main study but in the tissue level study only increased liver weights were seen. No treatment-related changes were seen in haematological parameters. Raised ALAT, gamma-GT and ALP levels were seen intermittently in male and female dose groups but there was no clear dose-related effect. The only significant histopathological finding was an increased incidence of pigmented macrophages in the lymph nodes of male rats receiving the oil at the 2% level. Residues of mineral hydrocarbon material were found in the liver and mesenteric lymph nodes of both male and female rats and in the kidneys of female rats in the tissue level study. In this study, the NOEL for the P70(H) oil was considered to be 0.2% since only minor effects, of doubtful biological significance, were seen at this dose level (Brantom, 1993). In a further 90-day study in F344 rats, the test materials were three mineral hydrocarbon waxes: a paraffinic, high-melting point, intermediate wax (also referred to as wax 64); a blended wax containing a 50:50 mixture of the low-melting point and high-melting point waxes used in the study by Worrell, 1992 (microcrystalline/ paraffin wax blend); and the LMPW tested by Worrell, 1992. The intermediate wax and the blended wax were fed in the diet to groups of 20 male and 20 female rats at levels of 0.02, 0.2 or 2%. The LMPW was fed at 2% only. Groups of 20 male and 20 female rats received control diet only. These animals comprised the main study. Further groups of 10 male and 10 female rats were fed either control diet only, 2% intermediate wax, 2% blended wax or 2% LMPW for 90 days, followed by an 85- or 86-day reversal period in which they received control diet only. This was the reversal study. Further groups of 5 male and 5 female rats received the same dose levels as in the reversal study for 96-97 days to provide tissue for measurement of mineral hydrocarbon content (tissue level study). Samples of liver were also taken from 5 female animals/group in the reversal study for measurement of mineral hydrocarbon levels (pooled sample). Methodology was as in the study by Worrell, 1992, except that histopathological examination was limited to liver and lymph nodes, any abnormal tissue, and to the mitral valve of the hearts from 5 male and 5 female animals from the control, 2% intermediate wax, 2% blended wax and 2% LMPW groups in the main and reversal studies. There were no significant treatment-related effects on body weight. Food intake was increased in male and female rats receiving 2% LMPW and 2% blended wax in the main study and in female rats receiving 0.2% blended wax. Increased liver, mesenteric lymph node and/or spleen weights were seen with all waxes in the 0.2% or 2% groups. Increased mesenteric lymph node weight was also seen in the 0.02% intermediate wax group. In the reversal study, the weights of these organs were still frequently slightly increased in all treated groups. Among haematology parameters, there were some sporadic decreases in haemoglobin and MCH concentrations. In male rats, increased monocyte and neutrophil levels and decreased lymphocyte levels were recorded in the groups receiving 2% blended wax or LMPW. In females, monocyte levels were markedly increased at all dose levels in the intermediate wax groups, and at 2% blended wax and LMPW. Increases in eosinophil and neutrophil counts also occurred. Platelet levels were decreased in both male and females at all but the lowest dose level. In the reversal study, no effects were seen in male rats but, in female rats, WBC levels were raised and platelet levels remained decreased. Transaminase levels were increased in male and female rats receiving the intermediate and blended waxes at the 0.2 and 2% levels and in male rats receiving 0.02% intermediate wax. Large (1-2 fold) increases in transaminase levels were seen in the LMPW groups. These enzymes remained elevated in both sexes receiving blended wax and LMPW in the reversal study. Large increases were recorded in gamma-GT levels in female rats only in the 2% blended wax and LMPW groups in the main study and in all wax groups in the reversal study. At histopathological examination, increased incidences of granuloma and/or microgranuloma were seen in the liver in all the 0.2% and 2% dose groups, often with an increased incidence of centrilobular or periportal vacuolation. Increased incidences of microgranuloma persisted in the reversal study in all groups except the females receiving intermediate wax. Significant levels of histiocytosis were seen in the cervical lymph nodes of male and female rats receiving the blended wax or LMPW at 2% and in female rats receiving 2% intermediate wax in the main study. In the reversal study, the incidence remained significantly increased only in female rats receiving intermediate wax or LMPW. In the mesenteric lymph nodes, histiocytosis was seen in all dose groups in the main study and the incidence remained significantly increased compared to controls in all dose groups in the reversal study. The lesion in the mitral valve, reported in the study by Worrell, 1992, was also seen in this study. Birefringent material, identified as mineral hydrocarbon material, was also seen in the mitral valve in certain animals in this study. The incidences among those rats examined (2% dose level only) are shown in Table 1. Tissue levels of mineral hydrocarbons were determined in the liver, kidneys, spleen and mesenteric lymph nodes in the tissue level study. Markedly increased levels were seen in both males and females in all tissues examined except in the kidneys of males fed intermediate wax. In the reversal study in female animals, hepatic levels of mineral hydrocarbons were considerably less (84%, 80% and 91% less for the LMPW, intermediate wax and blended wax groups, respectively). No NOEL could be identified in this study for any of the waxes tested (Brantom & Coatsworth, 1993; Freeman et al., 1989; Simpson & Smith, 1992). A further 90-day study was carried out on oil P15(H), the objectives of which were to reproduce liver and lymph node findings attributed to some of the mineral oils in previous studies and to provide insight into the comparative sensitivities of different rat strains to a representative oil. Only female rats were used as these had shown the greatest sensitivity to mineral oils in previous studies. Groups of 35 female F344 rats received either control diet or diet containing 2% P15(H) oil. A further group of 15 F344 rats received diet containing 0.2% P15(H) oil. Groups of 15 female CD Sprague-Dawley rats received control diet, 0.2 or 2% P15(H) oil. In the F344 rat control and high-dose groups, interim sacrifices of 10 animals/group were carried out at 30 or 61 days. Five animals/group were used for mineral hydrocarbon analysis at end of test (tissue level study). Methodology was similar to that in the study by Worrell, 1992, except that histopathological examination was limited to liver, mesenteric lymph nodes and any abnormal tissues. Table 1. Incidence of effects of different waxes on the mitral valve (2% dose level) Control Intermediate Blended LMPW Main study Mitral valve lesion M 0/5 0/5 4/5 5/5 F 1/5 2/5 5/5 5/5 Bifringent material M 0/5 0/5 5/5 5/5 F 0/5 5/5 5/5 5/5 Reversal study Mitral valve lesion M 0/5 2/5 4/5 5/5 F 0/5 3/5 5/5 5/5 Bifringent material M 0/5 1/5 0/5 0/5 F 0/5 1/5 0/5 3/5 There were no consistent, treatment-related effects on body-weight gain nor on food consumption in either strain. Increased liver weights were seen in all F344 rat groups receiving P15(H) oil, and increased mesenteric lymph node and spleen weights were also recorded in rats dosed at the 2% level. There were no such effects in Sprague-Dawley rats. WBC counts were increased in F344 rats in the 2% group at days 30 and 61 but not at the end of test (EOT). The proportion of neutrophils was increased and that of lymphocytes decreased in the 2% F344 rat group at day 61 and at EOT. Decreased RBC counts, haemoglobin and haematocrit values were also seen in F344 rats in the 2% group at day 61 and EOT, and in the 0.2% group at EOT. No treatment-related haematological changes were seen in Sprague-Dawley rats. F344 rats receiving 0.2% or 2% P15(H) oil showed an increase in gamma-GT at EOT. Intermittent increases in cholesterol levels were also seen in F344 rats but no effects were seen on clinical chemistry parameters in Sprague-Dawley rats. Among F344 rats, microgranulomas were present in the livers of rats fed 2% P15(H) oil at day 61 and at EOT but not at day 30, and in rats fed 0.2% at EOT. Treatment-related effects in the mesenteric lymph nodes were microgranulomas (in the 0.2% group at EOT, 2% group at day 61 and EOT), histiocytosis (2% group at day 30) and focal infiltrates of eosinophils (2% group at day 30). Among Sprague-Dawley rats, there was a slightly increased incidence of minimal, multifocal, chronic inflammation in the 2% dose group at EOT. In contrast to F344 rats, discrete microgranulomas were not observed in either dose group. Tissue levels of mineral hydrocarbons were determined in the liver, mesenteric lymph node, kidney and spleen. Increased levels were seen in particular in the liver, with a dose-related increase compared to controls in both strains of rats. The level of mineral hydrocarbon material was equivalent in the 2% Sprague-Dawley and the 0.2% F344 groups. Mineral hydrocarbon levels in the mesenteric lymph node were raised to a similar extent in F344 and Sprague-Dawley rats. No accumulation of mineral hydrocarbon was seen in the spleen or kidney. In this study, Sprague-Dawley rats were clearly less susceptible to the toxic effects of the test oil than F344 rats, although mineral hydrocarbon material was absorbed by both strains. No NOEL was apparent in F344 rats; the NOEL in Sprague-Dawley rats was probably 2% (Exxon Biomedical Sciences, 1993). 2.2 Observations in humans In a case report, tissues samples were taken at autopsy from a 55-year old man who died from coronary failure. Small granulomas were seen in the axillary and apical lung areas which, on microscopic examination, showed the presence of giant macrophage cells containing 'needle-like' inclusions consisting of long-chain n-alkanes. Similar crystalline inclusions were found in other organs, mainly in reticulo-endothelial cells. No significant difference was found in the amounts of neutral lipids and polar lipids in control and patient tissues. However. analysis by GLC identified the presence of abnormal compounds in lipid extracts from the patient tissues, which had the same GLC retention time as n-alkanes with 29, 31 and 33 carbon chain length. These n-alkanes were detected in most of the tissues examined except brain, but were not detected in control organs. In the organs studied, the highest concentrations of n-alkanes were found in lung granuloma and in lumbo-aortic lymph nodes. Intermediate levels were found in the liver and adrenal glands; very low levels were detected in the aorta, myocardium and adipose tissue. The amount of alkanes detected by GLC correlated with the histopathological findings. It was concluded that these alkanes were characteristic of vegetable cuticular waxes and, therefore, their source was deemed to be dietary. The patient was known to have consumed about one kg of unpeeled apples per day (for 18 years) corresponding to about 10 mg of n-alkanes daily (Salvayre et al, 1988; Duboucher et al, 1988). 3. COMMENTS At the present meeting, the Committee reviewed the results of four recent 90-day studies in F344 rats on a range of mineral oils and waxes representative of materials currently in use. The materials tested were as follows: Mineral oils N10(A), N15(H), P15(H), N70(A), N70(H), P70(H) and P100(H). Paraffin waxes Low-melting-point wax (LMPW) and intermediate-melting-point wax (IMPW). Microcrystalline waxes High-melting-point wax (HMPW) and high-sulfur wax (HSW). The dose levels used were 0.002%, 0.02%, 0.2% or 2% in the diet (equivalent to 20, 200, or 2000 mg/kg bw/day), except for the IMPW, for which the lowest dose level was 0.02%, equivalent to 20 mg/kg bw/day. In these studies, neither HSW nor HMPW accumulated in any tissues or produced any effects. The P100(H) oil produced no effects but did accumulate in the liver to a small extent at the highest dose level. The P70(H) oil accumulated in the liver, kidney and mesenteric lymph nodes when administered at the highest dose level, but the only treatment-related effect, which was seen at this level only, was described in the study report as an increased incidence of pigmented macrophages in the lymph nodes. With all the other materials, there was evidence of accumulation of test material and effects indicative of a reaction to a foreign body at one or more dose levels. The types of effects seen were similar in nature and included focal histiocytosis; increases in the weight of the liver, lymph nodes, spleen and kidney; granulomas or microgranulomas in the liver; haematological changes typical of a mild, chronic inflammatory reaction; and biochemical changes indicative of mild hepatic damage. These effects were similar to those seen in the two 90-day "Shell" studies with mineral oil considered by the Committee at its thirty-seventh meeting. The incidence of inflammatory lesions in the mitral valve of the heart was significantly increased in rats fed 0.2% or 2% LMPW and occasionally in rats fed other materials. Such lesions were also seen occasionally in control rats. The Committee considered that the significance and treatment-related incidence of these lesions could be clarified by a re-examination of the histological data on all treated and concurrent control groups in the recent studies and in historical controls. The Committee considered that, although the types of effects seen were essentially foreign body reactions, it was possible that a prolonged inflammatory response of this type could result in functional changes in the immune system and that this aspect required further investigation. The NOELs in these studies are given in Table 2. Except for the P70(H) oil and those materials showing no effects, the NOELs were based on an increased incidence of histiocytosis in the lymph nodes at the next highest dose level. For the P70(H) oil, the NOEL was based on an increased incidence of pigmented macrophages in the lymph nodes at the 2% dose level. Two of the studies included a reversal period of either 28 or approximately 90 days, but most of the toxicological effects were still evident at the end of this period. The Committee noted that there was limited evidence that the severity of some of these effects had decreased during this phase, although it appeared that a period longer than 90 days would be required in order to determine whether the effects produced by these materials are fully reversible. The Committee recalled that much earlier rat studies on mineral oils and waxes, of duration ranging from 90 days to lifetime and utilizing strains of rat other than F344, had shown no adverse toxicological effects. Although many of these studies had design and/or reporting deficiencies, the lack of significant findings in other strains suggested to the Committee that the F344 rat may be especially sensitive. In one of the studies considered at the meeting, the responses of F344 and Sprague-Dawley rats to P15(H) oil were compared, and it was found that the latter were less susceptible to the toxic effects of the test materials, although absorption and organ weight and histopathological changes were evident in both strains. The Committee also noted the limited data available on mineral hydrocarbons from human studies, which showed that mineral oil-induced lesions similar to those seen in rats have been identified in human tissues (Annex 1, reference 102). Vacuoles of accumulated mineral oil have been found in the liver, spleen and lymph nodes. Some studies reported the presence of an accompanying inflammatory or granulomatous reaction, while others reported no tissue reaction to the accumulated material. However, none of the studies contained detailed information about the individuals' history of use of liquid paraffin as a medicine nor their dietary intake. It was therefore not possible to assess the level of intake of mineral oil associated with hydrocarbon accumulation in humans. Food grade mineral oils and paraffin and microcrystalline waxes are complex mixtures of hydrocarbons and other materials. It was evident from the results of the new 90-day studies that the absorption and subsequent toxicity of these materials are associated with their physical properties rather than the crude oil source or refining method. The oils and waxes with a high NOEL contain a higher proportion of hydrocarbon components of high relative molecular mass (high carbon number) and have higher viscosities than those with a low NOEL, which contain a greater proportion of hydrocarbon components of low molecular weight (low carbon number). Mineral hydrocarbons are specified, on the whole, by means of ranges of physical and chemical parameters. The results of the studies considered at the present meeting were therefore applicable not only to the particular materials tested but also to other mineral hydrocarbons having physical and chemical parameters falling within the same ranges. 4. EVALUATION The Committee allocated ADIs for oils and waxes as set out in Table 2. A temporary group ADI of 0-0.01 mg/kg bw was allocated to mineral oils falling within the specifications for the N10(A), N15(H), P15(H), N70(A) and N70(H) oils and a temporary ADI of 0-1 mg/kg bw was allocated to mineral oils meeting the specification of the P70(H) oil. The Committee requires information about the compositional factors in mineral oils that influence the absorption and toxicology for review in 1998. It also requires a study of at least 1 year's duration on one of these materials in F344 rats, which should include an assessment of immune function at appropriate time periods and an investigation of the kinetics of accumulation of the material, and particularly whether a plateau is reached. A reversal period of 1 year should also be included, in order to determine whether the granulomatous hepatic lesions observed in rats in the 90-day studies are fully reversible. The results of this study, together with all relevant background data, including the physical and chemical parameters of the materials tested, should be submitted for review in 1998. For those materials that caused no adverse effects at the highest level tested, the Committee concluded that, although the NOEL was derived from a study of only 90 days' duration, the dose administered was sufficiently high to provide reassurance that accumulation of these substances was unlikely to occur following longer-term administration. An ADI of 0-20 mg/kg bw could therefore be established for mineral oils meeting the specifications for the P100(H) oil and a group ADI1 of 0-20 mg/kg bw could be established for waxes meeting the specifications for the HSW and HMPW. Insufficient information was available for the Committee to re-evaluate petroleum jelly This substance should be considered at a future meeting. Table 2. NOELs and ADIs for mineral oils, paraffin waxes and microcrystalline waxes tested in 90-day studies in F334 rats. Substance NOEL ADI (mg/kg bw) (mg/kg bw/day) LMPW < 2 ADI withdrawn1 IMPW < 2 ADI withdrawn1 N10(A) oil 2 0-0.012 N15(H) oil < 2 0-0.012 P15(H) oil 2 0-0.012 N70(A) oil 2 0-0.012 N70(H) oil 2 0-0.012 P70(H) oil 200 0-13 P100(H) oil 2000 0-20 HSW 2000 0-204 HMPW 2000 0-204 1 Previous ADI "not specified" 2 Temporary group ADI 3 Temporary ADI 4 Group ADI 5. 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A 90 day feeding study in the rat with six different white mineral oils (N15(H), N70(H), N70(A), P15(H), N10(A) and P100(H)), three different mineral waxes (a low melting point wax, a high melting point wax and a high sulphur wax) and coconut oil. Unpublished report No. 1010/3/92 from BIBRA Toxicology International, Carshalton, Surrey, UK. Submitted to WHO by CONCAWE. Brussels, Belgium.
See Also: Toxicological Abbreviations