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MOH accumulation in F344 rats
Science of the Total Environment 615 (2018) 1095–1098
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
MOH accumulation in F344 rats
Editor: Adrian Covaci
Dear Sir, It has been with great interest that we have read the two recent papers published in this Journal (Barp et al., 2017a; Barp et al., 2017b), on the effects of exposure to mineral hydrocarbons - MHC (what the authors call MOSH1) using the Fisher F344 rat as a model. One of us has previously made a comparison between the granuloma findings in the F344 and humans, especially on the types of hepatic granuloma caused by ingestion of mineral oil hydrocarbons (Fleming et al., 1998). While we cannot comment on the sophisticated analytical methods used, we would like nonetheless to provide our point of view about some of the arguments presented by the authors from the perspective of a human liver pathologist. I. “Strong accumulation is not of toxicological concern per se. However, as the MOSH concentrations in human tissues were probably severely underestimated when extrapolated from animal experiments involving high doses….”. Page 1227; 4.3 Practical consequences (Barp et al., 2017a)
It is reasonable to suppose that accumulation is per se not of toxicological concern because it is not an endpoint in itself. Thus, to be of pathological significance, the key question is whether any accumulation is associated with any adverse effects. It has been known for many years that MOSH is retained in the human liver and in other tissues. Early descriptions documented focal collections of vacuoles in liver and spleen, with smaller vacuoles within macrophages and larger vacuoles in multinuclear giant cells (Boitnott and Margolis, 1966; Boitnott and Margolis, 1970; Stryker, 1941). It was assumed that these vacuoles represented MOSH which had been dissolved out of the cells during preparing the tissues for microscopy. In the liver, the lesions were mainly portal, although clusters of vacuoles and macrophages were observed in the hepatic parenchyma, often around the central vein. These lesions were (and are) called
1
The key papers that describe the human pathology of mineral hydrocarbons do not talk about “MOSH”. Pragmatically however, to allow direct reference to these papers, the term MOSH will be used. The reader should thus be aware of MOSH as a broad term for alkanes commonly found in oils and waxes.
https://doi.org/10.1016/j.scitotenv.2017.07.275 0048-9697/© 2017 Elsevier B.V. All rights reserved.
lipogranulomas and are morphologically very different from the severe lesions seen in the F344 rats – these latter are formed of epithelioid cells. In 1982, Dincsoy et al. examined the incidence of lipogranulomas in human liver from both biopsies and autopsies (Dincsoy et al., 1982). There was a lack of association between the lesions and liver steatosis, ruling out simple reaction to presence of fat in hepatocytes. In the non-fatty livers, there was also a significantly lower incidence in 1952–3, in comparison with 1978–80 (1.7% versus 4.6%). Extracts of the autopsy livers were analyzed and the presence of MOSH of similar composition to commercial white oil grades was found. The authors concluded that the incidence of these lesions in non-fatty livers was probably associated with exposure to MOSH in food. Importantly, there was no evidence that the lesions were of clinical significance. Subsequently, Cruickshank, in 1984, examined samples of liver, spleen, bone marrow and lymph nodes from 200 autopsies performed in 1970–72 for the presence of lipogranulomas. The incidence was much higher than previously reported, at approximately 45%, and interestingly, showed marked geographical variation (Cruickshank and Thomas, 1984). In 1985, Wanless and Geddie extensively reviewed the previous literature and reported the results of their analysis of 465 autopsies performed between 1974 and 1976. Lipogranulomas were observed in liver at an incidence of 48%. A correlation in the incidence and severity of lipogranulomas with increasing age and maleness was observed. No correlation was found between the incidence of lipogranulomas and the occurrence of liver steatosis, diabetes mellitus, obesity or any liver disease. Again, importantly, there was no evidence of clinical significance, despite some mild accompanying fibrosis (Wanless and Geddie, 1985). In summary, several investigations have identified lesions (lipogranulomas) in up to 40–50% of human livers and these have been associated with the presence of MOSH in the tissues. It has been postulated that the lipogranulomas may be related to laxative and/or food mineral hydrocarbon ingestion. Crucially, in all reports, there has been no evidence of any clinical significance associated with the presence of these lesions and thus MOSH in tissues, including the liver. Again, note that these lesions are morphologically quite different from the epithelioid granulomas seen in the Fischer 344 rats. II. The animal data available might not even enable to adequately support the safety of the present human exposure, and end points other than granuloma formation and potential consecutive inflammation should be investigated” Page 1227; 4.3 Practical consequences (Barp et al., 2017a).
The available animal data are not restricted to studies performed with the F344 rat. Early investigations were primarily aimed at identifying carcinogenic potential of oral exposure to petroleum waxes (consisting of n-alkanes) where no toxicity was identified using SD rats (Shubik et al., 1962). Subsequent sub-chronic studies on medicinal grade
1096
MOH accumulation in F344 rats
mineral oil (consisting mostly of isoparaffin and cycloparaffin) were carried out in Beagle dogs and Sprague-Dawley (SD) and Long-Evans rats (Firriolo et al., 1995; Smith et al., 1995). From these studies, it became evident that the F344 rat responds quite differently to the exposure of MOSH when compared to other rat strains (Griffis et al., 2010) or dogs, namely that the F344 lesions are follicular epithelioid granulomas with associated lymphocytic inflammation and hepatocyte necrosis. Accordingly, the key questions are: whether there are granuloma lesions in human livers of similar morphology to the F344 rats; is there any evidence linking them to MOSH and; is there any evidence of harm? To attempt to answer these questions, four further questions need addressing: a.) b.) c.) d.)
What is the frequency of granuloma occurrence in human liver? What types are they? What are the causes? What is the outcome?
a.) Frequency of hepatic granuloma: Numerous publications have documented the frequency of the occurrence of granulomas in human liver and the range is large (2–25%), but the majority of reports suggest that around 5–10% of all liver biopsies contain granulomas (Burt et al., 2007; Klatskin, 1977; McCluggage and Sloan, 1994). b.) Types of hepatic granuloma: Liver granuloma is not a disease in itself. Granulomas are a type of inflammatory response with many causes. What is observed is the result of an ‘insult’ which produces an inflammatory response of the granuloma type. Around 100 “insults/causes” have been associated with liver granulomas (see below). As mentioned above, the literature on the association between granuloma and mineral hydrocarbon exposure identifies two types of “granuloma” in human livers. The first is the “lipogranuloma” described under point I.; small vacuolated macrophages with little or no lymphocytic inflammation or fibrosis. The second type is formed of epithelioid cells, with occasional giant cells, lymphocytic inflammation, necrosis and varying fibrosis. The name “granuloma” should be reserved for the latter (Denk et al., 1994). This lesion is similar in morphology to the F344 lesions. It is sometimes not clear from the literature that this distinction has been observed, but Dincsoy (Dincsoy et al., 1982) and Gaya (Gaya et al., 2003) specifically excluded lipogranulomas from their analysis. The latter paper found epithelioid granulomas in 63 out 1662 cases (3.8%), but excluded 24 cases of lipogranuloma (1.5%). Furthermore, as the publications quoted under point I. have shown that around 40–50% of livers contain “lipogranuloma”, the fact that a granuloma frequency of 5–10% is quoted in most papers suggests that in these cases true epithelioid granulomas are being reported, not lipogranulomas. Accordingly, it is probably reasonable to assume that the frequency of F344-like epithelioid granulomas in human livers is around 5–10%. c.) Causes of hepatic granuloma: There is a vast range of causes hepatic granuloma reported in the literature (Burt et al., 2007). The number approaches 100, with infectious causes (world-wide) representing around 50%. Of the rest, Sarcoidosis is associated with around 35% of cases, Primary Biliary Cholangitis (PBC) around 5% and drugs around 2% - it should be noted that around 100 drugs have been implicated in liver granulomas. However, the key finding is that in a significant number of cases of liver granulomas, a cause cannot be found – idiopathic granulomatous hepatitis. It is said that these patients tend to be middle-aged/elderly males with cholestatic liver function tests, fever, weight loss, weakness and myalgia. These signs and symptoms can last years. The
frequency of such unexplained cases varies, but the range in most papers is 10–25% of livers with granulomas (Cunningham et al., 1982; Klatskin, 1977; Sartin and Walker, 1991). The largest reported frequency is from the Mayo Clinic where 50% of granulomas were unexplained (Sartin and Walker, 1991). The 10–20% livers with unexplained granulomas have by definition been investigated thoroughly but the trigger(s) is not known. The reason for the unusually high frequency in this latter series is not clear, but the authors included only referral patients and only patients with clinical or biochemical abnormalities. They excluded autopsies and chance findings at biopsy. It is of interest that the proportion of unexplained granuloma has not changed much over the last 30 years despite the increasing sensitivity and specificity of diagnostic investigations – one would have expected that this increasing diagnostic capability would have reduced the proportion of undiagnosed granulomas. Could some/all of these unexplained cases be atypical reactions to MOSH - rather like the F344 rats that show a genetically determined increased sensitivity to MOSH in comparison to SD rats - and if yes what is the outcome? d.) Outcome: The outcome reflects the underlying disease. Thus, in infectious cases, eradication of the micro-organism will result in healing, usually with insignificant residual fibrosis. Sarcoidosis usually does not cause significant liver disease although it can occasionally produce very severe fibrosis and/or biliary disease. PBC is a slowly progressive condition which, if untreated, can result in cirrhosis if the duration of the disease is long enough (20 or more years). In contrast, the outcome of the unexplained granulomas is accepted to be excellent (Zoutman et al., 1991). While it is reported that around 50% receive steroids, at 10 years there is no evidence of clinically progressive liver disease and symptoms and signs have resolved. In summary, accumulation of MOSH is in itself, not an endpoint and should not be evaluated in isolation without a careful look at the accompanying pathology. Moreover, the appropriateness of the animal models to the human situation is unclear. Even if some/all of the humans with epithelioid granulomas of unexplained origin are hypersensitivity reactions to MOSH exposure (inferring that these individuals respond like F344 rats), the outcome is accepted to be excellent. III. “If exposure to wax components triggered granuloma formation, exposure to these must have exceeded a critical threshold. No more recent investigations on the occurrence of granuloma in humans have been published, but in the livers and spleens analyzed for MOSH in 2013 (Barp et al., 2014) virtually no granulomas were observed (A. Reiner, personal communication, co-author of Barp et al., 2014). This suggests decreased exposure to waxes and that the dose threshold initiating granuloma formation is somewhere between the present exposure and that several decades ago”. Page 330. 4.3. Relevance for humans (Barp et al., 2017b).
One of the interesting points about the above paragraph is that a key finding by Barp et al., 2014 that the subjects examined had virtually no granulomas. However, this is only reported 3 years after the publication and not in the actual report of 2014 as one would expect. If it had been published then, it could perhaps have allowed a more detailed description of the circumstances at the time. So, a key question is what are the data to show that liver granulomas are now rare? The Barp et al., 2014 paper that they quote had 37 autopsies with very limited data about the deceased, age range and sex ratio. We would want to see much more data. The one observation that can be made from the data presented is that the individual with the highest
MOH accumulation in F344 rats
MOSH in liver (900 mg/kg) was not the oldest (90 years) as one would expect if accumulation was an additive effect, but in fact a female subject of 65 years. From what is stated in Barp et al., 2017b, there was no granuloma in this person's liver. If MOSH was a simple toxin, then one would have expected that if any liver was going to contain granulomas, it would be this one. However, in the absence of any further information, one cannot explore possible correlations between MOSH accumulation and increasing age, levels and granuloma incidence in liver, especially in such a small number of individuals. As additional information, one would consider the following questions: a., Were there any selection biases in the cases – the older literature showed a geographical and age association with liver granulomas (Cruickshank, 1984; Wanless and Geddie, 1985)? b., Did the deceased have a particular disease distribution or not? c., Was there any information on exposure to MOSH? and equally important, d., what were the levels and types of MOSH in the few cases with granulomas compared to the negative ones? For all the reasons outlined in the three points above, it seems to us that to answer to the question of the relevance of MOSH in human livers will not come from yet further experiments in animals. One needs a population-based investigation of the occurrence of granulomas in the liver and a correlation with the amount and type of MOSH found in the same livers, with a full set of data about the individual (life style, weight, diseases, etc). There are no published data. However, by making some very broad assumptions, including that all unexplained human liver granulomas are caused by MOSH exposure, one can make a “guesstimate” of the possible incidence by extrapolation from known data. It must be again emphasized that granuloma is itself not a disease, but an inflammatory response to an ‘insult’. For the guesstimate we are assuming that all the unexplained granuloma are not the lipogranuloma associated with MOSH exposure, but an atypical inflammatory reaction to mineral hydrocarbon exposure. In Oxford, there are around 400 liver biopsies/year. Thus, based on point II., around 20–40 cases will have granulomas. Of these, 2–10 will contain unexplained granulomas – that we will assume are an atypical reaction to MOSH. The number of liver biopsies per year in the UK is unknown, but there are around 20 centres with specialist liver units which will have significant numbers of liver biopsies. If one assumes that each has around 400 cases (as in Oxford), then the total number of biopsies a year in the UK would be about 8000. If one then assumes that the rest of the hospitals in the UK perform around the same number in total, then there would be around 16,000 liver biopsies a year in the UK - this may be a considerable over-guesstimate. Accordingly, for a population of 63 million, there would be around 800–1600 biopsies/year with granulomas, and around 80–400 biopsies with unexplained granulomas. The population of the EU is about 500 million (~ 8 × the population of the UK). So, by extrapolation, there could be around 640–3200 biopsies a year with unexplained granulomas, of which none, some, or all might be hypersensitivity reactions to MOSH. In a worst-case scenario, if one assumed that 5% of these cases showed progression to significant liver disease, then there would be between 32 and 160 such cases/year across the whole of the EU. Three things must be emphasized regarding the above speculation: First, these numbers are guesstimates where we have adopted the speculative option with the largest hazard implication, namely that the unexplained granuloma are different from the known lipogranuloma associated with MOSH exposure and represent those occasional cases of individuals who are hypersensitive to MOSH. These numbers have then been extrapolated, with the resultant danger of magnification of errors. Second, they assume that the pattern of liver disease is the same across the EU, which may very well not be correct. As mentioned above, while lipogranulomas have been reported in up to 40–50% of human livers, these lesions increase with age and are more common
1097
in the USA and Australia than the UK and Europe (Cruickshank and Thomas, 1984; Wanless and Geddie, 1985). Third, the numbers are based on unfounded assumptions about both causality and progression – namely that all unexplained granulomas are caused by a reaction to MOSH and that 5% progress. There are no data to suggest that mineral hydrocarbons are implicated in the cause of the unexplained granulomas and all the evidence is that unexplained granulomas do not result in significant liver disease. Conclusion There is no convincing evidence that the accumulation of MOSH (alkanes) and the severe granulomas seen in livers of the F344 rat after ingestion of wax or mineral oil, translates into similar toxicity in humans. Even in a worst-case scenario (based on unsubstantiated assumptions and numerical guesstimates), the implied numbers across the EU are low and progression is very unlikely. Accordingly, it seems unlikely that liver disease due to accumulation of MOSH is a significant public health hazard for humans. References Barp, L., Kornauth, C., Wuerger, T., et al., 2014. Mineral oil in human tissues, part I: concentrations and molecular mass distributions. Food Chem. Toxicol. 72:312–321. https://doi.org/10.1016/j.fct.2014.04.029. Barp, L., Biedermann, M., Grob, K., et al., 2017a. Accumulation of mineral oil saturated hydrocarbons (MOSH) in female Fischer 344 rats: comparison with human data and consequences for risk assessment. Sci. Total Environ. 575:1263–1278. https:// doi.org/10.1016/j.scitotenv.2016.09.203. Barp, L., Biedermann, M., Grob, K., et al., 2017b. Mineral oil saturated hydrocarbons (MOSH) in female Fischer 344 rats; accumulation of wax components; implications for risk assessment. Sci. Total Environ. 583:319–333. https://doi.org/10.1016/ j.scitotenv.2017.01.071. Boitnott, J., Margolis, S., 1966. Mineral oil in human tissues. 2. Oil droplets in lymph nodes of porta hepatis. Bull. Johns Hopkins Hosp. 118 (5), 414. Boitnott, J.K., Margolis, S., 1970. Saturated hydrocarbons in human tissues. 3. Oil droplets in the liver and spleen. Johns Hopkins Med. J. 127 (2), 65–78. Burt, A., Portmann, B., MacSween, R., 2007. Liver pathology associated with diseases of other organs or systems. MacSween's Pathology of the Liver, 5th ed. Churchill Livingstone Elsevier, Philadelphia, PA, p. 917. Cruickshank, B., 1984. Follicular (mineral oil) lipidosis: I. Epidemiologic studies of involvement of the spleen. Hum. Pathol. 15 (8), 724–730. Cruickshank, B., Thomas, M.J., 1984. Mineral oil (follicular) lipidosis: II. Histologic studies of spleen, liver, lymph nodes, and bone marrow. Hum. Pathol. 15 (8), 731–737. Cunningham, D., Mills, P., Quigley, E., et al., 1982. Hepatic granulomas: experience over a 10-year period in the West of Scotland. QJM 51 (2), 162–170. Denk, H., Scheuer, P., Baptista, A., et al., 1994. Guidelines for the diagnosis and interpretation of hepatic granulomas. Histopathology 25 (3), 209–218. Dincsoy, H.P., Weesner, R.E., MacGee, J., 1982. Lipogranulomas in non-fatty human livers. A mineral oil induced environmental disease. Am. J. Clin. Pathol. 78 (1), 35–41. Firriolo, J.M., Morris, C.F., Trimmer, G.W., Twitty, L.D., Smith, J.H., Freeman, J.J., 1995. Comparative 90-day feeding study with low-viscosity white mineral oil in Fischer-344 and Sprague-Dawley-derived CRL:CD rats. Toxicol. Pathol. 23 (1):26–33. https:// doi.org/10.1177/019262339502300104. Fleming, K.A., Zimmerman, H., Shubik, P., 1998. Granulomas in the livers of humans and fischer rats associated with the ingestion of mineral hydrocarbons: a comparison. Regul. Toxicol. Pharmacol. 27 (1 Pt 2):75–81 (doi:S0273–2300(97)91109–0 [pii]https://doi.org/10.1006/rtph.1997.1109). Gaya, D., Thorburn, D., Oien, K., Morris, A., Stanley, A., 2003. Hepatic granulomas: a 10 year single centre experience. J. Clin. Pathol. 56 (11), 850–853. Griffis, L.C., Twerdok, L.E., Francke-Carroll, S., et al., 2010. Comparative 90-day dietary study of paraffin wax in Fischer-344 and Sprague-Dawley rats. Food Chem. Toxicol. 48 (1):363–372. https://doi.org/10.1016/j.fct.2009.10.024. Klatskin, G., 1977. Hepatic granulomata: problems in interpretation. Mount Sinai J. Med. New York 44 (6), 798–812. McCluggage, W., Sloan, J., 1994. Hepatic granulomas in Northern Ireland: a thirteen year review. Histopathology 25 (3), 219–228. Sartin, J.S., Walker, R.C., 1991. Granulomatous hepatitis: a retrospective review of 88 cases at the Mayo Clinic. Mayo Clinic Proceedings. vol 66. Elsevier, pp. 914–918. Shubik, P., Saffiotti, U., Lijinsky, W., et al., 1962. Studies on the toxicity of petroleum waxes. Toxicol. Appl. Pharmacol. 4 (Suppl), 1–62. Smith, J., Bird, M., Lewis, S., Freeman, J., Hogan, G., Scala, R., 1995. Subchronic feeding study of four white mineral oils in dogs and rats. Drug Chem. Toxicol. 18 (1), 83–103. Stryker, W.A., 1941. Absorption of Liquid Petrolatum (“Mineral Oil”) From the Intestine: A Histologic and Chemical Study. Wanless, I.R., Geddie, W.R., 1985. Mineral oil lipogranulomata in liver and spleen. A study of 465 autopsies. Arch. Pathol. Lab. Med. 109 (3), 283–286.
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Zoutman, D.E., Ralph, E.D., Frei, J.V., 1991. Granulomatous hepatitis and fever of unknown origin: an 11-year experience of 23 cases with three years' follow-up. J. Clin. Gastroenterol. 13 (1), 69–75.
Kenneth Fleming Green Templeton College, University of Oxford 2 The Winnyards, Cumnor, Oxford, OX2 9RJ, UK Corresponding author. E-mail address: kenneth.fl[email protected]
Juan-Carlos Carrillo Shell International B.V.; Carel van Bylandtlaan 16, 2596 HR, The Hague, The Netherlands University of Oxford, 2 The Winnyards, Oxford, Oxon OX2 9RJ, United Kingdom 1 July 2017
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
MOH accumulation in F344 rats
Editor: Adrian Covaci
Dear Sir, It has been with great interest that we have read the two recent papers published in this Journal (Barp et al., 2017a; Barp et al., 2017b), on the effects of exposure to mineral hydrocarbons - MHC (what the authors call MOSH1) using the Fisher F344 rat as a model. One of us has previously made a comparison between the granuloma findings in the F344 and humans, especially on the types of hepatic granuloma caused by ingestion of mineral oil hydrocarbons (Fleming et al., 1998). While we cannot comment on the sophisticated analytical methods used, we would like nonetheless to provide our point of view about some of the arguments presented by the authors from the perspective of a human liver pathologist. I. “Strong accumulation is not of toxicological concern per se. However, as the MOSH concentrations in human tissues were probably severely underestimated when extrapolated from animal experiments involving high doses….”. Page 1227; 4.3 Practical consequences (Barp et al., 2017a)
It is reasonable to suppose that accumulation is per se not of toxicological concern because it is not an endpoint in itself. Thus, to be of pathological significance, the key question is whether any accumulation is associated with any adverse effects. It has been known for many years that MOSH is retained in the human liver and in other tissues. Early descriptions documented focal collections of vacuoles in liver and spleen, with smaller vacuoles within macrophages and larger vacuoles in multinuclear giant cells (Boitnott and Margolis, 1966; Boitnott and Margolis, 1970; Stryker, 1941). It was assumed that these vacuoles represented MOSH which had been dissolved out of the cells during preparing the tissues for microscopy. In the liver, the lesions were mainly portal, although clusters of vacuoles and macrophages were observed in the hepatic parenchyma, often around the central vein. These lesions were (and are) called
1
The key papers that describe the human pathology of mineral hydrocarbons do not talk about “MOSH”. Pragmatically however, to allow direct reference to these papers, the term MOSH will be used. The reader should thus be aware of MOSH as a broad term for alkanes commonly found in oils and waxes.
https://doi.org/10.1016/j.scitotenv.2017.07.275 0048-9697/© 2017 Elsevier B.V. All rights reserved.
lipogranulomas and are morphologically very different from the severe lesions seen in the F344 rats – these latter are formed of epithelioid cells. In 1982, Dincsoy et al. examined the incidence of lipogranulomas in human liver from both biopsies and autopsies (Dincsoy et al., 1982). There was a lack of association between the lesions and liver steatosis, ruling out simple reaction to presence of fat in hepatocytes. In the non-fatty livers, there was also a significantly lower incidence in 1952–3, in comparison with 1978–80 (1.7% versus 4.6%). Extracts of the autopsy livers were analyzed and the presence of MOSH of similar composition to commercial white oil grades was found. The authors concluded that the incidence of these lesions in non-fatty livers was probably associated with exposure to MOSH in food. Importantly, there was no evidence that the lesions were of clinical significance. Subsequently, Cruickshank, in 1984, examined samples of liver, spleen, bone marrow and lymph nodes from 200 autopsies performed in 1970–72 for the presence of lipogranulomas. The incidence was much higher than previously reported, at approximately 45%, and interestingly, showed marked geographical variation (Cruickshank and Thomas, 1984). In 1985, Wanless and Geddie extensively reviewed the previous literature and reported the results of their analysis of 465 autopsies performed between 1974 and 1976. Lipogranulomas were observed in liver at an incidence of 48%. A correlation in the incidence and severity of lipogranulomas with increasing age and maleness was observed. No correlation was found between the incidence of lipogranulomas and the occurrence of liver steatosis, diabetes mellitus, obesity or any liver disease. Again, importantly, there was no evidence of clinical significance, despite some mild accompanying fibrosis (Wanless and Geddie, 1985). In summary, several investigations have identified lesions (lipogranulomas) in up to 40–50% of human livers and these have been associated with the presence of MOSH in the tissues. It has been postulated that the lipogranulomas may be related to laxative and/or food mineral hydrocarbon ingestion. Crucially, in all reports, there has been no evidence of any clinical significance associated with the presence of these lesions and thus MOSH in tissues, including the liver. Again, note that these lesions are morphologically quite different from the epithelioid granulomas seen in the Fischer 344 rats. II. The animal data available might not even enable to adequately support the safety of the present human exposure, and end points other than granuloma formation and potential consecutive inflammation should be investigated” Page 1227; 4.3 Practical consequences (Barp et al., 2017a).
The available animal data are not restricted to studies performed with the F344 rat. Early investigations were primarily aimed at identifying carcinogenic potential of oral exposure to petroleum waxes (consisting of n-alkanes) where no toxicity was identified using SD rats (Shubik et al., 1962). Subsequent sub-chronic studies on medicinal grade
1096
MOH accumulation in F344 rats
mineral oil (consisting mostly of isoparaffin and cycloparaffin) were carried out in Beagle dogs and Sprague-Dawley (SD) and Long-Evans rats (Firriolo et al., 1995; Smith et al., 1995). From these studies, it became evident that the F344 rat responds quite differently to the exposure of MOSH when compared to other rat strains (Griffis et al., 2010) or dogs, namely that the F344 lesions are follicular epithelioid granulomas with associated lymphocytic inflammation and hepatocyte necrosis. Accordingly, the key questions are: whether there are granuloma lesions in human livers of similar morphology to the F344 rats; is there any evidence linking them to MOSH and; is there any evidence of harm? To attempt to answer these questions, four further questions need addressing: a.) b.) c.) d.)
What is the frequency of granuloma occurrence in human liver? What types are they? What are the causes? What is the outcome?
a.) Frequency of hepatic granuloma: Numerous publications have documented the frequency of the occurrence of granulomas in human liver and the range is large (2–25%), but the majority of reports suggest that around 5–10% of all liver biopsies contain granulomas (Burt et al., 2007; Klatskin, 1977; McCluggage and Sloan, 1994). b.) Types of hepatic granuloma: Liver granuloma is not a disease in itself. Granulomas are a type of inflammatory response with many causes. What is observed is the result of an ‘insult’ which produces an inflammatory response of the granuloma type. Around 100 “insults/causes” have been associated with liver granulomas (see below). As mentioned above, the literature on the association between granuloma and mineral hydrocarbon exposure identifies two types of “granuloma” in human livers. The first is the “lipogranuloma” described under point I.; small vacuolated macrophages with little or no lymphocytic inflammation or fibrosis. The second type is formed of epithelioid cells, with occasional giant cells, lymphocytic inflammation, necrosis and varying fibrosis. The name “granuloma” should be reserved for the latter (Denk et al., 1994). This lesion is similar in morphology to the F344 lesions. It is sometimes not clear from the literature that this distinction has been observed, but Dincsoy (Dincsoy et al., 1982) and Gaya (Gaya et al., 2003) specifically excluded lipogranulomas from their analysis. The latter paper found epithelioid granulomas in 63 out 1662 cases (3.8%), but excluded 24 cases of lipogranuloma (1.5%). Furthermore, as the publications quoted under point I. have shown that around 40–50% of livers contain “lipogranuloma”, the fact that a granuloma frequency of 5–10% is quoted in most papers suggests that in these cases true epithelioid granulomas are being reported, not lipogranulomas. Accordingly, it is probably reasonable to assume that the frequency of F344-like epithelioid granulomas in human livers is around 5–10%. c.) Causes of hepatic granuloma: There is a vast range of causes hepatic granuloma reported in the literature (Burt et al., 2007). The number approaches 100, with infectious causes (world-wide) representing around 50%. Of the rest, Sarcoidosis is associated with around 35% of cases, Primary Biliary Cholangitis (PBC) around 5% and drugs around 2% - it should be noted that around 100 drugs have been implicated in liver granulomas. However, the key finding is that in a significant number of cases of liver granulomas, a cause cannot be found – idiopathic granulomatous hepatitis. It is said that these patients tend to be middle-aged/elderly males with cholestatic liver function tests, fever, weight loss, weakness and myalgia. These signs and symptoms can last years. The
frequency of such unexplained cases varies, but the range in most papers is 10–25% of livers with granulomas (Cunningham et al., 1982; Klatskin, 1977; Sartin and Walker, 1991). The largest reported frequency is from the Mayo Clinic where 50% of granulomas were unexplained (Sartin and Walker, 1991). The 10–20% livers with unexplained granulomas have by definition been investigated thoroughly but the trigger(s) is not known. The reason for the unusually high frequency in this latter series is not clear, but the authors included only referral patients and only patients with clinical or biochemical abnormalities. They excluded autopsies and chance findings at biopsy. It is of interest that the proportion of unexplained granuloma has not changed much over the last 30 years despite the increasing sensitivity and specificity of diagnostic investigations – one would have expected that this increasing diagnostic capability would have reduced the proportion of undiagnosed granulomas. Could some/all of these unexplained cases be atypical reactions to MOSH - rather like the F344 rats that show a genetically determined increased sensitivity to MOSH in comparison to SD rats - and if yes what is the outcome? d.) Outcome: The outcome reflects the underlying disease. Thus, in infectious cases, eradication of the micro-organism will result in healing, usually with insignificant residual fibrosis. Sarcoidosis usually does not cause significant liver disease although it can occasionally produce very severe fibrosis and/or biliary disease. PBC is a slowly progressive condition which, if untreated, can result in cirrhosis if the duration of the disease is long enough (20 or more years). In contrast, the outcome of the unexplained granulomas is accepted to be excellent (Zoutman et al., 1991). While it is reported that around 50% receive steroids, at 10 years there is no evidence of clinically progressive liver disease and symptoms and signs have resolved. In summary, accumulation of MOSH is in itself, not an endpoint and should not be evaluated in isolation without a careful look at the accompanying pathology. Moreover, the appropriateness of the animal models to the human situation is unclear. Even if some/all of the humans with epithelioid granulomas of unexplained origin are hypersensitivity reactions to MOSH exposure (inferring that these individuals respond like F344 rats), the outcome is accepted to be excellent. III. “If exposure to wax components triggered granuloma formation, exposure to these must have exceeded a critical threshold. No more recent investigations on the occurrence of granuloma in humans have been published, but in the livers and spleens analyzed for MOSH in 2013 (Barp et al., 2014) virtually no granulomas were observed (A. Reiner, personal communication, co-author of Barp et al., 2014). This suggests decreased exposure to waxes and that the dose threshold initiating granuloma formation is somewhere between the present exposure and that several decades ago”. Page 330. 4.3. Relevance for humans (Barp et al., 2017b).
One of the interesting points about the above paragraph is that a key finding by Barp et al., 2014 that the subjects examined had virtually no granulomas. However, this is only reported 3 years after the publication and not in the actual report of 2014 as one would expect. If it had been published then, it could perhaps have allowed a more detailed description of the circumstances at the time. So, a key question is what are the data to show that liver granulomas are now rare? The Barp et al., 2014 paper that they quote had 37 autopsies with very limited data about the deceased, age range and sex ratio. We would want to see much more data. The one observation that can be made from the data presented is that the individual with the highest
MOH accumulation in F344 rats
MOSH in liver (900 mg/kg) was not the oldest (90 years) as one would expect if accumulation was an additive effect, but in fact a female subject of 65 years. From what is stated in Barp et al., 2017b, there was no granuloma in this person's liver. If MOSH was a simple toxin, then one would have expected that if any liver was going to contain granulomas, it would be this one. However, in the absence of any further information, one cannot explore possible correlations between MOSH accumulation and increasing age, levels and granuloma incidence in liver, especially in such a small number of individuals. As additional information, one would consider the following questions: a., Were there any selection biases in the cases – the older literature showed a geographical and age association with liver granulomas (Cruickshank, 1984; Wanless and Geddie, 1985)? b., Did the deceased have a particular disease distribution or not? c., Was there any information on exposure to MOSH? and equally important, d., what were the levels and types of MOSH in the few cases with granulomas compared to the negative ones? For all the reasons outlined in the three points above, it seems to us that to answer to the question of the relevance of MOSH in human livers will not come from yet further experiments in animals. One needs a population-based investigation of the occurrence of granulomas in the liver and a correlation with the amount and type of MOSH found in the same livers, with a full set of data about the individual (life style, weight, diseases, etc). There are no published data. However, by making some very broad assumptions, including that all unexplained human liver granulomas are caused by MOSH exposure, one can make a “guesstimate” of the possible incidence by extrapolation from known data. It must be again emphasized that granuloma is itself not a disease, but an inflammatory response to an ‘insult’. For the guesstimate we are assuming that all the unexplained granuloma are not the lipogranuloma associated with MOSH exposure, but an atypical inflammatory reaction to mineral hydrocarbon exposure. In Oxford, there are around 400 liver biopsies/year. Thus, based on point II., around 20–40 cases will have granulomas. Of these, 2–10 will contain unexplained granulomas – that we will assume are an atypical reaction to MOSH. The number of liver biopsies per year in the UK is unknown, but there are around 20 centres with specialist liver units which will have significant numbers of liver biopsies. If one assumes that each has around 400 cases (as in Oxford), then the total number of biopsies a year in the UK would be about 8000. If one then assumes that the rest of the hospitals in the UK perform around the same number in total, then there would be around 16,000 liver biopsies a year in the UK - this may be a considerable over-guesstimate. Accordingly, for a population of 63 million, there would be around 800–1600 biopsies/year with granulomas, and around 80–400 biopsies with unexplained granulomas. The population of the EU is about 500 million (~ 8 × the population of the UK). So, by extrapolation, there could be around 640–3200 biopsies a year with unexplained granulomas, of which none, some, or all might be hypersensitivity reactions to MOSH. In a worst-case scenario, if one assumed that 5% of these cases showed progression to significant liver disease, then there would be between 32 and 160 such cases/year across the whole of the EU. Three things must be emphasized regarding the above speculation: First, these numbers are guesstimates where we have adopted the speculative option with the largest hazard implication, namely that the unexplained granuloma are different from the known lipogranuloma associated with MOSH exposure and represent those occasional cases of individuals who are hypersensitive to MOSH. These numbers have then been extrapolated, with the resultant danger of magnification of errors. Second, they assume that the pattern of liver disease is the same across the EU, which may very well not be correct. As mentioned above, while lipogranulomas have been reported in up to 40–50% of human livers, these lesions increase with age and are more common
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in the USA and Australia than the UK and Europe (Cruickshank and Thomas, 1984; Wanless and Geddie, 1985). Third, the numbers are based on unfounded assumptions about both causality and progression – namely that all unexplained granulomas are caused by a reaction to MOSH and that 5% progress. There are no data to suggest that mineral hydrocarbons are implicated in the cause of the unexplained granulomas and all the evidence is that unexplained granulomas do not result in significant liver disease. Conclusion There is no convincing evidence that the accumulation of MOSH (alkanes) and the severe granulomas seen in livers of the F344 rat after ingestion of wax or mineral oil, translates into similar toxicity in humans. Even in a worst-case scenario (based on unsubstantiated assumptions and numerical guesstimates), the implied numbers across the EU are low and progression is very unlikely. Accordingly, it seems unlikely that liver disease due to accumulation of MOSH is a significant public health hazard for humans. References Barp, L., Kornauth, C., Wuerger, T., et al., 2014. Mineral oil in human tissues, part I: concentrations and molecular mass distributions. Food Chem. Toxicol. 72:312–321. https://doi.org/10.1016/j.fct.2014.04.029. Barp, L., Biedermann, M., Grob, K., et al., 2017a. Accumulation of mineral oil saturated hydrocarbons (MOSH) in female Fischer 344 rats: comparison with human data and consequences for risk assessment. Sci. Total Environ. 575:1263–1278. https:// doi.org/10.1016/j.scitotenv.2016.09.203. Barp, L., Biedermann, M., Grob, K., et al., 2017b. Mineral oil saturated hydrocarbons (MOSH) in female Fischer 344 rats; accumulation of wax components; implications for risk assessment. Sci. Total Environ. 583:319–333. https://doi.org/10.1016/ j.scitotenv.2017.01.071. Boitnott, J., Margolis, S., 1966. Mineral oil in human tissues. 2. Oil droplets in lymph nodes of porta hepatis. Bull. Johns Hopkins Hosp. 118 (5), 414. Boitnott, J.K., Margolis, S., 1970. Saturated hydrocarbons in human tissues. 3. Oil droplets in the liver and spleen. Johns Hopkins Med. J. 127 (2), 65–78. Burt, A., Portmann, B., MacSween, R., 2007. Liver pathology associated with diseases of other organs or systems. MacSween's Pathology of the Liver, 5th ed. Churchill Livingstone Elsevier, Philadelphia, PA, p. 917. Cruickshank, B., 1984. Follicular (mineral oil) lipidosis: I. Epidemiologic studies of involvement of the spleen. Hum. Pathol. 15 (8), 724–730. Cruickshank, B., Thomas, M.J., 1984. Mineral oil (follicular) lipidosis: II. Histologic studies of spleen, liver, lymph nodes, and bone marrow. Hum. Pathol. 15 (8), 731–737. Cunningham, D., Mills, P., Quigley, E., et al., 1982. Hepatic granulomas: experience over a 10-year period in the West of Scotland. QJM 51 (2), 162–170. Denk, H., Scheuer, P., Baptista, A., et al., 1994. Guidelines for the diagnosis and interpretation of hepatic granulomas. Histopathology 25 (3), 209–218. Dincsoy, H.P., Weesner, R.E., MacGee, J., 1982. Lipogranulomas in non-fatty human livers. A mineral oil induced environmental disease. Am. J. Clin. Pathol. 78 (1), 35–41. Firriolo, J.M., Morris, C.F., Trimmer, G.W., Twitty, L.D., Smith, J.H., Freeman, J.J., 1995. Comparative 90-day feeding study with low-viscosity white mineral oil in Fischer-344 and Sprague-Dawley-derived CRL:CD rats. Toxicol. Pathol. 23 (1):26–33. https:// doi.org/10.1177/019262339502300104. Fleming, K.A., Zimmerman, H., Shubik, P., 1998. Granulomas in the livers of humans and fischer rats associated with the ingestion of mineral hydrocarbons: a comparison. Regul. Toxicol. Pharmacol. 27 (1 Pt 2):75–81 (doi:S0273–2300(97)91109–0 [pii]https://doi.org/10.1006/rtph.1997.1109). Gaya, D., Thorburn, D., Oien, K., Morris, A., Stanley, A., 2003. Hepatic granulomas: a 10 year single centre experience. J. Clin. 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MOH accumulation in F344 rats
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Kenneth Fleming Green Templeton College, University of Oxford 2 The Winnyards, Cumnor, Oxford, OX2 9RJ, UK Corresponding author. E-mail address: kenneth.fl[email protected]
Juan-Carlos Carrillo Shell International B.V.; Carel van Bylandtlaan 16, 2596 HR, The Hague, The Netherlands University of Oxford, 2 The Winnyards, Oxford, Oxon OX2 9RJ, United Kingdom 1 July 2017