1 Introduction

Humans are exposed to MOH e.g. through foodstuffs and cosmetics. The main reasons for their occurrence are the use of mineral oil and wax products as food additives, release agents and lubricants (EFSA 2009; 2013a; b) as well as cosmetic ingredients (Petry et al. 2017) and plasticisers in elastomeric and polymeric materials (Bartsch et al. 2016). Apart from these intentional uses, there is also food contamination via the environment, by technological processes and migration from food packaging (EFSA 2012).

Autopsy studies from the mid-1960s showed that exposure to MOH in food causes deposits in human tissues, specifically in the mesenteric lymph nodes (MLNs), spleen, and liver (Boitnott and Margolis 1966; 1970; Liber and Rose 1967; Rose and Liber 1966). The deposits turned out to be complex mixtures of saturated hydrocarbons bearing the chemical signature of mineral oil and consisting predominantly of branched and cyclic compounds (Boitnott and Margolis 1966; 1970). Collectively, these compounds have been named mineral oil saturated hydrocarbons (MOSH), to distinguish them from saturated hydrocarbons of plant origin, and mineral oil aromatic hydrocarbons (MOAH) (Biedermann et al. 2009). It was not until this century that new human data became available from autopsy and biopsy studies that involved the use of advanced analytical methods (Barp et al. 2014; Biedermann et al. 2015; Concin et al. 2008). Although the dietary exposure to MOH has decreased in the last decades (Boitnott and Margolis 1966; EFSA 2012; EFSA 2023; Heimbach et al. 2002), these recent studies confirmed the presence of MOSH in MLNs, spleen and liver, but additionally adipose tissue was identified as a quantitatively important target of MOSH deposition (Barp et al. 2014; Biedermann et al. 2015; Concin et al. 2008).

Similar to the human situation, repeated dietary administration of MOSH caused deposition in MLNs, spleen, liver and adipose tissue of rats and pigs (Baldwin et al. 1992; Barp et al. 2017a, b; Firriolo et al. 1995; Griffis et al. 2010; Scotter et al. 2003; Smith et al. 1996; Trimmer et al. 2004; Tulliez 1986; Tulliez et al. 1975). The degree of deposition depends on species, strain, sex, hydrocarbon structure, carbon number, dose and tissue type. Studies in female F-344 rats additionally determined the reversibility of MOSH deposition during a post-exposure recovery phase (Barp et al. 2017a; Smith et al. 1996; Trimmer et al. 2004). It was shown that total MOSH levels in liver decreased, with apparent half-life estimates ranging from one to a few months (although the elimination rates for the readily metabolized constituents are very likely shorter, while other MOSH might have longer half-lives) (Pirow et al. 2019). There were indications for a reversibility of MOSH deposition in spleen (Barp et al. 2017a). In adipose tissue, however, the MOSH level continued to increase during the recovery period, which suggests accumulation of MOSH and a post-exposure transfer from other tissues, including – possibly – the liver and the gastrointestinal tract.

The results from animal studies raised concerns about a long-term or even irreversible accumulation of MOSH in humans. To address this issue, we performed a statistical re-analysis of published biopsy and autopsy data regarding the age-dependence of MOSH levels in human tissue. Multiple linear regression was used to identify additional predictors, such as cosmetics usage and sex that could be associated with MOSH levels in human tissue. Finally, we derived effect sizes for statistically significant predictors and discussed the physiological causality and toxicological relevance.

2 Materials and methods

2.1 Biopsy and autopsy study data

The MOSH levels in human tissue were taken from 2 studies: A biopsy study (Concin et al. 2008) measured the MOSH concentration in subcutaneous fat sampled from 2005 to 2006 in 144 Austrian women (aged 19–47). Subcutaneous fat was removed from the abdominal wall during elective caesarean section. The data were approximately log-normally distributed with a median concentration of 52.5 mg/kg (range: 10–260 mg/kg, n = 142 subjects). Assuming an average fat content of 84% in human adipose tissue (Thomas 1962), this value corresponds to a median MOSH content of 44 mg/kg. Barp et al. (2014) analysed tissue samples from 37 patients (aged 25–91) autopsied in Austria in 2013. The MOSH concentrations were measured in MLNs, subcutaneous abdominal adipose tissue, liver, spleen and lung, for some individuals also in kidney, heart and brain. Again, the data in MLNs, subcutaneous abdominal adipose tissue, liver and spleen were approximately log-normally distributed with a median of 166 mg/kg in the MLNs (range: 21–1390 mg/kg), 87 mg/kg in adipose tissue (range: 17–493 mg/kg), 71 mg/kg in liver (range: 14–901 mg/kg), 28 mg/kg in spleen (range: 6–1400 mg/kg), and 7 mg/kg in lung (range: <2–91 mg/kg). The data for MLNs, subcutaneous abdominal adipose tissue, liver, and spleen were subjected to statistical re-analysis in the present study.

2.2 Statistical analysis

Multiple linear regression was applied to predict the MOSH concentration in post-mortem tissues (MLNs, adipose tissue, liver, spleen) on the basis of the predictor variables age, sex, and body mass index (BMI). Since the data were approximately log-normally distributed, the data were log-transformed to approach a normal distribution. Predictor variable selection was applied based on Akaike’s Information Criterion (AIC) to identify the “best” subset from the initial set of candidate variables, which was then used in a reduced model to predict the MOSH concentration in a given tissue.

The data set on MOSH concentrations in subcutaneous fat of women undergoing elective caesarean section had already been subjected to multiple linear regression analysis (Concin et al. 2011). As in the autopsy data set, the log-transformed MOSH concentration had been used as outcome variable. This analysis had identified age, pre-pregnancy BMI, country of main residence (Austria vs. others), number of previous childbirths (> 2 vs. ≤ 2), use of sun cream (SC) in the present pregnancy as well as use of hand cream (HC) and lipstick (LS) in daily life as relevant predictors for the MOSH concentration in subcutaneous fat. These predictors were included in the linear model with slight modifications as detailed below.

First, the BMI was replaced by the inverted body mass index (iBMI), which is approximately normally distributed (Nevill et al. 2011). Second, the continuous predictor variables age and iBMI were centred (by subtracting the respective mean) to give the intercept a meaningful interpretation. Third, the categorical predictors related to cosmetics usage were combined in a 4-level factor coding the increasing number of applicable use categories (0: none, 1: LS or HC or SC, 2: LS + HC or LS + SC or HC + SC, 3: LS + HC + SC). The factor level “LS + HC + SC”, for example, referred to the subgroup of “multi-category users”; it applied to women who indicated the use of products from all 3 categories (i.e., lipstick, hand cream, sun cream). The latter modification aimed at quantifying potential differences in MOSH concentrations between multi-category users and non-users.

All analyses were performed in the statistical computing environment R (R Core Team 2023). Scatter plots were created using the R package ‘lattice’ (Sarkar 2008). Predictor effect displays were added to the graphs to visually check the plausibility of the model estimates and to ease their interpretation.

3 Results

The MOSH concentrations in post-mortem tissue samples (MLNs, adipose tissue, liver, spleen) from the 37 autopsy patients were plotted in relation to age, conditional on tissue type and sex (Fig. 1). Multiple linear regression analysis identified age and/or sex as statistically significant predictors, dependent on tissue type. BMI turned out to be a redundant predictor and was removed from the linear model by the predictor selection algorithm. MOSH concentrations in MLNs and adipose tissues showed a 1.2–1.4-fold increase per decade, pointing to a very long-term accumulation in both tissues. Women had, on average, a 2.2–2.5-fold higher MOSH concentration in liver, spleen, and adipose tissue compared to men. In contrast, the MOSH concentration in MLNs was not statistically different between women and men. There was no evidence for an age-dependence of the MOSH concentrations in liver and spleen.

Fig. 1
figure 1

Age and sex dependency of MOSH concentration in MLNs, adipose tissue, liver, and spleen from human autopsy samples. MOSH concentration in MLNs and adipose tissue increased with age as indicated by the upward-sloping lines. There was no evidence of an age dependency of MOSH concentration in liver and spleen; dotted and dashed horizontal lines indicate the geometric mean levels for women and men. Effect sizes comprising the fold-increase per decade and the fold-increase for women relative to men are provided with 95% confidence intervals given in parenthesis. The data of 37 autopsy patients is shown (Barp et al. 2014). The grey-filled diamond symbol in B indicates the median concentration in adipose tissue of pregnant women at the mean age of 31 years (Concin et al. 2008). Symbols with orange background indicate patients with high-grade steatosis. Numbers associated with symbols refer to data of patients which are discussed in the main text

Full size image

The MOSH concentration in abdominal subcutaneous fat from 142 women at the time of caesarean delivery was plotted in relation to age and the inverse of pre-pregnancy BMI, conditional on cosmetics use (Fig. 2). Multiple linear regression quantified the influence of 5 predictor variables on MOSH concentration: age, iBMI, country of main residence, number of previous childbirths, cosmetics use (Table 1). MOSH levels in tissue fat of the pregnant women increased 1.5-fold per decade (Fig. 2A). In addition, MOSH concentrations in tissue fat increased with inverted BMI, meaning that it decreased with BMI (Fig. 2B). For example, on average, women with a BMI of 30 had a 1.2-fold lower MOSH level compared to women with a reference BMI of 23. Since the BMI is highly correlated with body fat mass, this might be explained by an absolute amount of MOSH being distributed (or diluted) in a larger amount of tissue fat. Women very likely experienced such a “dilution effect” during pregnancy, since pregnancy is associated with maternal fat mass gain (Pitkin 1976). This physiological mechanism could contribute to explain why the median MOSH content in the adipose tissue of the pregnant women (44 mg/kg) was below the regression line describing the age dependence of MOSH concentrations in adipose tissue of autopsy patients (Fig. 1B). Table 2 shows the effect size for the categorical predictors like country of main residence, number of previous childbirths, cosmetics use. Women that used cosmetic products from all groups (i.e., lipstick, hand cream and sun cream) had on average a 2.1-fold higher MOSH concentration in abdominal subcutaneous fat than non-users (Fig. 2).

Fig. 2
figure 2

MOSH concentration in abdominal subcutaneous fat of pregnant women at the time of caesarean delivery in relation to age, pre-pregnancy BMI, and cosmetics use. For cosmetics, three categories were considered: daily application of hand cream (HC) and lipstick (LS), and sun cream (SC) during present pregnancy; the number of applicable use categories (e.g., 0: none, 3: LS + HC + SC) is indicated by different symbols and colours. (A) Age dependence of MOSH concentration. Effect sizes comprising the fold-increase per decade and the fold-increase for multi-category users (LS + HC + SC) relative to non-users are provided with 95% confidence intervals given in parenthesis. (B) Dependence of MOSH concentration on inverted BMI. The fold-decrease for an increase in BMI from 23 to 30 is given. Dotted lines in both panels show the predicted average MOSH concentration in relation to age (A) and inverted BMI (B) for non-users and multi-category users with a BMI of 23 (A) and an age of 31 years (B), respectively. The predicted relationships apply to women with a main residence in Austria and for ≤ 2 previous childbirths (reference condition). The data of 142 women is shown (Concin et al. 2008)

Full size image
Table 1 Result of the multiple linear regression analysis quantifying the influence of predictor variables on MOSH concentration (log10-transformed scale) in abdominal subcutaneous fat of 142 pregnant women at the time of caesarean delivery. The estimate, standard deviation (SE) and associated p-value of the model coefficients are given. The intercept is the average MOSH concentration (log10-transformed scale) under the reference condition: women at the age of 31 years, with a BMI of 23 kg/m2, not resident in Austria, with ≤ 2 previous childbirths, and not using any cosmetics of the product groups lipstick (LS), hand cream (HC), and sun cream (SC)
Full size table
Table 2 Effect size for categorical predictor variables regarding the MOSH concentration in abdominal subcutaneous fat of 142 pregnant women at the time of caesarean delivery. Given are the estimate and 95% confidence interval (CI) for the fold-increase in MOSH concentration (original scale) under the alternative condition with respect to the reference condition (see Table 1 legend)
Full size table

4 Discussion

Statistical re-analysis of the data from 2 studies in humans provided evidence of a very long-term accumulation of MOSH in adipose tissue. This finding is consistent with results from animal studies (Barp et al. 2017a). From a physiological perspective, this suggests that adipocytes lack both the ability to metabolise alkanes to the corresponding fatty alcohols and subsequently to fatty acids as well as the ability to distribute incorporated MOSH back to the systemic circulation. In quantitative terms, the biopsy study suggests a 1.5-fold increase of MOSH per decade, and the autopsy study a 1.2-fold increase per decade. A similar increase (1.2-fold per decade) can be calculated from the mean age and median MOSH level in adipose tissue of pregnant women (31 years old, 44 mg/kg) and autopsy patients (67 years old, 87 mg/kg).

For complete retention, a diminishing fold-increase with age, e.g., by a factor of 1.3 at age 30 to 40, factor 1.2 at age 50 to 70, and factor 1.14 at age 70 to 80 years would be expected under constant exposure. A non-diminishing fold-increase could imply that older subjects experience a higher absolute retention compared to younger subjects. As a more likely explanation, the stronger increase in absolute terms reflects the decrease of exposure over time, i.e. older individuals still retain MOSH from a higher exposure during an earlier period of their life. Note that data refers to the sum of a broad range of MOSH with different elimination rates, resulting in a re-concentration of the hydrocarbons of lowest elimination rate (if any).

Like the adipose tissue, the MLNs showed a 1.4-fold increase in MOSH concentration per decade. MLNs are the first-pass organ for highly lipophilic substances, such as dietary lipids that are absorbed into intestinal lymphatics (Trevaskis et al. 2015). Animal studies with oral administration of linear, branched, and cyclic alkanes, which were used as model compounds for MOSH, showed these to follow the intestinal absorption pathway of dietary lipids (Albro and Fishbein 1970; Albro and Thomas 1974; Savary and Constantin 1967; Tulliez and Bories 1975; Vost and Maclean 1984). Therefore, the presence of MOSH in human MLNs is physiologically plausible and implies an exposure via the oral route. The long-term accumulation of MOSH in MLNs can be explained by their function as a “garbage collector”, which involves the filtering and sequestration of waste products and foreign material. The absence of a sex difference in MOSH concentrations suggests a similar oral exposure between men and women.

The autopsy study provided no evidence of a long-term MOSH accumulation in liver and spleen. This suggests a steady-state between uptake and elimination processes. It also implies that former exposures to MOH (i.e., earlier in lifetime of the subjects) and the decreasing exposure during the last decades is not reflected, i.e. that the retained MOSH are likely from a more recent uptake. How “recent” depends on the half-life of the elimination process, for which human data are not available. There are, however, estimates from animal studies. Apparent half-lives in the range of 23 to 122 days (Pirow et al. 2019) have been estimated from the post-exposure recovery phase of studies in female F344 rats with dietary exposure to a broad MOSH mixture (Barp et al. 2017a) and 2 white mineral oil products (Trimmer et al. 2004).

Metabolic elimination is a relevant process in liver, and studies with different model compounds for MOSH have demonstrated that different oxidative pathways are involved in biotransformation, depending on alkane type (EFSA 2012). In addition, it is possible that MOSH are also be eliminated together with triglycerides and cholesterol from liver via loading and secretion of very low density lipoproteins (VLDLs).

The autopsy data revealed that, on average, women had a 2.2–2.5-fold higher MOSH concentration in the liver, spleen and adipose tissue compared to men. A slower MOSH metabolism and/or a higher MOSH exposure of women relative to men could account for these differences. The higher MOSH concentration in liver of females compared to males agrees with findings from animal studies with F344 rats (Baldwin et al. 1992; Smith et al. 1996), indicating that sex-specific differences in metabolism exist. The animal studies also showed a higher MOSH concentration in the MLNs of females compared to males, which suggests a sex difference in intestinal absorption. However, animal studies usually involve the administration of high doses and that the relative absorption of MOSH (and model substances for MOSH) decreases with increasing dose (Pirow et al. 2019). Therefore, it is possible that the sex difference in intestinal absorption is manifested only at high doses, and not for low background levels at which humans are exposed. The absence of a sex difference in the MOSH concentration of human MLNs would be in line with the conclusion of a similar oral exposure between men and women.

The data of the six autopsy patients with the highest MOSH concentrations in liver can be used to discuss potential, additional correlations with physiological and pathological characteristics (Fig. 1; see symbols with associated numbers 1–6). All 6 subjects had MOSH concentrations in the MLNs above the regression line (Fig. 1A), indicating a lifetime oral exposure to MOSH above the average. Subjects 1–3 had a high-grade steatosis (fatty liver disease), which could have additionally contributed to the high MOSH concentrations in liver (Fig. 1C), given the propensity of MOSH to distribute into tissue lipids. Subjects 1 and 6 had a low BMI of 20 and 22, respectively, which likely contributed to the high concentrations in adipose tissue (Fig. 1B), since the BMI is positively correlated with body fat mass (the amount of MOSH is distributed in a smaller amount of tissue fat). Finally, the high MOSH concentrations in the liver of all 6 subjects went along with elevated MOSH concentrations in spleen (Fig. 1D), possibly reflecting exchange processes between both organs.

The percentage of body fat in women is, on average, approximately 10% higher than in men. It seems possible that the sex difference in the absolute amount of MOSH in adipose tissue was even more than 2.2-fold. We estimated the amount of body fat in the autopsy patients from body weight and percentage body fat. The latter was predicted from BMI, age, and sex, using the equation of Jackson et al. (2002). Since the data on body fat amount were approximately log-normally distributed, we calculated the geometric means for men and women. By taking the ratio of both values, the autopsied females had on average a 1.2-fold higher amount of body fat compared to males. Therefore, the total amount of MOSH in adipose tissue was 2.6-fold higher in women compared to men.

The use of certain cosmetic products was a relevant predictor for MOSH concentration in subcutaneous fat of women. White mineral oil products are used as ingredients in various cosmetic product groups and serve, e.g., as vehicles, viscosity regulators and moisturisers. Women using products from the categories lipstick, hand cream and sun cream had on average a 2.1-fold higher MOSH concentration in their subcutaneous fat than non-users. This finding might suggest a dermal MOSH uptake. However, given the poor dermal absorption of saturated hydrocarbons (Petry et al. 2017) as well as the high intestinal MOSH absorption at low doses via the pathway for dietary lipids and the high MOSH levels in MLNs, a relevant dermal uptake is not plausible. Instead, an oral uptake seems to be more likely, mainly owing to direct ingestion from MOSH-containing lipsticks and other lip-care products.

It has been known since the last century that high MOSH levels in human tissue can be associated with histopathological effects (Boitnott and Margolis 1966, 1970), visible as oil droplets termed “lipogranuloma” (Bellamy and Burt 2018; Cruickshank 1984; Cruickshank and Thomas 1984; Dincsoy et al. 1982; Fleming and Carrillo 2018; Fleming et al. 1998; Klatskin 1977; Wanless and Geddie 1985). Main target tissues were the abdominal lymph nodes, spleen and liver. The described liver effects in humans have regained attention after studies in female F-344 rats with oral administration of certain mineral oil products and waxes have revealed inflammatory granulomatous changes in liver. However, the MOSH-related formation of lipogranulomas in human liver is morphologically distinct from the lesions seen in F-344 rats (Carlton et al. 2001; Fleming and Carrillo 2018; Fleming et al. 1998; Miller et al. 1996; Smith et al. 1996). In fact, the granuloma observed in F344 rats could be associated with the poor elimination of wax-type hydrocarbons, possibly their crystallisation, which seems to be specific to F344 rats (Barp et al. 2017b). The EFSA CONTAM Panel in its 2012 opinion noted that “the clinical significance of the lipogranulomas [in humans] is not known, but their presence had not been associated with inflammatory responses, and not reported to be associated with clinical abnormalities” (EFSA 2012).

5 Conclusion

Since the 1990s, MOSH exposure and levels in tissue have decreased due to the reduced use of mineral oil products in food and food processing (Boitnott and Margolis 1966; EFSA 2012; EFSA 2023; Heimbach et al. 2002). Despite such good news, it is still mandatory that efforts continue to minimise dietary MOSH exposure. Some MOSH have a strong tendency to bioaccumulate with no obvious adverse health effects. However, as we are just beginning to gain a better understanding of the fate of absorbed MOSH and their potential toxicity, further studies would be beneficial in support of this current working hypothesis.

Among the cosmetics, lipsticks and lip care products are of particular concern, as they are ultimately ingested (Niederer et al. 2016). To ensure a high level of consumer safety, quality specifications of the respective oils and waxes as cosmetic ingredients have been issued (Cosmetics Europe 2004; Cosmetics Europe 2014); manufactures should comply with these recommendations and ensure that cosmetics are not a relevant MOSH exposure source.