Critical Reviews in Toxicology

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Mineral oil in food, cosmetic products, and in

products regulated by other legislations

Ralph Pirow, Annegret Blume, Nicole Hellwig, Matthias Herzler, Bettina

Huhse, Christoph Hutzler, Karla Pfaff, Hermann-Josef Thierse, Tewes Tralau,
Bärbel Vieth & Andreas Luch

To cite this article: Ralph Pirow, Annegret Blume, Nicole Hellwig, Matthias Herzler, Bettina Huhse,
Christoph Hutzler, Karla Pfaff, Hermann-Josef Thierse, Tewes Tralau, Bärbel Vieth & Andreas
Luch (2019) Mineral oil in food, cosmetic products, and in products regulated by other legislations,
Critical Reviews in Toxicology, 49:9, 742-789, DOI: 10.1080/10408444.2019.1694862

To link to this article: https://doi.org/10.1080/10408444.2019.1694862

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CRITICAL REVIEWS IN TOXICOLOGY
2019, VOL. 49, NO. 9, 742–789
https://doi.org/10.1080/10408444.2019.1694862

REVIEW ARTICLE

Mineral oil in food, cosmetic products, and in products regulated by other

legislations
Ralph Pirow, Annegret Blume, Nicole Hellwig, Matthias Herzler, Bettina Huhse, Christoph Hutzler, Karla Pfaff,
Hermann-Josef Thierse, Tewes Tralau, B€arbel Vieth and Andreas Luch
Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany

ABSTRACT ARTICLE HISTORY

For a few years, mineral oils and their potential adverse health effects have been a constant issue of Received 14 July 2019
concern in many regulatory areas such as food, cosmetics, other consumer products, and industrial Revised 13 November 2019
chemicals. Analytically, two fractions can be distinguished: mineral oil saturated hydrocarbons (MOSH) Accepted 15 November 2019
and mineral oil aromatic hydrocarbons (MOAH). This paper aims at assessing the bioaccumulative
KEYWORDS
potential and associated histopathological effects of MOSH as well as the carcinogenic potential of Mineral oil; saturated
MOAH for consumer-relevant mineral oils. It also covers the absorption, distribution, metabolism, and hydrocarbons; aromatic
excretion of MOSH and MOAH upon oral and dermal exposures. The use and occurrence of consumer- hydrocarbons; carcinogen-
relevant, highly refined mineral oils in food, cosmetics and medicinal products are summarized, and icity; accumulation;
estimates for the exposure of consumers are provided. Also addressed are the challenges in character- granuloma; liver; spleen;
izing the substance identity of mineral oil products under REACH. Evidence from more recent autopsy lymph nodes; food;
and biopsy studies, along with information on decreasing food contamination levels, indicates a low cosmetics; pharmaceuticals;
risk for adverse hepatic lesions that may arise from the retention of MOSH in the liver. With respect to REACH; CLP;
MOAH, at present there is no indication of any carcinogenic effects in animals dermally or orally consumer safety
exposed to highly refined mineral oils and waxes. Such products are used not only in cosmetics but
also in medicinal products and as additives in food contact materials. The safety of these mineral oil-
containing products is thus indirectly documented by their prevalent and long-term use, with a simul-
taneous lack of clinical and epidemiological evidence for adverse health effects.

Abbreviations: AD: Atopic dermatitis; ADI: Acceptable daily intake; ADME: Absorption, Distribution,
Metabolism, and Excretion; AfA: Application for Authorization; AhR: Arylhydrocarbon Receptor; ALT:
Alanine aminotransferase; AMP: Antimicrobial peptide; ANS: EFSA Panel on Food Additives and Nutrient
Sources added to Food; AST: Aspartate aminotransferase; B[a]P: Benzo[a]pyrene; BfR: German Federal
Institute for Risk Assessment; CL: Candidate List (under REACH); CLH: harmonized classification and
labeling; CLP: European regulation on Classification, Labeling and Packaging of substances and mix-
tures; CMR: Carcinogenic, Mutagenic, toxic for Reproduction; Concawe: A division of the European
Petroleum Refiners Association; CONTAM: EFSA Panel on Contaminants in the Food Chain; Cosmetics
Europe: European trade association for the cosmetics and personal care industry; DMSO: Dimethyl sulf-
oxide; ECHA: European CHemicals Agency; EFSA: European Food Safety Authority; EPA: U.S.
Environmental Protection Agency; FAO: Food and Agriculture Organization of the United Nations; FCM:
food contact material; FID: Flame ionization detection; GC: Gas chromatography; GC–FID: Gas chroma-
tography coupled to flame ionization detection; GC
GC: Comprehensive (two-dimensional) gas chro-
matography; GC
GCMS: Comprehensive gas chromatography coupled to mass spectrometry;
GC
GC–ToF–MS: Comprehensive gas chromatography coupled to time-of-flight mass spectrometry;
GC–MS: Gas chromatography coupled to mass spectrometry; GHS: Globally harmonized system of clas-
sification and labeling of chemicals; GGT: Gamma glutamyl transferase; HCBM: hydrocarbon block
method; HDL: High density lipoprotein; HMPW: High melting point wax; NMR: Nuclear magnetic reson-
ance (spectroscopy); HRBO: Highly refined base oils; HRF: Human relevance framework; HTWO: Hydro-
treated white oil; HVMO: High viscosity (white) mineral oils; IARC: International Agency for Research on
Cancer; IL: Interleukin; IMPW: Intermediate melting point wax; INCI: International Nomenclature of
Cosmetic Ingredients; I/P: Initiation/Promotion; JECFA: Joint FAO/WHO Expert Committee on Food
Additives; L-E: Long-Evans (rat strain); LC: Liquid chromatography; LC–GC–FID: Liquid chromatography
coupled to gas chromatography and flame ionization detection; LDL: Low density lipoprotein; LOAEC:
Lowest observed adverse effect concentration; LMPW: Low melting point wax; MAK: German Senate
Commission for the investigation of health hazards of chemical compounds in the work area; MLN:
Mesenteric lymph node; MMAD: Mass median aerodynamic diameter; MoA: Mode of action; MOAH:
Mineral oil aromatic hydrocarbons; MOE: Margin of exposure; MOH: Mineral oil hydrocarbons; MOSH:
Mineral oil saturated hydrocarbons; MS: Mass spectrometry; MSCA: Member States Competent
Authority; MVMO: Medium viscosity (white) mineral oils; NPV: Negative predictive value; NF: National

CONTACT Ralph Pirow [email protected] Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Max-
Dohrn-Straße 8-10, Berlin 10589, Germany
ß 2020 German Federal Institute for Risk Assessment (BfR). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/),
which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.
 CRITICAL REVIEWS IN TOXICOLOGY 743

formulary; NMSC: Non-melanoma skin cancer; NOAEC: No observed adverse effect concentration;
NQO1: NAD(P)H quinone oxidoreductase; Nrf2: Nuclear factor erythroid 2-related factor 2; OECD:
Organization for Economic Co-operation and Development; OLBO: Other lubricant base oils; OTWO:
oleum-treated white oil; PAC: Polycyclic aromatic compounds; o/w: oil-in-water (emulsion); PAH:
Polycyclic aromatic hydrocarbon; PAO: Poly-a-olefin; PET: polyethylene terephthalate; PetCo: Attribute
related to petroleum or coal streams; Ph. Eur.: European Pharmacopeia; POSH: Polyolefin oligomeric
saturated hydrocarbons; RAC: Risk Assessment Committee (at ECHA); REACH: Registration, Evaluation,
Authorization, and restriction of CHemicals; SAICM: Strategic approach to international chemicals man-
agement; SD: Sprague Dawley (rat strain); SCOEL: Scientific Committee on Occupational Exposure
Limits; SEAC: Committee for Socio-Economic Analysis (at ECHA); SML: Specific migration limit; SVHC:
Substance of very high concern; Th: T-helper (cells); USP: United States Pharmacopeia; UVCB: Attribute
related to substances of unknown or variable composition, complex reaction products or biological
materials; WHO: World Health Organization; w/o: water-in-oil (emulsion)

Table of contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743 Appendix 2: Literature search for ADME, tissue levels,
Mineral oil analytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 745 and histopathological effects of MOSH upon oral
Toxicology of mineral oils . . . . . . . . . . . . . . . . . . . . . . . . . . . 747 exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786
Literature search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747 Appendix 3: Literature search for gastrointestinal absorp-
MOSH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747 tion of MOAH and PAHs . . . . . . . . . . . . . . . . . . . . . . . . . 787
Dermal absorption of MOSH . . . . . . . . . . . . . . . . . . . 747 Appendix 4: Literature search for dermal absorption of
Intestinal absorption of MOSH. . . . . . . . . . . . . . . . . . 749 MOAH and PAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
Distribution, metabolism, and excretion of MOSH 751 Appendix 5: Literature search for (dermal) carcinogenicity
MOSH levels in animal tissues . . . . . . . . . . . . . . . . . . 752 of PAHs and MOAH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 788
MOSH levels in human tissues . . . . . . . . . . . . . . . . . . 756
Effects associated with the accumulation of MOSH
in animal tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . 759 Introduction
Histopathological lesions associated with the depos-
ition of MOSH in human tissues . . . . . . . . . . . . . 761 Mineral oils are derived from crude petroleum oil by a number
MOAH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764 of processing steps that start with atmospheric distillation fol-
Gastrointestinal and dermal absorption of MOAH lowed by vacuum distillation of the atmospheric residue
(Figure 1) (IARC 1984; Mackerer et al. 2003; EFSA 2012; IARC
and PAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
2012). The vacuum-distillate fractions, which contain hydrocar-
Carcinogenicity of PAHs and MOAH . . . . . . . . . . . . . 764
bons with boiling points (at atmospheric pressure) in the
Evaluation of the inhalation toxicity of pharmaceutical
range of about 300–600  C, and the deasphalted vacuum resi-
white oil by the german MAK commission . . . . . . . . . 767 due are fed into refinery processes such as solvent extraction,
Evaluation of mineral oil hydrocarbons in food by the hydrotreatment, and dewaxing (i.e. removal of n-alkanes, also
EFSA CONTAM panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 768 called deparaffinization) (IARC 1984). These refinery streams
Activities of surveillance and regulatory authorities on (also known as base oils or lubricant base oils) can be further
MOH in food and food contact materials . . . . . . . . . . 770 purified, blended, and formulated to yield the finished mineral
Mineral oil hydrocarbons in cosmetic products . . . . . . . 770 oil products. The by-product of the dewaxing step (called slack
Mineral oils in dermatology . . . . . . . . . . . . . . . . . . . . . . . . . 771 wax) is deoiled to yield microcrystalline waxes and (hard) par-
Mineral oils in medicinal products . . . . . . . . . . . . . . . . . . . 773 affin waxes which can be further refined by, e.g. hydrotreat-
Mineral-oil based substances under REACH and CLP . . 774 ment and blending. A well-known blend of mineral oils and
Identification of mineral oil-based substances . . . . . . . . 774 waxes is petrolatum, which is also called petroleum jelly or
Classification for hazardous properties . . . . . . . . . . . . . . 775 Vaseline. Table 1 shows the physical properties of highly
Registration and evaluation under REACH . . . . . . . . . . . 775 refined mineral oils, paraffin waxes, and microcrystalline waxes.
Mineral oil products can be classified into lubricants and
Restriction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775
products intended for non-lubricant purposes (IARC 1984,
SVHC identification and authorization . . . . . . . . . . . . . . . 776
2012). Lubricant products include, for example, automotive
SVHC roadmap 2020 and PetCo group . . . . . . . . . . . . . . 776
and industrial engine oils and greases, hydraulic oils, metal-
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 777 working fluids, but also food machinery lubricants and textile
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778 oils. On the other hand, “non-lubricant” products are used,
Declaration of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778 for example, as extender oils for rubber, carriers for printing
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 778 inks, processing aids in the manufacturing of rubber, plastics,
Appendix 1: Literature search for dermal absorption and textiles, and as batching oils for the softening of natural
of MOSH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 785 fibers such as jute. Highly refined mineral oils and waxes,
744 R. PIROW ET AL.

Figure 1. Simplified refinery diagram showing the principal processing steps leading to mineral oils and waxes. Foodgrade and pharmaceutical grade qualities are
shown bold. The IP 346 method as a gatekeeper and qualifier for refining efficiency is indicated. Dewaxing not necessary for naphthenic base oils and white oils
manufacture. Adapted from Concawe (2017).

Table 1. Physical properties of highly refined mineral hydrocarbons comprising Mineral oils are complex substances of saturated and aromatic
white mineral oils, paraffin waxes and microcrystalline waxes.
hydrocarbons predominantly with carbon numbers ranging from
Average
molecular C15 to C50 (Table 1) (IARC 1984). By using certain analytical tech-
Viscosity (mm2/s)
weight Average carbon niques, two fractions can be distinguished: mineral oil saturated
Name at 40  C at 100  C (g/mol) number range hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons
White mineral oils (MOAH). MOSH are composed of straight and branched open-
N7H 7 2.2 265 14–32
N10A 13.3 3.1 320 15–30 chain alkanes (paraffins) and largely alkylated cycloalkanes (naph-
N15H 16.6 3.4 330 17–30 thenes) (Biedermann et al. 2009; EFSA 2012). The proportion of
P15H 15.0 3.5 350 18–30
N70A 76.4 7.9 410 21–35
straight-chain alkanes (n-alkanes, also called paraffin waxes)
N70H 68.0 7.6 420 22–37 among the MOSH is low because a dewaxing step is included in
P70H 69.5 8.6 485 27–43 the manufacturing of mineral oils. MOAH include mono- and/or
P100H 99.8 11.0 510 28–45
S-C25 18 14–34 polyaromatic hydrocarbons that are highly alkylated and also
L-C25 80 25–45 often partially hydrogenated (EFSA 2012).
Paraffin waxes The term “MOSH” was introduced by Biedermann et al.
LMPW solid 3.3 375 19–42
IMPW solid 6.3 450 21–49 (2009) for petroleum-derived (i.e. petrogenic) saturated hydrocar-
W solid 23–45 bons to make a distinction from those of plant origin. The term
Microcrystalline waxes “MOAH” was originally coined by the same authors (Biedermann
HMPW solid 15.4 630 22–80
et al. 2009) referring to contaminated food in order to distin-
Adapted from Miller et al. (1996), EFSA (2012), McKee et al. (2012), Barp et al.
(2017b) and Cravedi et al. (2017). guish the aromatic hydrocarbons of petrogenic origin from the
Mineral-oil type abbreviation: N: naphthenic (crude oil source); P: paraffinic; H: polycyclic aromatic hydrocarbons (PAHs) that are typically
hydro-treated (catalytic hydrogenation); A: acid-treated; number (15, 70,
100): approximate viscosity (mm2/s) at 40  C.
formed by pyrogenic processes (i.e. incomplete combustion of
organic compounds at high temperatures) (Moret and Conte
2000). PAHs in the narrower sense of the chemical nomenclature
and blends thereof, are used as food additives, additives for are unsubstituted fused ring systems containing at least two
food contact materials, cosmetic ingredients, and excipients benzene rings. These are often called parent PAHs to distinguish
for pharmaceutical formulations (Table 2). them from alkylated (e.g. methylated) derivatives.
 CRITICAL REVIEWS IN TOXICOLOGY 745

Table 2. Consumer-relevant product groups of mineral oils and waxes and their use in the EU as food additives (Annex II of Regulation (EC) No 1333/2008),
additives for plastics in food contact (Annex I of Regulation (EU) No 10/2011), cosmetic ingredients, and excipients in medicinal products (Monographs of the
European Pharmacopeia). Cosmetic ingredient names follow the international nomenclature of cosmetic ingredients (INCI).
Additive for plastics in food Excipient in medicinal
contact (FCM Cosmetic Ingredient products
Substance Food Additive (E number) substance number) (CAS number) (monograph number)
Medicinal white oil / White High viscosity mineral oil (95) Paraffinum liquidum Paraffin, liquid (0239)
mineral oil (E 905a) Medium viscosity (8012-95-1, 8042-47-5) Paraffin, light liquid (0240)
mineral oil ()
Microcrystalline wax Microcrystalline wax (94) Microcrystalline wax Microcrystalline wax
(E 905) (63231-60-7) (2399 Draft)
Cera microcristallina
(63231-60-7, 64742-42-3)
Hydrogenated microcrystalline
wax (64742-60-5, 92045-76-6)
Hydrogenated microcrystalline
cera (92045-76-6)
Fully refined paraffin / (93) Paraffin (8002-74-2) Paraffin, hard (1034)
hard paraffin
White petrolatum Petrolatum (8009-03-8) Paraffin, white soft (1799)
Yellow petrolatum Petrolatum (8009-03-8) Paraffin, yellow soft (1554)
FCM: food contact material.

Mineral oils can occur in consumer products and foodstuff hydrocarbons by use of technical lubricants. This topic, how-
for different reasons. They are intentionally used as ingredients ever, is outside the scope of this review.
in cosmetic products (Petry et al. 2017), as extender oils in
elastomeric and polymeric materials (e.g. tool handles) (Bartsch
Mineral oil analytics
et al. 2016), and as food additives (e.g. glazing agents, preser-
vatives, anti-foaming agents, release agents, anti-dusting The structural diversity of hydrocarbons in mineral oil
agents) (EFSA 2009, 2013a, 2013b). Contamination can result is immense (EFSA 2012). The composition of mineral oils is
from mineral oils used in printing inks for printing of food therefore extremely complex. A widely used method for quan-
packaging which can evaporate and migrate via the gas phase titative mineral oil analysis is on-line coupled liquid chroma-
into the packaged food (EFSA 2012). Food contamination may tography coupled to gas chromatography and flame ionization
also result from the migration of mineral oil from paper and detection (LC–GC–FID) (Biedermann and Grob 2012a). This
cardboard made of recycled fibers (Biedermann and Grob method has been successfully applied to quantify MOSH and
2010; EFSA 2012). In addition, food and consumer products MOAH fractions as a sum parameter in food and cardboard
can become contaminated by mineral oils and other petrol- packaging (Biedermann and Grob 2010, 2012b; EFSA 2012),
eum-derived substances (e.g. diesel fuel) via the environment and in cosmetic products (Niederer et al. 2015). A structural
and by technological processes (e.g. by contact with food characterization of selected mineral oil constituents is, how-
machinery lubricants) (EFSA 2012). ever, not possible with this technique. Instead, comprehensive
Mineral oil hydrocarbons (MOH) and their potential adverse two-dimensional gas chromatography (GC
GC) (Biedermann
health effects are a recurring issue of concern in many regula- and Grob 2010, 2012a) in combination with mass spectrometry
tory areas such as food, food contact materials, cosmetics, and (GC
GC–MS) (Biedermann and Grob 2015; Bartsch et al.
2016) enables the characterization of structural elements and
industrial chemicals. The aim of this paper is to review the toxi-
to determine the degree of substitution of aromatic hydrocar-
cology of consumer-relevant mineral oils, specifically the bioac-
bons in mineral oil. Analysis at the individual substance level,
cumulative potential and associated histopathological effects of
however, cannot be achieved with the currently existing ana-
MOSH as well as the carcinogenic potential of MOAH. The
lytical techniques.
absorption, distribution, metabolism, and excretion (ADME) of
To quantify MOSH and MOAH in cosmetic products and
MOSH and MOAH are briefly summarized. The paper describes
other matrices, a chromatographic pre-separation of both frac-
the use and occurrence of mineral oils in food, cosmetics and tions by LC is required (Biedermann and Grob 2012a). The differ-
pharmaceuticals. A chapter on analytics is included to describe ent mineral oil components elute from a normal phase column
the state-of-the-art technology for the monitoring of mineral in the following order: high and low molecular weight paraffins,
oils in food and consumer products. This review also covers naphthenes as last eluting MOSH, highly alkylated benzenes as
the exposure of consumers to mineral oils, the potential health first eluting MOAH, and alkylated polyaromatics as finally eluting
risks associated with this exposure, and the regulatory meas- MOAH. Quantitative analysis is then carried out by GC–FID. On-
ures taken to limit and to reduce these risks. Also addressed line coupled LC–GC–FID combines the pre-separation of MOSH
are the challenges in characterizing the substance identity of and MOAH by high performance LC, involving simultaneous up-
mineral oil products under the European chemicals legislation. concentration by solvent trapping and re-condensation, with GC
Finally, the paper briefly summarizes the activities at the analysis and quantification via the universal FID detector.
European Chemicals Agency (ECHA) with respect to the identifi- On-line coupled LC–GC–FID provides separate chromato-
cation and regulation of hazardous mineral oil substances. grams for MOSH and MOAH which show unresolved humps,
Consumers may also be exposed directly to mineral oil sometimes with discrete peaks on top representing individual
746 R. PIROW ET AL.

compounds such as n-alkanes for the MOSH fraction and This removal procedure eliminates n-alkanes of biogenic
alkylated benzenes for the MOAH fraction. The volatility origin, but also n-alkanes of mineral origin. It is applied when
range and shape of the hump reveals information on the n-alkanes in plant samples or oil samples are present in very
source of the mineral oil. An identification of individual peaks high concentration so that their peaks would overlay and
is possible by analyzing the samples further, for example, via mask a MOSH hump of minor concentration (Fiselier et al.
GC–MS or GC
GC–MS. For the correct evaluation and inter- 2009; Fiselier and Grob 2009). When natural n-alkanes (in e.g.
pretation of MOSH and MOAH chromatograms, well-trained food samples) are present in lower concentrations, it is not
and experienced analytical staff and knowledge about the necessary to remove them from the sample in order to be
sample and its history are mandatory. able to achieve a proper quantitation of MOSH. The removal
Individual constituents of the cosmetic or food matrix may step was not applied for the analysis of MOSH in biological tis-
interfere with the on-line coupled LC–GC–FID analysis if they sues and breast milk (Noti et al. 2003; Concin et al. 2008; Barp
co-elute with MOSH or MOAH fractions. Since MOSH and et al. 2014; Biedermann et al. 2015; Barp et al. 2017a, 2017b).
MOAH are relatively non-polar, it is unlikely that more polar The coupling of two orthogonal GC columns (GC
GC)
allows for the characterization of ring systems and their degree
substances interfere with their analysis. Individual substances
of alkylation in the mineral oil mixture (Figure 2). This informa-
such as certain fragrances with terpene-like structures may
tion can be estimated from the position in the two-dimensional
appear as single peaks on top of the MOAH hump. In addition,
chromatogram. Combination of GC
GC with time-of-flight
natural polyolefins and unsaturated poly-a-olefins (PAOs) have
mass spectrometry (GC
GC–ToF–MS) allows for the identifica-
to be considered as possible interferences for MOAH humps.
tion of partially hydrogenated ring systems. However, an exact
For the quantitative analysis of MOAH chromatograms, single
quantification of the total MOAH fractions or individual ring sys-
peaks sitting on the top of the hump are subtracted from the tems by GC
GC–ToF–MS is not possible. For substances with a
total area of the MOAH hump. If necessary for the correct inte- medium or high degree of alkylation, a resolution down to the
gration of the MOAH hump, olefinic and polyolefinic interfer- level of single constituents cannot be achieved technically. This
ences can be removed by chemical modification such as is because of the immense number of potential isomers.
epoxidation. Interferences with MOSH humps may result from Several classical methods exist for the analysis of PAHs in
n-alkanes of plant origin and saturated synthetic hydrocarbons mineral oil products such as proton nuclear magnetic reson-
such as polyolefin oligomeric saturated hydrocarbons (POSH). ance (1H-NMR) spectroscopy or the measurement of the UV
A separate quantification of MOSH and synthetic hydrocarbons absorbance of a dimethyl sulfoxide (DMSO) extract. 1H-NMR
is not always possible, so that only the total amounts of both is correlated to the content of aromatic protons, whereas all
can be quantified. n-Alkanes can be removed from the MOSH methods that use DMSO extracts focus on the most polar
fraction by chromatography on activated aluminum oxide prior and therefore DMSO-extractable compounds in the MOAH
to on-line LC–GC–FID analysis. fraction: aromatic ring systems with low alkylation degree.

Figure 2. GC
GC–FID chromatogram of a complex mixture of mineral oils to which internal standards and the 16 polycyclic aromatic hydrocarbons selected by
the U.S. Environmental Protection Agency (EPA-PAHs) were added. In the first dimension of the GC
GC, a moderately polar stationary phase was used, and a non-
polar phase in the second dimension. Both MOSH and MOAH are visible. The solid line interconnects the centers of the signals representing the n-alkanes (for
some, the carbon numbers are indicated); between them are little branched iso-alkanes. Numerous multibranched hydrocarbons (incl. pristane and phytane) and
cycloalkanes (incl. n-alkyl cyclopentanes and -hexanes, four-ring stearanes and five-ring hopanes) are prominent. The grayish background is formed by a cloud of
unresolved, highly isomerized components, most of which being naphthenes. MOAH appear as parallel bands of given number of aromatic rings. The (non-alky-
lated) PAH (mainly EPA) are located at the bottom of the chromatogram (dotted line). Internal standards in italics: Cycy: cyclohexyl cyclohexane; Cho: cholestane;
MNs: 1- and 2-methylnaphthalene; Tbb: tri-tertbutylbenzene; Per: perylene; DEHB: di(2-ethylhexyl)benzene; 18B: n-octadecylbenzene. (Figure taken from
Biedermann et al. (2017), # 4694221061510).
 CRITICAL REVIEWS IN TOXICOLOGY 747

These kind of assays are used, inter alia, for impurity testing Excised porcine back skin (3–4 mm thick) was used to
according to the European Pharmacopeia (Ph. Eur.). study the dermal absorption of 14C-radiolabelled n-hexade-
Consistency of the results of these classical methods with the cane (n-C16) and 3H-labeled n-docosane (n-C22) in different
state-of-the-art analytical methods remains to be demon- cosmetic vehicles in Franz-type diffusion cells (Brown et al.
strated, as they provide a different readout compared to the 1995). The vehicles comprised petrolatum, polydecene, white
MOAH results obtained with online LC–GC–FID, which refer mineral oil, soya oil, and a water-in-oil (w/o) cream. Test for-
to the total content of aromatic compounds. mulations were applied at a dermal load of 3–5 mg/cm2.
Analysis of the radioisotope distribution some 24 h later indi-
cated absorption of 4–42%, 0.4–1.2% and 0.3–1.0% for stra-
Toxicology of mineral oils tum corneum, the viable epidermis and the dermis,
The bioaccumulative potential of MOSH and the carcinogenic respectively. No radioactivity was detected in the recep-
potential of MOAH represent two of the main toxicological tor fluid.
concerns associated with mineral oils. This chapter focuses Split-thickness cadaver skin was used to study the dermal
on these two aspects but additionally addresses the ADME absorption of 14C-radiolabelled dotriacontane (C32) in petrol-
(absorption, distribution, metabolism, excretion) properties of atum in Franz-type diffusion cells (Intarakumhaeng et al.
these substances and the histopathological changes associ- 2018). The petrolatum was applied to the skin with solvent
ated with the accumulation of MOSH in tissues. The rele- deposition using hexane or hexane/pentane mixtures under
vance of effects observed in animal experiments for human finite-dose conditions. A dose range of 0.05–0.2 mg/cm2 was
health is also being discussed. chosen to mimic the amounts of cosmetic ingredients depos-
ited on the skin surface after personal care products rinsing.
The amounts of the model permeant dotriacontane meas-
Literature search ured in the receptor chamber 1 h, 24 h, and 72 h were below
the detection limit.
The starting point for the literature search was in most cases
Three microscopy studies (Ghadially et al. 1992; Brown
the comprehensive scientific opinion of the European Food
et al. 1995; Mao-Qiang et al. 1995) determined the penetra-
Safety Authority (EFSA) on mineral oil hydrocarbons in food
tion depth of petrolatum into intact and acetone-treated skin
(EFSA 2012). Relevant references cited by EFSA on the bioaccu-
of hairless mice. Skin wiping with acetone was used to dis-
mulative potential and associated histopathological effects of
rupt the skin barrier by selectively removing the stratum cor-
MOSH (in animals and humans) as well as on carcinogenic
neum lipids. Petrolatum was applied once for 2–6 h, or
potential of MOAH and PAHs were included in the initial list of
repeatedly twice daily for three days. Skin biopsies were
references. Additionally included from the EFSA opinion were
taken and processed for electron microscopy and fluores-
references on the ADME of MOSH and MOAH upon dermal
cence microscopy. The latter required lipid-soluble dyes,
and oral exposure. Systematic literature searches were subse-
either as a tracer or as histochemical stain. In both acetone-
quently conducted in the Science Citation Index Expanded
compromised and intact skin, under conditions of single and
database of Web of Science to find additional, relevant refer-
repeated exposure, petrolatum remained restricted to the
ences. The details of the literature search in Web of Science
stratum corneum, inducing lacunae formation in the lamellar
(e.g. search options, set of queries, number of returned
lipid layers.
records, number of included and excluded references) as well Radioisotope studies complemented the microscopy stud-
as the lists of included/excluded studies and of additionally ies in hairless mice. One study (Brown et al. 1995) analyzed
included references are provided in the Appendices 1 to 5. the absorption of 3H-radiolabelled n-docosane (n-C22) into
acetone-treated skin. Petrolatum and 1% petrolatum in pro-
MOSH pylene glycol:ethanol (7:3 v/v) served as vehicles. Test formu-
lations were applied to the flanks at a dermal load of
Dermal absorption of MOSH 5.4–6 mg/cm2. The animals were sacrificed after 2.5 h, and
Different techniques were employed to determine the extent excess test substance was removed from the skin surface.
of skin penetration and percutaneous absorption of individual Excess surface material, dissected layers of the skin, subcuta-
model compounds and complex mixtures in in vitro and neous fat, and blood were analyzed. In the case of petrol-
in vivo studies using animal and human skin. This section con- atum, 7.8% of the applied label was found in the stratum
siders studies with dermal application of neat substances and corneum, 1.4% in the viable epidermis, 0.2% in the dermis,
of substances present in cosmetic formulations (Nash et al. and 0.1% in subcutaneous fat. For petrolatum in the propyl-
1996; Petry et al. 2017). Studies with n-alkane-containing jet ene glycol:ethanol mixture, a higher proportion (45%) of the
fuels (Singh et al. 2002; Jakasa et al. 2015) were excluded since radioactivity partitioned into the stratum corneum. The pro-
these contain n-alkanes and aromatics (carbon range: C6–C17) portion in the viable epidermis remained essentially
with skin-irritating and penetration-enhancing properties. In unchanged (1.2%), whereas that in the dermis and subcuta-
the studies reviewed below, the amount of the applied neat neous fat increased to 1.2% and 0.4%, respectively. Blood did
substance and formulation is indicated as dermal load (mg/ not contain any detectable levels of radioactivity
cm2) to enable comparison with the real-life use of cosmetic above background.
products. For a face cream, for example, the typical daily value Whole-body autoradiography was used to study the per-
is 2.72 mg/cm2 (SCCS 2016; Petry et al. 2017). cutaneous absorption of 14C-labeled n-octadecane (n-C18) in
748 R. PIROW ET AL.

hairless mice (Suzuki et al. 1978). Octadecane was applied to

the back, either neat (1.25 mg/cm2) or in a 5% hydrophilic
ointment (8 mg formulation per cm2). After 48 h incubation
under occlusive conditions, the animals were anesthesized,
shock-frozen, and sagittal sections were then prepared. The
radioactivity was localized on the applied regions, no radio-
activity was detected in the systemic compartments. The sub-
stance was also applied neat to the shaved backs of guinea
pigs (dermal load: 1.95 mg/cm2). In skin biopsies, the distribu-
tion was analyzed by microautoradiography. After 24 h of
treatment under occlusive conditions, the radioactivity con-
centrated in the sebaceous glands of the hair follicles with
some spreading into the surrounding dermis.
In another radioisotope study, neat 14C-radiolabelled n-
hexadecane (n-C16) was applied to the backs of guinea pigs
at a dermal load of 3.85 mg/cm2 (Rossmiller and Hoekstra
1966). The animals were sacrificed after 48 h, and the residual
test material was removed from the skin surface. The treated
skin was excised and the stratum corneum plus viable epider-
mis was separated from the dermis by mechanical stretching.
15.2% of the radioactivity was found in the stratum corneum
plus viable epidermis, 0.1% in the underlying dermis, and a
combined total of 0.1% in the liver and kidneys. Blood did
not contain measurable amounts of 14C. Applying hexade-
cane in a 1:1 dilution with white mineral oil markedly
reduced the dermal penetration, yielding 4.4% radioactivity
in the stratum corneum plus viable epidermis.
Human skin from surgery was used to study the penetra-
tion of 14C-labeled n-octadecane (n-C18) from various oint-
ments, including w/o and o/w creams, and petrolatum (Zesch
and Bauer 1985). Test formulations were applied at a dermal Figure 3. Penetration profiles of MOSH into the horny layer of the skin. (A)
load of 1–3 mg/cm2. The skin samples were mounted in a Distribution of radioactivity in stratum corneum, as determined using tape strips
penetration chamber. After application times of 0.5 to 16.7 h, at 30 and 100 min after dermal application of 14C-labeled n-octadecane in pet-
rolatum to human skin. (B) MOSH concentration in relation to stratum corneum
the excess substance was removed from the skin, and each depth, as measured by confocal Raman microscopy at 60 min after dermal appli-
specimen was separated into individual layers by tape strip- cation of petrolatum or paraffin oil to the skin of the inner forearm of human
ping of the stratum corneum and by horizontal slicing of the volunteers. Data were compiled from Zesch and Bauer (1985) (A) and Choe
et al. (2015) (B).
viable epidermis and dermis using a freeze-microtome. The
distribution and concentration of octadecane in the stratum
dermal loads of 2 mg/cm2. Fluorescence laser scanning
corneum were virtually identical for all vehicles. The concen-
microscopy was used to analyze the distribution of dye-
tration profiles showed a continuous decrease towards
labeled MOSH (Patzelt et al. 2012). After a short application
deeper layers of the stratum corneum (Figure 3(A)). The viable
time of 30 min, excess amounts of the test substances were
epidermis and the dermis did not contain detectable levels.
wiped off the skin surface. In case of mineral oil, only a very
The dermal penetration of petrolatum was studied in
human subjects using a lipid-soluble tracer dye (Strakosch weak fluorescence signal was obtained from the skin surface
1943). Petrolatum was applied to the anterior region of the and the lipid layers around the uppermost layers of corneo-
thigh, forearms, back or abdomen at a dermal load of 17 mg/ cytes. Petrolatum was exclusively detected on the skin sur-
cm2. Histological sections were prepared from skin biopsies face. With confocal Raman microscopy, the penetration
taken 24 h after application. The distribution of the dye- process can be monitored and the thickness of the horny
labeled petrolatum was analyzed microscopically and layer can be determined without the use of a tracer dye
checked by counter-staining. Petrolatum was localized in the (Stamatas et al. 2008; Choe et al. 2014; Choe et al. 2015;
upper horny layer, and in the upper half of the hair shafts Choe et al. 2017). The stratum corneum of untreated forearm
and sebaceous glands, which anatomically belong to the der- skin was measured to be 19.2 mm thick (Choe et al. 2014;
mis. No penetration through the stratum corneum was Choe et al. 2015). 60 min after application of the test substan-
observed in this study from 1943. ces, the concentration profiles showed an exponential
Noninvasive optical methods were employed to analyze decrease towards deeper layers (Figure 3(B)), with a penetra-
the skin penetration of petrolatum and white mineral oil in tion depth of 6–7 mm (Choe et al. 2014; Choe et al. 2015).
human subjects (Stamatas et al. 2008; Patzelt et al. 2012; Petrolatum had a pronounced swelling effect, increasing the
Choe et al. 2014; Choe et al. 2015; Choe et al. 2017). Test stratum corneum thickness by 32% due to the absorption of
substances were applied to the inner forearm, mainly at water by corneocytes.
 CRITICAL REVIEWS IN TOXICOLOGY 749

Overall, percutaneous absorption of MOSH is low, if not, 1984). This is in line with the log PO/W of these compounds
extremely low. If MOSH are enabled to pass the stratum cor- being well above 5 (Trevaskis et al. 2008; Trevaskis et al.
neum barrier, as in the case of acetone-compromised skin tis- 2015). There is also experimental evidence in rats that n-C16
sue in hairless mice, they are able to reach the subcutaneous or, perhaps more likely, its oxidation product(s) can be
fat layer within 2.5 h (Brown et al. 1995). The extremely high absorbed to some extent into the hepatic portal vein (Savary
octanol-water partition coefficient (e.g. estimated log PO/W for and Constantin 1967; Albro and Fishbein 1970a).
n-docosane: 11.15) favors partitioning into lipophilic compart- Gastrointestinal emulsification and bile salt-mediated micellar
ments. Easy mobilization from adipose tissue is unlikely given solubilization are required for the transport across the intes-
the findings from animal experiments with oral exposure (see tinal mucosa (Savary and Constantin 1967). Co-administration
Section “MOSH levels in animal tissue”). Subcutaneous injec- of dietary lipids such as triglycerides and fatty acids promotes
tion of a water-in-oil emulsion (Freund’s incomplete adjuvant) the micellar solubilization of n-C16 and enhances its uptake
containing light mineral oil (with 14C-radiolabelled n-hexade- by the lymphatic system (Savary and Constantin 1967).
cane) and a surfactant (mannide monooleate) in rats and The extent of intestinal absorption was determined in rats
monkeys revealed the radioactivity to be very slowly cleared by subtracting the amount excreted in feces from the admin-
from the site of injection (Bollinger 1970). Intradermal injec- istered dose. This approach is assumed to be valid since
tion of Freund’s incomplete adjuvant in guinea pigs showed there is no evidence either for a (relevant) biliary excretion of
that some of the injected MOSH traveled along lymphatic unmetabolized hydrocarbons (Albro and Fishbein 1970a,
vessels and appeared as oils droplets in the regional lymph 1970b; Albro and Thomas 1974; Le Bon et al. 1988; Halladay
nodes draining the site of intradermal injection. Local persist- et al. 2002) or for hydrocarbon oxidation by the gut flora
ence of MOSH, the possible transport to regional lymph (Mitchell and Hu €bscher 1968; Albro and Fishbein 1970a;
nodes, and a mechanism to load the plasma lipoproteins and Albro and Thomas 1974). Experiments with dietary and gav-
albumin with MOSH (see Section “Distribution, metabolism, age administration of increasing doses of shorter-chain n-
and excretion of MOSH”) should be taken into consideration alkanes (n-C17, n-C18) (Albro and Fishbein 1970a; Popovic
when addressing the systemic availability of dermally et al. 1973; Tulliez and Bories 1978, 1979) revealed a rela-
applied MOSH. tively constant maximum uptake in the lower dose range
In summary, MOSH readily partition into the uppermost and a decrease at higher doses (Figure 4(A)), indicating a sat-
cell layers of the stratum corneum. Although many studies uration of solubilization and absorption processes. The co-
seemingly fail to indicate a transport of MOSH through the administration of absorption-enhancing agents (dietary lipids,
stratum corneum one should note that this can also be attrib- synthetic emulsifier) very likely contributed to the study-spe-
uted to the experimental design, particularly in studies with cific differences in the dose-absorption curves, whose max-
short application times in conjunction with rather low analyt- imum levels ranged from 45% (Albro and Fishbein 1970a) up
ical sensitivity. Radioisotope studies in hairless mice with to almost 100% (Popovic et al. 1973; Tulliez and Bories 1978).
acetone-compromised skin, in guinea pigs, and with excised Application of fixed doses of differently sized n-alkanes (n-C14
pork skin demonstrated a weak permeation of the horny to n-C32) showed the absorption to be negatively correlated
layer by experimental MOSH surrogates, that is, radiolabelled with carbon number (Albro and Fishbein 1970a; Popovic
shorter-chain n-alkanes (C16–C22). It should be noted, how- et al. 1973; Tulliez and Bories 1975a) (Figure 4(B)).
ever, that the skin of hairless mice and guinea pig is more Discrepancies between studies in the carbon number-
permeable than human skin (Jung and Maibach 2015) and dependent absorption curves are very likely caused by differ-
that a radioisotope study with human skin using labeled n- ences in the experimental procedures (dose, method of
C18 did not show any permeation of the stratum corneum, administration, use of absorption-enhancing agents).
even after 16.7 h, whereas longer time periods were not Constant dosing of linear, branched, and cyclic alkanes of
addressed. Given the slow penetration into the horny layer, similar carbon numbers did not indicate a marked difference
the unfavorable partitioning into aqueous epidermal and der- in bioavailability (Albro and Fishbein 1970a; Tulliez and
mal tissues, and the potential impact of removal processes Bories 1975a) (Figure 4(B)).
(contact with other surfaces, skin washing, desquamation and Not only individual compounds were tested but also min-
regeneration), it therefore seems unlikely that toxicologically eral oils. In a toxicokinetic study (Halladay et al. 2002), F-344
relevant amounts of MOSH from, e.g. skin creams become and SD rats received two doses (34 and 340 mg/kg bw) by
bioavailable in humans. gavage of a low-viscosity white mineral oil (presumably
P15H, see Table 3) in olive oil containing radiolabelled 1-eico-
sanylcyclohexane (cyclic C6 with C20 side chain) as surrogate
Intestinal absorption of MOSH marker for MOSH. The fecal excretion data indicated an
Upon ingestion, MOSH follow the intestinal absorption path- absorption of 24 (F-344) and 30% (SD) at the low dose, and
way of dietary lipids. Experiments in rats with gastrointestinal values of 8 (F-344) and 12% (SD) at the high dose. Further
administration of n-hexadecane (n-C16), n-octadecane (n-C18), data comes from studies involving dietary exposure to min-
and phytane (branched C20), which were used as model com- eral oil. In a study with rats and pigs (Tulliez 1986), the ani-
pounds for MOSH, showed these to be absorbed from the mals were fed two types of white mineral oil, denoted A and
small intestine into the lymphatic system (Savary and B, with average carbon numbers of 20 and 28. The diet con-
Constantin 1967; Albro and Fishbein 1970a; Albro and taining 0.1% mineral oil of type A or B led to an absorption
Thomas 1974; Tulliez and Bories 1975a; Vost and Maclean rate in rats of 52% and 35%, respectively, whereas in pigs,
750 R. PIROW ET AL.

Figure 4. Intestinal absorption of model compounds of MOSH in rats in relation to dose and carbon number. Distinct colors are used to distinguish the data from
different studies. (A) Dose dependency of absorption of shorter-chain n-alkanes (n-C17, n-C18) and dodecylcyclohexane (C18). The graph labels have an index refer-
ence number (in bold) linking to study details described below and provide information on carbon number, administration procedure (d: diet, g: gavage), and the
use co-administered absorption-enhancing agents (e: synthetic emulsifier, p: peanut oil). (B) Absorption of linear, branched, and cyclic alkanes in relation to the
number of carbon atoms. Branched alkanes comprised 2,2,4,4,6,8,8-heptamethylnonane (C16), pristane (C19), and phytane (C20). The graph labels contain the index
reference number (in bold) and provide information on the applied dose (mg/kg bw per day), administration procedure, and the use of absorption-enhancing
agents such as triolein (t). Experimental information for A and B: 1 – single dose of n-octadecane (A) or of an equal-weight mixture of 3–4 unsaturated and satu-
rated aliphatic hydrocarbons, mixed with a branched C30 alkane (squalene) given to male CD rats (B) (Albro and Fishbein 1970a); 2 – single dose given to male
albino rats (Popovic et al. 1973); 3 – single dose given to Wistar rats (Tulliez and Bories 1975a, 1978); 4 – single dose given to male Wistar rats (Tulliez and Bories
1979); 5 – single dose given to rats (Savary and Constantin 1967); 6 – single dose given to male Wistar rats (Le Bon et al. 1988); 7 – administration of 10 mg/kg bw
per day of plant wax paraffins containing 90% n-C29 given to male Osborne-Mendel rats over a 5-day period.

Table 3. Classification of highly refined mineral hydrocarbons (intended for use in food).
JECFA class Average
for medium- and Viscosity relative Carbon number
low-viscosity at 100  C molecular at 5% distillation
Name mineral oils (mm2/s) mass point
Microcrystalline wax 11 500 25
E 905 11 650 25
High-melting-point wax
Low-melting-point wax No specification
Low-melting-point wax 3.3 380 22
High-viscosity mineral oil >11 500 28
P100 11 520 29
Medium-viscosity mineral oil I 8.5–11 480–500 25
P70 9.0 480 27
Medium-viscosity liquid petroleum 8.7 480 25
P70H 8.6 480 27
Low-viscosity mineral oil II 7.0–8.5 400–480 22
N70H 7.7 420 23
Low-viscosity mineral oil III 3.0–7.0 300–400 17
P15H 3.5 350 17
N15H 3.5 330 17
Adapted from JECFA (2002, 2012) and EFSA (2009, 2013b, 2013a).
Mineral oil type abbreviation: N: naphthenic (crude oil source); P: paraffinic; H: hydro-treated (catalytic hydrogenation); number (15, 70, 100) ¼ approximate vis-
cosity (mm2/s) at 40  C.

the percentages were 38% and 20%, respectively. The rates retention characteristics of liver and spleen suggested that
decreased in both species when the mineral oil content in intestinal absorption of MOSH decreased above C32 and
the diet was increased to 1%, indicating an absorption limita- approached zero at around C40. After 30 days of exposure,
tion. Pigs receiving a diet containing 0.65% of a third type of 10.9% of ingested MOSH were recovered from the rats’ body
white mineral oil with an average carbon number of 16 over (excluding the gastrointestinal tract). This retained percent-
a period of ten days showed an absorption of 88% age refers to the total of the administered MOSH mixture,
(Tulliez 1986). including the constituents of lowest and highest carbon
Further insights into the intestinal absorption of mineral number which did not show up in the tissues. For the most
oils comes from the measurement of MOSH in tissue. In a efficiently retained constituents, the percentage was even
recent study, Barp et al. (2017a) exposed female F-344 rats to higher (17.2%). These figures are lower bound estimates for
a diet containing 0.004% of a broad MOSH mixture with a the extent of intestinal absorption. The true absorption is
carbon number range from C14 to C50. The test item was dis- higher due to elimination of hydrocarbons that are more
solved in olive oil before incorporating it into the diet. The amenable to metabolism. Higher dietary concentrations of
 CRITICAL REVIEWS IN TOXICOLOGY 751

MOSH decreased the percentage of MOSH retained in the 3–6 h post-dosing. The terminal phase half-life derived from
body, again indicating an absorption limitation. these blood concentration-time profiles was relatively long
To summarize, ingested MOSH follow the absorption path- (43–86 h) (Halladay et al. 2002; Boogaard et al. 2012), reflect-
way of dietary lipids. The fraction of absorbed dose of MOSH ing a relatively slow elimination from the blood compart-
is negatively correlated with carbon number and dose. Co- ment. Based on blood concentration-time profiles and
administration of dietary lipids enhances the absorption. hepatic MOSH concentrations, F-344 rats exhibited a higher
Exposure of rats to a diet containing a MOSH mixture of low systemic exposure than SD rats. This difference presumably
concentration (0.004% or 40 ppm) results in absorption rates resulted from a higher metabolic activity in the latter, since
well above 20% for constituents that are most efficiently fecal excretion data (Halladay et al. 2002) indicated a slightly
retained in the tissues. It is noted that this low concentration higher absorption in SD rats compared to F-344 rats (see
is comparable to the highest average background concentra- Section “Intestinal absorption of MOSH”). In female human
tion in certain food categories such as vegetable oils and volunteers, the oral administration of a gelatin capsule con-
canned fish (see Chapter “Evaluation of mineral oil hydrocar- taining 71 mg of a low-viscosity white mineral oil failed to
bons in food by the EFSA CONTAM Panel”). give detectable blood levels of MOSH (Boogaard et al. 2012),
presumably because the dose was too low. A toxicokinetic
study of the blood of patients undergoing colonoscopy after
Distribution, metabolism, and excretion of MOSH taking liquid paraffin as laxative (for dosage, see Chapter
Ingested alkanes may already be metabolized whilst travers- “Mineral oils in medicinal products”) might be a more suit-
ing the intestinal mucosa. Metabolic studies demonstrated able approach to obtain plasma half-life readings for intestin-
that the intestinal mucosa of rats (McWeeny (1957), cited in ally absorbed MOSH.
Mitchell and Hu €bscher (1968)), guinea pigs (Mitchell and The fate of intestinally absorbed MOSH (incl. model com-
H€ubscher 1968) and rabbits (Ichihara et al. 1981), but also pounds and n-alkanes) in the systemic circulation is deter-
the rumen tissue of goats (McCarthy 1964), has the capacity mined by the uptake and metabolism in the liver, and the
to oxidize n-C16 and n-C18 into the corresponding fatty acids. distribution to other tissues, preferentially to adipose tissue
Following intestinal lymphatic uptake, alkanes are trans- (Pokrovskii et al. 1969) (for MOSH levels in selective tissues,
ported as solutes in the lipid moieties of lymph chylomicrons see Sections “MOSH levels in animal tissues” and “MOSH levels
towards systemic circulation. In rats, duodenal co-administra- in human tissues”). Metabolism is the dominant elimination
tion of n-alkanes (n-C16, n-C18) and triglycerides showed these process as there is no evidence for the excretion of non-
hydrocarbons to be associated with the nonpolar triglyceride metabolized alkanes via the urinary tract (Tulliez and Bories
core of lymphatic chylomicrons (Vost and Maclean 1984). 1975a, 1978, 1979; Le Bon et al. 1988) and virtually none for
Intravenous injection of these chylomicrons in rats revealed a the biliary excretion of the unchanged form (Albro and
rapid clearance of hydrocarbons from plasma chylomicrons Fishbein 1970a, 1970b; Albro and Thomas 1974; Le Bon et al.
caused, inter alia, by the uptake into the liver and the trans- 1988; Halladay et al. 2002). Also, an in vitro study found no
fer to other plasma lipoprotein classes (Vost and Maclean evidence for a relevant extrahepatic metabolism of n-hexade-
1984). Remarkably, hydrocarbon clearance was closely corre- cane in lung and kidney microsomes of mice (Kusunose et al.
lated with chylomicron triglyceride clearance. A study on the 1969). Exhalation of smaller, more volatile hydrocarbons could
lipid composition of human serum detected MOSH in low- be an additionally route of elimination, given the detection of
and high-density lipoprotein classes (LDL, HDL) and in the n-alkanes (up to n-C20) in human breath and indoor air
albumin-containing fraction (Skipski et al. 1967); the MOSH in (Phillips et al. 1999; Phillips et al. 2000). However, the alkane
the albumin-containing fraction had a carbon number range concentrations and the alveolar gradient (concentration in
of C17–C35 with n-alkanes being most prominent. Removal of breath minus concentration in ambient room air) were in the
n-alkanes yielded a residual hydrocarbon fraction distributed picomolar range which renders elimination via exhalation less
between C26 and C33. Since human serum albumin exhibits relevant than metabolic elimination.
six binding sites for long-chain fatty acids (Peters 1995) it Depending on the type of alkane, different metabolic
may contribute to the distribution of n-alkanes throughout pathways are involved in the biotransformation (Tulliez 1986;
the body in a similar way as the usage of LDL and HDL as EFSA 2012). Linear alkanes undergo terminal (x) oxidation via
possible MOSH transporters. alkyl/fatty alcohols to their corresponding fatty acids (Figure
Information on the plasma half-life of intestinally absorbed 5(A)), which then enter the b-oxidation pathway or are incor-
MOSH comes from toxicokinetic studies in rats with single- porated into lipids (McCarthy 1964; Kolattukudy and Hankin
dose administration of white mineral oils and individual 1966; Savary and Constantin 1967; Kusunose et al. 1969;
model compounds. Two studies, in female F-344 and SD rats, Popovic 1970; Ichihara et al. 1981; Popovic et al. 1982; Perdu-
with gavage administration of low-viscosity white mineral oils Durand and Tulliez 1985; Cravedi et al. 2011; Cravedi and
(e.g. P15H, see Table 3) in olive oil were performed to estab- Perdu 2012). The metabolism of cycloalkanes was studied
lish blood concentration-time profiles for MOSH in the car- with mono- and bicyclic model compounds. The n-alkyl side
bon number range of C19–C24 (Boogaard et al. 2012) and for chain of cyclohexanes (cyclic C6 with C12 or C20 n-alkyl side
radiolabelled 1-eicosanylcyclohexane (cyclic C6 with C20 side chains) is subjected to x-oxidation followed by b-oxidation
chain), which was added to the mineral oil as a surrogate to yield cyclohexyl alkanoic acids (Figure 5(B)), which may
marker for MOSH (Halladay et al. 2002). In the blood the eventually become the target of further transformations prior
hydrocarbons reached a peak concentration essentially within to urinary excretion (Tulliez and Peleran 1977a, 1977b; Tulliez
752 R. PIROW ET AL.

Figure 5. Metabolic pathways of n-alkanes (A), cycloalkanes (B), and branched alkanes (C) in mammals.

and Bories 1979; Tulliez et al. 1981; Halladay et al. 2002). The Obviously, the high degree of isomerization due to possible
unsubstituted bicyclic compound decalin (C10) is hydroxy- branching of the alkyl group seemed to prevent a fast/effi-
lated to different decalols which are conjugated and excreted cient metabolic break down. Further analysis focused on
via urine (Elliott et al. 1966; Olson et al. 1986; Dill et al. 2003). hydrocarbons of the same carbon number. The composition
The fate of branched alkanes was elucidated in studies with of retained C26 naphthenes, which mainly comprised 1–4 ring
pristane (C19) and phytane (C20), which undergo terminal (x) systems, indicated a facilitated elimination of compounds
or subterminal (x–1) oxidation (Figure 5(C)) (Albro and with a major straight alkyl chain. The analysis of C24 mono-
Thomas 1974; Le Bon et al. 1988). Whereas the former leads cyclic naphthenes revealed an efficient metabolism of n-alky-
to fatty acids entering the b-oxidation route, the latter yields lated compounds with elimination efficiency decreasing with
tertiary alcohols, which are considered as a metabolic dead increasing alkyl chain branching or multi-substitution.
end precluding any further oxidation (Albro and Thomas To summarize, the fate of intestinally absorbed MOSH is
1974; Le Bon et al. 1988). Depending on branching determined by the uptake and metabolism in the liver, and
patterns, a-oxidation could step in if b-oxidation of fatty acid the partitioning to other tissues, preferentially to adipose tis-
derivatives is prevented by a tertiary carbon center at the sue. Biotransformation is the dominant elimination process.
b-position (Hansen 1968). Studies with individual model compounds showed that differ-
Information on the metabolism of complex MOSH mix- ent oxidative pathways could be involved in biotransform-
tures can be derived from GC
GC analysis of hydrocarbons ation, depending on the type of alkanes. For complex MOSH
that are retained in tissue. In a recent study, Barp et al. mixtures that are retained in F-344 rat liver, there is evidence
(2017a) exposed female F-344 rats to a diet containing a of some degree of selective facilitated elimination of simple-
broad MOSH mixture, ranging from about C14 to C50 and structured hydrocarbons such as small multibranched paraf-
being largely devoid of n-alkanes above C21, over a period of fins and n-alkylated naphthenes.
90 days, followed by a 30-day post-exposure period.
Hydrocarbons retained in liver ranged from C16 to C40. Subtle MOSH levels in animal tissues
differences between the MOSH composition of the adminis- MOSH levels in animal tissues were analyzed in studies with
tered mixture and the hydrocarbons retained in tissue pro- single and repeated oral administration of single model com-
vided evidence of some degree of selective elimination from pounds and petroleum products, including white mineral oils,
the liver (via metabolism and also possibly by exchange proc- paraffin waxes, and microcrystalline waxes (Table 1).
esses between liver and blood). These differences were Single dietary administration of linear, branched, and cyc-
revealed by MS analysis of characteristic fragment ions being lic alkanes (C16 to C32) to Wistar rats showed a differential
selective for (i) multibranched paraffins, (ii) monoalkylated retention pattern in tissues, depending on alkane type, car-
cyclopentanes and cyclohexanes, (iii) C26 naphthenes with bon number, and tissue type (Tulliez and Bories 1975a, 1978,
1–4 rings, and (iv) alkylated C24 monocyclic naphthenes, 1979; Le Bon et al. 1988). Repeated administration of a diet
respectively. Among multi-branched paraffins retained in containing 0.1% shorter-chain cyclic and linear alkanes (cyclic
liver, smaller constituents, including pristane and phytane, C6 with C12 n-alkyl side chain, n-C20) to Wistar rats over
were largely absent (Barp et al. 2017a), suggesting an effect- 3 months resulted in low concentrations (10.7–29.6 ppm) in
ive elimination. In the GC
GC–MS plot related to monoalky- liver and high levels (1029–1134 ppm) in adipose tissue
lated cyclopentanes and cyclohexanes, the series of distinct (Tulliez and Bories 1975b). A similar picture was observed in
signals for n-alkyl monocyclics was virtually absent, leaving a 10-day feeding study with pigs receiving a diet containing
behind a cloud of unresolved, highly isomerized material. 0.65% white mineral oil composed of shorter-chain MOSH
 CRITICAL REVIEWS IN TOXICOLOGY 753

(average carbon number: 16, 60% linear alkanes and 40% varying types of petroleum products are being used, because
branched-plus-cyclic hydrocarbons) (Tulliez 1986). The levels the MOSH composition can be totally different (see below).
of linear and branched-plus-cyclic alkanes were low in liver (6 Test items causing high MOSH concentrations in liver also
and 15 ppm) and high in adipose tissue (1640 and 570 ppm). caused similar levels in the MLNs; in contrast, MOSH levels in
The low liver levels suggest efficient metabolism of shorter- adipose tissue were about an order of magnitude lower than
chain compounds. those in liver (Smith et al. 1996). The accumulation of MOSH
Conversely, when the mineral oil constituents had higher in liver was reversible. F-344 rats receiving a control diet for
carbon numbers and a higher proportion of branched and 28 days after a 90-day treatment with low-viscosity oils
cyclic hydrocarbons a different picture emerged. Pigs receiv- showed a reduction of liver MOSH contents by 10–30%
ing a diet containing 3.23% white mineral oil (average carbon (females, Figure 6(A)) and 35–50% (males). For paraffin waxes,
number: 20) over 3 months had high MOSH concentrations a reduction by 80–90% was observed after an 85-day post-
in liver (1353 ± 377 ppm, N ¼ 5 animals) and low levels in sub- exposure period (Figure 6(A)). These reductions can be dir-
cutaneous (293 ± 47 ppm) and back fat (256 ± 53 ppm) ectly related to elimination half-lives if the underlying process
(Tulliez, Bories, and Peleran 1975). Among the other tissues, can be assumed to follow a mono-exponential decay. For
the spleen had very low levels (65 ± 15 ppm) whereas the example, a reduction by 80% (corresponding to a relative
mesenteric lymph nodes (MLNs) showed the highest value decrease from 1 to 0.2) in 85 days would then translate into
(1286 ppm, N ¼ 1). Chromatographic analyses revealed that an elimination half-life of 37 days (¼ 85/log2 0.2).
liver accumulated MOSH with higher and adipose tissue Several studies exist in which female F-344 rats were
those with lower carbon numbers (Tulliez, Bories, and exposed subchronically to diets of different concentrations
Peleran 1975). ranging from 0.004% to 2% (Figures 6–8) (Firriolo et al. 1995;
It should be noted that analytical techniques have rapidly Griffis et al. 2010; McKee et al. 2012; Barp et al. 2017a,
evolved during the past decades widening the scope and 2017b). Here, the MOSH levels in liver increased less than
increasing resolution as well as sensitivity. Therefore, from proportional with dietary concentration. For example, a 10-
fold increase in the dietary concentration of P7H (McKee
the today’s perspective, the mid-1970s study results on
et al. 2012), P15H (Firriolo et al. 1995), and LMPW (Griffis
MOSH content, composition, and carbon number range are
et al. 2010) from 0.2% to 2% caused an increase of liver
associated with a certain degree of uncertainty.
MOSH levels by 1.3–1.5-fold only (Figures 6(B), 7(B,C)). At
In the literature, several studies exist in which rats were
lower dietary levels, this sub-linearity was still noticeable: A
exposed subchronically or chronically to white mineral oils
2.5-fold increase in the concentration of two pharmaceutical-
and to paraffin and microcrystalline waxes via feed (Baldwin
grade mineral oils (S-C25 and L-C25, Ph. Eur. Paraffinum perli-
et al. 1992; Firriolo et al. 1995; Smith et al. 1996; Scotter et al.
quidum and P. liquidum, something close to P15 and P70 oils
2003; Trimmer et al. 2004; Griffis et al. 2010; Barp et al.
according to viscosity at 40  C of 18 and 80 mm2/s, respect-
2017a, 2017b). They provide information on the MOSH levels
ively) from 0.04% to 0.1% (Barp et al. 2017b) raised the
in liver and other tissues in dependence on the special kinds
MOSH concentration in liver by only 1.9-fold (Figure 6(C)).
of mineral oils and waxes, dietary concentrations, strains and
The under-proportional increase suggests saturation of the
sexes, and exposure times. In addition, they provide informa-
transport system due to a limitation in digestive solubiliza-
tion on the reversibility of accumulation effects and on the
tion and intestinal absorption of MOSH.
time to reach steady-state levels of MOSH. Comparative studies in rats revealed pronounced sex and
In a 90-day study (Smith et al. 1996) F-344 rats were fed strain differences. Female F-344 rats appear to have a predis-
with diets containing 2% mineral oil or wax of various types. position to retain high MOSH levels in liver in response to
The feeding of low-viscosity (e.g. N15H, P15H, N70H) and the dietary exposure to low-viscosity and medium-viscosity
medium-viscosity (P70H) oils caused elevated MOSH levels oils, and low and intermediate melting point waxes. Much
(1000–4300 ppm) in female livers (Figure 6(A)). Male livers lower levels occurred in male F-344 rats (Figure 7(A))
had 4–5 times lower levels. Females exposed to a high-vis- (Baldwin et al. 1992) and female SD rats (Figure 7(B)) (Firriolo
cosity oil (P100H) had MOSH concentrations (600 ppm) being et al. 1995) when being exposed to low-viscosity and
only slightly elevated above the background levels found in medium-viscosity oils. Remarkably, female SD rats ingesting
the liver of controls (200 ppm). Administration of paraffin the (n-alkane-rich) low melting point wax showed hepatic
waxes with low and intermediate melting points (LMPW, MOSH levels below the limit of quantification (Figure 7(C))
IMPW) induced the highest MOSH levels (7600–16900 ppm) (Griffis et al. 2010).
in female livers (Figure 6(A)). Levels in male livers were about In female F-344 rats, the strong retention in liver following
1.5–2 times lower. For microcrystalline wax (HMPW: high exposure to paraffin waxes (e.g. LMPW) indicates the inability
melting point wax), MOSH levels in liver were not signifi- to efficiently metabolize absorbed n-alkanes consisting of a
cantly different from control values. It is important to note certain carbon number range (Smith et al. 1996; Griffis et al.
that paraffin waxes are defined by a high content of n-alka- 2010; Barp et al. 2017b; Cravedi et al. 2017). Even very low
nes, a feature that distinguishes them from mineral oils. This concentrations (0.02–0.12 ppm) of natural n-alkanes in the
has important toxicokinetic consequences, best exemplified range of C29–C33, that were present in control feed, were
by the difference in accumulation between LMPW and found to be enriched in liver of untreated control animals
P100H. This example illustrates that merely comparing the (Cravedi et al. 2017). It was hypothesized by the authors that
level of MOSH in tissue can be misleading, especially when these wax components crystallized in the liver tissue, which
754 R. PIROW ET AL.

Figure 6. MOSH concentration in the liver of female F-344 rats in relation to administered mineral oil/paraffin wax type and dietary concentration. Data were
compiled from Smith et al. (1996) (A), McKee et al. (2012) (B), and Barp et al. (2017b) (C). The mineral oil/paraffin wax types and average carbon number ranges are
indicated. Data in (A) show the concentrations after a 90-day treatment (white filled symbols) as means þ standard deviation as well as the corresponding concen-
trations following a post-exposure period of 28 days (gray-filled circles) and 85 days (gray-filled rectangles). Mineral oil nomenclature: N: naphthenic (crude oil
source); P: paraffinic; A: acid-treated; H: hydro-treated (catalytic hydrogenation); number (7, 10, 15, 25, 70, 100): approximate viscosity (mm2/s) at 40  C. Paraffin wax
nomenclature: LMPW: low melting point wax; IMPW: intermediate melting point wax; HMPW: high melting point wax. Abbreviations adopted from Barp et al.
(2017b): S-C25: presumably a light liquid paraffin oil (Ph. Eur. Paraffinum perliquidum), L-C25: presumably a liquid paraffin oil (Ph. Eur. Paraffinum liquidum),
L-C25W: a 1:1 (w/w) mixture of L-C25 and a paraffin wax (W) (see Table 1).

Figure 7. MOSH concentration in the liver of rats in relation to administered mineral oil type, dietary concentration, sex, and strain. Data were compiled from
Firriolo et al. (1995) (A), Baldwin et al. (1992) (B), and Griffis et al. (2010) (C). Data are given as means ± standard deviation. Mineral oil nomenclature: OTWO: oleum-
treated white oil; HTWO: hydro-treated white oil. For other abbreviations, see legend of Figure 6.
 CRITICAL REVIEWS IN TOXICOLOGY 755

lives. Accordingly, it would take 1.2–1.7 years to reach the

steady state when feeding these types of mineral oils.
MOSH accumulation was also studied by Barp et al.
(2017a) in a subchronic study in female F-344 rats which
received a diet containing a broad MOSH mixture, ranging
from about C14 to C50 and being largely devoid of n-alkanes
above C21, at concentrations of 0.004, 0.04 and 0.4%. The test
item was prepared by combining two pharmaceutical-grade
white oils (S-C25 and L-C25, something close to P15 and P70
oils), a high-quality process oil (P100H-type), and the vacuum
distillate of a pharmaceutical-grade light white oil (Ph. Eur. P.
perliquidum). The resulting MOSH mixture displayed a chro-
matographic profile covering a wide carbon-number range.
The test item was dissolved in soybean oil before incorporat-
ing it into the diet. Amounts and composition of the MOSH
were analyzed in liver, spleen, adipose tissue, and the carcass
after 30, 60, 90 and 120 days of exposure, and after 90-day
exposure plus 30-day recovery period.
For all three dietary concentrations, the MOSH levels in
liver increased rapidly until day 30 and then slower until it
reached a plateau between day 90 and 120 (Barp et al.
2017a) (Figure 8(B)). During the 30-day recovery period, hep-
atic MOSH levels decreased by 60, 54, and 34% (corrected for
hepatic MOSH levels in the control group) in the low, inter-
mediate, and high-exposure groups, respectively. These
reductions correspond to half-lives of 23, 26, and 50 days.
Accordingly, the time to reach a quasi-steady-state level was
115–250 days, which agrees well with the observation of a
plateau after 90 days of exposure. Compared to the MOSH
levels in liver, those in spleen and adipose tissue were
Figure 8. MOSH concentration in the liver of female F-344 rats in relation to approximately one order of magnitude lower and did not
exposure and recovery duration. (A) Data (means ± standard deviation) were reach a steady state during the 120 days of exposure. The
compiled from a 2-year study with dietary exposure to P70H or P100H (Trimmer
et al. 2004). Included are data from control animals, and from the recovery
post-exposure reduction of splenic MOSH levels at low food
group which received the treated diet for 12 months and then a control diet for concentration was 72%. At intermediate and high food con-
the following 12 months. For mineral oil type abbreviation, see legend of Figure centrations, however, splenic MOSH levels decreased by only
6. (B) Data of a 120-day feeding study in which animals received different doses
of a mineral oil mixture (Barp et al. 2017a). MOSH concentration is on a log10-
11% during the post-exposure period. In adipose tissue, the
transformed scale. Data for the recovery groups and the control animals MOSH concentration still increased during the recovery
are included. period, suggesting an accumulation of MOSH and a post-
exposure transfer from other tissues (e.g. from liver via lipo-
would be an additional factor contributing to the slow bio- proteins), including – possibly – the gastrointestinal tract.
transformation (Barp et al. 2017b; Cravedi et al. 2017). Overall, the 30-day recovery period led to the elimination of
The time-course of MOSH retention and the elimination of 40–50% of the retained MOSH from the body.
MOSH during a recovery period was monitored in a chronic To determine the most strongly retained hydrocarbons in
study with female F-344 rats (Trimmer et al. 2004). Animals terms of carbon numbers, Barp et al. (2017a) compared the
received a diet containing P70H or P100H in a concentration LC–GC–FID chromatograms of the dietary MOSH mixture
of 2.5%, corresponding to a dose of 1200 mg/kg bw/day. with those of liver, spleen, and adipose tissue. The carbon
After 3 months, the MOSH levels in liver increased to approxi- number distribution of hydrocarbons was calibrated using n-
mately 900 and 1600 ppm for P70H and P100H, respectively alkanes as markers. The authors selected the tissue chromato-
(Figure 8(A)). Further increase was slow, reaching 1400 and grams of the recovery group (90-day exposure plus 30-day
2270 ppm, respectively, after two years. This suggests that recovery) as these singled out the retained hydrocarbons at
the MOSH levels reach a steady state within two years. best. The data for the lowest dietary concentration (0.004%)
Reversibility was assessed by a twelve-month recovery phase were chosen since the liver chromatograms indicated a limit-
following twelve months of exposure. During this post-exposure ing absorption of hydrocarbons above C24 at higher dietary
period, MOSH levels in liver decreased towards the levels in the MOSH levels. For data analysis purposes, the x-axes of the
control group in a mono-exponential manner (Figure 8(A)) with chromatograms were binned using a segment width of one
half-lives of 91 and 122 days for P70H and P100H, respectively. carbon number unit in terms of the n-alkane calibration. The
Based on these elimination kinetics, the time for MOSH to reach processed profiles were finally used to calculate a so-called
a steady state level can be calculated. As a rule of thumb, a retention function (or transfer function) which translates the
quasi-steady state (97% of the final level) is reached after 5 half- dietary MOSH distribution (“input signal”) into a given tissue
756 R. PIROW ET AL.

Figure 9. Retention of MOSH in tissues of female F-344 rats in relation to carbon number. (A) Illustration of the retention function (¼ transfer function) which trans-
lates the molecular mass distribution of MOSH in food (“input signal”) into a given tissue distribution (“output signal”). The symbol k is a normalization constant,
the index i refers to the carbon number of n-alkanes. (B) Normalized retention functions for the MOSH distribution in liver, spleen, and adipose tissue. The retention
functions were calculated from tissue chromatograms of a recovery group (90-day exposure plus 30-day recovery) receiving to diet containing 0.004% of a broad
MOSH mixture. (Figure adapted from Barp et al. (2017a), # 4622010703947).

MOSH distribution (“output signal”) (Figure 9(A)). The max- and female SD rats. The latter had hepatic MOSH levels below
imum retention in liver and spleen was at C29, and at C15/16 the limit of quantification upon dietary exposure to (n-alkane-
in adipose tissue (Figure 9(B)). Both the preferential seques- rich) low melting point wax. In female F-344 rats exposed to
tration of shorter-chain compounds in fat, and the more effi- moderate dietary concentrations of  0.4% of a broad MOSH
cient metabolism of these compounds in liver, very likely mixture, steady-state MOSH levels in liver were reached after
contributed to the different profiles in these tissues. 115–250 days. Conversely, adipose tissue seems to slowly accu-
Apart from the characteristic shapes of the retention pro- mulate MOSH.
files, the chromatogram of the MOSH in liver of female F-344
rats revealed an additional, subtle but toxicologically signifi-
cant, feature. The top and the downslope of the MOSH MOSH levels in human tissues
hump above C22 showed distinct peaks which were largely Information on the concentration and composition of MOSH
invisible in the broad MOSH mixture in the animals’ diet in human tissue became first available from autopsy studies
(Barp et al. 2017a). The same phenomenon was observed in performed in the mid-1960s (Boitnott and Margolis 1966b;
female F-344 rats exposed to the white oil L-C25 (Barp et al. Rose and Liber 1966; Liber and Rose 1967; Boitnott and
2017b). Further characterization by GC
GC analysis showed Margolis 1970). It was not until this century that new data
that the main peaks represented branched alkanes, whereas were obtained from autopsy and biopsy specimens using
the small peaks were identified as n-alkanes (Barp et al. advanced analytical methods (Noti et al. 2003; Concin et al.
2017b). These results indicate that n-alkanes and certain 2008; Barp et al. 2014; Biedermann et al. 2015).
branched alkanes from C27 to C35 are selectively enriched in The mid-1960s studies analyzed lymph nodes, spleen, and
the liver of female F-344 rats (Barp et al. 2017b). liver tissues from autopsies performed in the US (Boitnott
To summarize the results, rats and pigs show a differential and Margolis 1966b; Rose and Liber 1966; Liber and Rose
retention pattern of MOSH in tissues. Shorter-chain com- 1967; Boitnott and Margolis 1970). MS of saturated hydrocar-
pounds around n-C16 partition into and accumulate in adipose bons, which were isolated from all three tissues types, identi-
tissue. The liver retains MOSH with higher carbon numbers, fied complex mixtures of predominantly branched and cyclic
suggesting a more efficient oxidation of shorter-chain com- compounds, a composition typical of MOSH (Boitnott and
pounds. Female F-344 rats showed a maximum retention in Margolis 1966b, 1970). GC–FID analysis of splenic tissue
liver and spleen at C29. They retained high MOSH levels in revealed a largely unresolved hump ranging from C21 to C28
liver, and in the MLNs, upon dietary exposure to low-viscosity (Rose and Liber 1966; Liber and Rose 1967). Using column or
and medium-viscosity oils, and low and intermediate melting thin-layer chromatography, the MOSH concentrations in
point waxes. The highest MOSH levels in liver were caused by spleen and liver were determined gravimetrically (Rose and
dietary exposure to low melting point waxes, which – together Liber 1966; Boitnott and Margolis 1970). MOSH levels in
with findings in ‘untreated’ animals exposed to natural n-alka- spleen samples were in the range of 120–4500 ppm (N ¼ 26)
nes in control feed – indicate the inability to efficiently metab- (Boitnott and Margolis 1970) and 60–2660 ppm (N ¼ 13) (Rose
olize long n-alkanes (above C22) from both petrogenic and and Liber 1966; Liber and Rose 1967). Liver samples con-
natural origin. Compared to female F-344 rats, much lower tained MOSH concentrations ranging from <100 ppm to
MOSH levels in liver and MLNs occurred in male F-344 rats 4100 ppm (N ¼ 42) (Boitnott and Margolis 1970). It should be
 CRITICAL REVIEWS IN TOXICOLOGY 757

Figure 10. Concentration and molecular mass distribution of MOSH in MLN, fat tissue, liver, and spleen of autopsy samples. (A) Box-percentile plots (Harrell 2019)
of the MOSH concentrations in the different tissues. Data were extracted from Barp et al. (2014). Shown are the distributional characteristics comprising the 5th,
12.5th, 25th, 37.5th, 50th, 87.5th, and 95th percentiles. Filled circles represent the geometric means. Individual values are shown by gray-coloured tick marks. In
addition, the number of subjects (n) is given. (B–E) LC-GC-FID chromatograms showing the approximate molecular mass distribution of MOSH from MLN, fat tissue,
liver, and spleen, collected from the same subject. Numbers indicate the carbon number of n-alkanes. In addition, the MOSH concentration (ppm) is given.
(Chromatograms adapted from Barp et al. (2014), # 4622010018723).

noted that analytical techniques have been rapidly evolved considered to be of petrogenic origin (Noti et al. 2003;
during the past decades widening the scope and increasing Biedermann et al. 2015). MOSH in liver and spleen had similar
resolution as well as sensitivity. Therefore, from the today’s mass distributions, which were shifted to higher values, rang-
perspective, the mid-1960s study results on MOSH content ing from C18 to beyond C45, centered on C25–27 (Figure
and carbon number range are associated with a significant 10(D,E)) (Barp et al. 2014). The humps were completely
degree of uncertainty. devoid of skinny peaks indicative of n-alkanes, suggesting
In samples from 37 patients (25–91 years old) autopsied in efficient metabolism of these hydrocarbons in the human
Austria in 2013, Barp et al. (2014) determined the MOSH con- liver. A similar pattern, with mass distributions being different
centration in MLNs, subcutaneous abdominal adipose tissue, for adipose tissue and liver/spleen, was also observed in a
liver, spleen, and lung by on-line coupled LC–GC–FID. The subchronic study in rats: Female F-344 rats were exposed to
data were approximately log-normally distributed with a a diet containing a low concentration (0.004%) of a broad
median of 166 ppm in the MLNs (range: 21–1390 ppm), MOSH mixture relevant to human dietary exposures (Barp
87 ppm in adipose tissue (range: 17–493 ppm), 71 ppm in et al. 2017a). In rat liver and spleen, the maximum retention
liver (range: 14–901 ppm), 28 ppm in spleen (range: in terms of molecular mass was at C29 (Figure 9) which is
6–1400 ppm), and 7 ppm in lung (range: < 2–91 ppm) (Figure close to the peak of the mass distributions in human liver
10(A)). As in rats (Smith et al. 1996), the MLNs as the first site and spleen.
of contact following intestinal lymphatic uptake showed the Multiple linear regression (RP et al., unpublished data) was
highest MOSH concentrations, suggesting an exposure via applied to analyze the age and sex dependence of the MOSH
the oral route. concentration in the autopsy tissues. Women had, on aver-
The MOSH composition in MLNs and adipose tissue dis- age, a 2.2–2.5-fold higher MOSH concentration in liver,
played high similarity and varied little between subjects spleen, and adipose tissue compared to men. A slower MOSH
(Barp et al. 2014). The bulk of hydrocarbons (highly branched metabolism and/or a higher MOSH exposure of women rela-
and cyclic MOSH isomers) remained unseparated and formed tive to men could account for these differences. There was
a hump, ranging from C16 to C35, centered at C23/24 (Figure no evidence of an age dependence for the MOSH concentra-
10(B,C)). The tall skinny peaks on the downslope of the tions in liver and spleen, suggesting that a steady state had
humps above C24 indicated the presence of even- and odd- been reached. The MOSH levels in adipose tissue and MLNs
numbered n-alkanes. The peaks of the odd-numbered n-alka- exhibited a 1.2–1.4-fold increase per decade pointing to a
nes were more prominent than those of the even-numbered lifelong accumulation in both tissues. A constant fold
ones. A strong predominance of the former points to a bio- increase might seem to be implausible since, under condi-
genic origin (e.g. from plant waxes), whereas a balanced tions of constant exposure, it would imply that older subjects
occurrence of even- and odd-numbered n-alkanes is would experience a higher absolute increase compared to
758 R. PIROW ET AL.

younger subjects. However, this finding could still be com-

patible with a plausible linear accumulation process when
taking into account that a given tissue level of MOSH is the
result of cumulative past exposure and that, historically,
exposure to MOSH via the diet was higher in the past as it is
today (see Section “Histopathological lesions associated with
the deposition of MOSH in human tissues”).
Tissue-specific retention kinetics with MOSH levels reach-
ing a steady state in human liver but not in adipose tissue is
consistent with the F-344 rat model (see Section “MOSH lev-
els in animal tissues”). Since elevated MOSH levels in liver
can result in histopathological lesions (see Sections “Effects
associated with the accumulation of MOSH in animal tissues”
and “Histopathological lesions associated with the deposition
of MOSH in human tissues”), the crucial question is whether
the dose-concentration relationship for the F-344 rat can be
plausibly extrapolated to humans. A MOSH concentration of
220 ppm was measured in the liver of female F-344 rats fol-
lowing the subchronic dietary exposure to 2 mg/kg bw per Figure 11. Box-percentile plots of the MOSH concentration in subcutaneous fat
and milk fat of breast-feeding women. Data were extracted from Concin et al.
day of a broad MOSH mixture (0.004% in diet) (Barp et al. (2008). For further details, see legend to Figure 10(A).
2017a). Human autopsy livers contained a median concentra-
tion of 71 ppm. Interspecies extrapolation can be applied to (Noti et al. 2003) also noted a decreasing trend between the
derive a human equivalent oral dose from the rat data. By start and the end of a breast-feeding session.
assuming a linear dose-concentration relationship at low The MOSH concentrations shown in Figure 11 refer to the
doses (i.e. linear kinetics) and by using the default adjust- fat phase of tissue and breast milk. Day-4 and day-20 milk
ment factor of 4 for interspecies toxicokinetic differences, the samples had a mean fat content of 2.4% (w/v; range:
human equivalent oral dose for a liver concentration of 0.5–5.9%) and 3.7% (range: 0.5–8.5%) (Concin et al. 2008).
71 ppm would be 0.16 mg/kg bw per day (¼2
71/220/4). The MOSH content in human milk was therefore considerably
The EFSA CONTAM Panel (EFSA 2012) estimated a back- lower, with median levels of 0.72 ppm for day-4 milk and
ground dietary exposure of 0.03–0.32 mg/kg bw per day (see
0.37 ppm for day-20 milk. Human adipose tissue has an aver-
Chapter “Evaluation of mineral oil hydrocarbons in food by
age fat content of 84% (Thomas 1962), so that the median
the EFSA CONTAM Panel”). For MOSH in lip care products, an
MOSH content in adipose tissue would be 44 ppm (compared
oral exposure of 0.08–0.7 mg/kg bw per day was estimated in
to the 52.5 ppm in tissue fat).
a worst-case exposure scenario (see Chapter “Mineral oil
Multiple linear regression was used to identify independ-
hydrocarbons in cosmetic products” and BfR (2018)”). These
ent variables associated with the MOSH concentration in
exposure estimates are in line with the derived human
abdominal subcutaneous fat at the time of cesarean delivery
equivalent oral dose.
(Concin et al. 2011). Age, body mass index (BMI) prior to
In a biopsy study performed in Austria in 2005–2006,
pregnancy, and cosmetics use were significantly correlated
Concin et al. (2008) measured the MOSH concentrations in
with MOSH content, showing higher levels with increasing
subcutaneous fat and in the fat phase of breast milk sampled
from 144 women (19–47 years old). Subcutaneous fat was age, lower BMI, and the use of certain cosmetic products
removed from the abdominal wall during cesarean sections. (Concin et al. 2011). The observed negative correlation
Milk samples were collected on day 4 and day 20 after birth, between MOSH concentration in tissue fat and BMI (which is
at the end of breast-feeding sessions. MOSH chromatograms highly correlated with body fat mass) might be explained by
of day-4 milk and tissue fat were virtually identical. the fact that a given absolute amount of MOSH is distributed
Occasional deviations from the typical MOSH composition (or diluted) in a large volume of tissue fat. Three predictive
were observed, suggesting a local contamination most likely categories of cosmetics use were identified: hand cream and
caused by a mineral oil-containing nipple ointment (e.g. lipstick use in daily life, and sun cream use in the present
Vaseline). The data were approximately log-normally distrib- pregnancy (Concin et al. 2011).
uted. Subcutaneous fat had a median concentration of A re-analysis of the data was carried out to estimate the
52.5 ppm (range: 10–360 ppm, N ¼ 142 subjects) (Figure 11). effect sizes for age and cosmetics use (RP et al., unpublished
The median concentration in milk fat was 30 ppm in day-4 data). The MOSH levels in tissue fat of the pregnant women
milk (range: 10–355 ppm) and 10 ppm in day-20 milk (range: exhibited a 1.5-fold increase per decade. A similar effect size
<5–285 ppm) (Figure 11). A decrease over the breast-feeding (1.2-fold increase per decade) was obtained for the MOSH
period was also observed in another study performed in content in adipose tissue of the autopsied subjects. The
Switzerland around 2002 (Noti et al. 2003): the median con- same increase (1.2-fold per decade) can be calculated from
centration in milk fat decreased from 54 ppm on day 3–5 the mean age and median MOSH level in adipose tissue of
(range: 25–190 ppm, N ¼ 7 subjects) to 6 ppm (range: pregnant women (31 years, 44 ppm) and autopsy patients
<3–66 ppm, N ¼ 14 subjects) during month 1–13. This study (67 years, 87 ppm).
 CRITICAL REVIEWS IN TOXICOLOGY 759

To determine the effect size for cosmetics, the three previ- and females SD rats (Firriolo et al. 1995; Griffis et al. 2010)
ously identified categories of use were included in the re- were much less susceptible. There was also no evidence of
analysis (RP et al., unpublished data). Women, who indicated treatment-related increases in liver weight of L-E rats and
the use of items from all three product categories (i.e. hand beagle dogs receiving a lower-dose diet containing 0.1% of
cream, lipstick, and sunscreen), on average had a 2.1-fold different white mineral oils (Smith et al. 1995).
higher MOSH concentration in abdominal subcutaneous fat In MLNs of F-344 rats, oral exposure to mineral oils and
than non-users. This finding might suggest a dermal uptake waxes increased the incidence and severity of microgranulo-
of MOSH from cosmetic products. However, given the poor mas (Baldwin et al. 1992; Firriolo et al. 1995; Smith et al.
dermal absorption of MOSH (see Section “Dermal absorption 1996; Scotter et al. 2003; Griffis et al. 2010; McKee et al.
of MOSH”), the good intestinal absorption of low MOSH 2012). These are described as “discrete collections of macro-
doses via the absorption pathway for dietary lipids (see phages with medium-sized, vesicular nuclei and abundant
Section “Intestinal absorption of MOSH”), and the high MOSH eosinophilic, or sometimes faintly pigmented, granular to
levels in human MLNs (see also Section “Histopathological finely foamy cytoplasm”; “cell boundaries were indistinct”
lesions associated with the deposition of MOSH in human (Carlton et al. 2001). Such lipid-laden macrophages are also
tissues”), a relevant dermal uptake is not plausible. Instead, called foamy histiocytes (Bowdler 1990). In F-344 rats, micro-
an oral uptake is more likely, owing to direct ingestion (lip- granulomas “most commonly occurred in cortical and para-
stick material), hand-to-mouth contact, and the transfer of cortical zones, in or adjacent to subcapsular or cortical
MOSH from hands to food (see Chapter “Mineral oil hydrocar- sinuses” (Carlton et al. 2001) of MLNs. Histology indicates a
bons in cosmetic products”). microgranuloma associated histiocytosis (Smith et al. 1996;
To summarize, median MOSH levels in autopsy samples Scotter et al. 2003) which can be distinguished from a path-
(from 2013) were 166 ppm in MLNs, 87 ppm in adipose tissue, ology with a more diffuse and loose accumulation of macro-
71 ppm in liver, and 28 ppm in spleen. The high MOSH levels phages, located mostly in the medullary sinuses, which is
in MLNs as the first site of contact following intestinal lymph- called sinus histiocytosis (Carlton et al. 2001; Willard-
atic uptake suggest an uptake via the oral route. The MLNs Mack 2006).
and subcutaneous adipose tissue showed a similar MOSH The occurrence of microgranulomas in MLNs of female F-
composition and mass distribution, being different from the 344 rats was analyzed in six subchronic dietary studies
profiles obtained for liver and spleen. MOSH in these organs (Baldwin et al. 1992; Firriolo et al. 1995; Smith et al. 1996;
were characterized by a higher mass distribution and the vir- Scotter et al. 2003; Griffis et al. 2010; McKee et al. 2012). To
tual absence of n-alkanes, indicating that only a very distinct assess the effect size in the treatment groups, three different
and consistent fraction is retained from the total bioavailable measures were used (Figure 12). Baldwin et al. (1992) applied
material. A statistically significant, age-dependent increase in an incidence-severity rating scale with three levels. Firriolo
MOSH concentration was only found for the MLNs and adi- et al. (1995) determined the incidence (%) of microgranulo-
pose tissue, pointing to a lifelong accumulation in these tis- mas. Griffis et al. (2010) and others used an incidence-severity
sues. Women had, on average, a 2.2–2.5-fold higher MOSH score which is an aggregated measure calculated from inci-
concentration in liver, spleen, and adipose tissue compared dences and numerical severity grades (Figure 12).
to men. Across the different subchronic studies, the incidence and
severity of microgranoluma formation in MLNs of female F-
344 rats showed a clear dependence on dietary concentra-
Effects associated with the accumulation of MOSH tion and on the type of the test material (Figure 12).
in animal tissues Incidence and severity increased for lower molecular weight
The accumulation of MOSH in specific tissues of orally materials, including light white oil (P7H), certain low-viscosity
exposed F-344 rats was associated with increased organ mineral oils (N10A, N15H, P15H, N26A) and LMPW. Compared
weights, microgranuloma formation in MLNs, granulomatous to female F-344 rats, male F-344 rats (Baldwin et al. 1992;
inflammatory changes in liver, haematological changes, and Smith et al. 1996), other rat strains (Firriolo et al. 1995; Smith
clinical chemistry changes. et al. 1995; Griffis et al. 2010), and beagle dogs (Smith et al.
Light white oil (P7H) and certain low-viscosity mineral oils 1995) showed much less pronounced or no granulomatous
(N10A, N15H, P15H, P26A) caused pronounced dose-depend- alterations (Figure 12).
ent increases in organ weight. Female F-344 rats subchroni- There is no evidence from these subchronic studies, and
cally exposed to a diet containing 2% of one of these from chronic studies with medium- and high-viscosity white
materials showed increases up to 120–140% for liver, mineral oils (P70A/H, P70H, P100H) (Shoda et al. 1997;
110–200% for spleen, and 330–600% for the MLNs (Baldwin Trimmer et al. 2004), that the MLN microgranulomas in F-344
et al. 1992; Firriolo et al. 1995; Smith et al. 1996; Scotter et al. rats progress to more a severe pathology such as inflamma-
2003; McKee et al. 2012). The biggest weight gain occurred tory, necrotic or fibrotic lesions (Carlton et al. 2001; EFSA
in the MLNs, which represent the first site of contact follow- 2012; JECFA 2012). This is supported by findings of histiocy-
ing intestinal lymphatic uptake. LMPW induced even stronger tosis in MLNs of rats orally exposed to other high molecular
increases in organ weight: up to 110–180% for liver, weight, poorly soluble materials such as calcium lignosulfo-
140–230% for spleen, and 380–770% for the MLNs (Smith nate (Thiel et al. 2013), pentosan polysulphate (Abdo et al.
et al. 1996; Scotter et al. 2003; Griffis et al. 2010). Compared 2003), and copovidone (Mellert et al. 2004). The presence of
to female F-344 rats, male F-344 rats (Baldwin et al. 1992), foamy histiocytes in MLNs indicate concentration-dependent
760 R. PIROW ET AL.

Figure 12. Summary plot for the incidence and severity of granulomatous changes in the mesenteric lymph nodes (MLNs) and liver of rats and dogs after sub-
chronic exposure to highly refined mineral oils and waxes. A Cleveland dot plot (Sarkar 2008) was used to visualize the dietary concentrations of the tested mineral
oil/wax materials. The data of the control groups (C) are included. The different symbols relate to females (circles), males (diamonds), and both sexes (rectangles).
The coloring of the symbols according to the color key on top visualizes the incidence and severity of granulomatous changes. Depending on the study (see first-
author names and year at the right margin), different scales were used to classify or quantify effect magnitude: Three-level Incidence Severity Rating (ISR), Incidence
P Incidence Severity Score (ISS), and granulomas density. The ISS represents a mean score which, for example, was calculated in Griffis et al. (2010) as 100

(%),
(relative incidence
severity rating), where severity was rated as none, minimal, moderate or moderately-severe (score of 0, 1, 2, 3 or 4, respectively) (Griffis
et al. 2010). Depicted granuloma densities represent the medians of the treatment groups; individual density values ranged up to 800 granulomas per cm2. Data
were compiled from Baldwin et al. (1992), Firriolo et al. (1995), Smith et al. (1995, 1996), Scotter et al. (2003), Griffis et al. (2010), McKee et al. (2012), and Cravedi
et al. (2017). For the nomenclature of mineral oils and waxes, see legend of Figure 6. The exposure duration was 90 days in all studies except in Cravedi et al.
(2017) where animals were exposed for 120 days.

(overloaded) MOSH-lipid-laden, metabolically activated mac- certain mineral oils and waxes. Some studies subclassified
rophages which, according to the Joint FAO/WHO Expert these lesions by size as granulomas and microgranulomas
Committee on Food Additives (JECFA 2012), do not represent (Miller et al. 1996; Smith et al. 1996). The latter were defined
an adverse effect as such. The EFSA Panel on Contaminants as small collections of macrophages/histiocytes (three to five)
in the Food Chain (CONTAM Panel) considered that the pres- with a few number of lymphocytes in the periphery (Miller
ence of microgranulomas in MLNs is a nonspecific, adaptive et al. 1996; Smith et al. 1996). Granulomas predominantly
change of low toxicological concern. consist of aggregations of epithelioid macrophages, with or
Granulomatous lesions of variable size and morphology without the participation of other cell types (Baldwin et al.
occurred in the liver of F-344 rats due to the exposure to 1992; Firriolo et al. 1995; Fleming et al. 1998; Griffis et al.
 CRITICAL REVIEWS IN TOXICOLOGY 761

2010; Fleming and Carrillo 2018). Epithelioid macrophages Immunologically, there are two main types of granulomas:
are “enlarged macrophages with eosinophilic cytoplasm, immune granulomas caused by immunogenic agents or par-
indistinct cell borders, and vesicular eccentric nuclei, resem- ticles via T cell-mediated immune responses, and foreign
bling epithelial cells” (Fleming et al. 1998). Another descrip- body granulomas caused by non-immunogenic foreign
tion characterizes them as “macrophages with abundant material in the absence of T cell-mediated immune responses
cytoplasm and ill-defined cytoplasmic borders and generally (de Mello and Ferreira Alves 2018; Kumar et al. 2018).
round to oval vesicular nuclei which were more elongated in According to Adenuga et al. (2017), the lesions appear to
some cells” (Carlton et al. 2001). Depending on the severity occur as a result of the elicitation of a “foreign body”-type
of the lesion, these aggregates are variably enclosed by an immune response to MOSH in the liver of F-344 rats.
infiltrate of mononuclear inflammatory cells, mainly lympho- Alterations of haematological and clinical parameters asso-
cytes, may contain multinucleated Langhans-type giant cells, ciated with the histopathological lesions seen in F-344 rats
and occasionally exhibit necrosis and fibrosis (Baldwin et al. were treatment-related increases in white blood cell and neu-
1992; Firriolo et al. 1995; Smith et al. 1996; Fleming et al. trophil counts, and in serum levels of the liver enzymes ALT
1998; Scotter et al. 2003; Griffis et al. 2010). These aggregates (alanine aminotransferase), AST (aspartate aminotransferase),
are commonly called “epithelioid (cell) granulomas”. and GGT (gamma glutamyl transferase) (Baldwin et al. 1992;
The occurrence of granulomatous changes in the liver of Firriolo et al. 1995; Smith et al. 1996; Hoglen et al. 1998;
female F-344 rats was analyzed in seven subchronic dietary Griffis et al. 2010; McKee et al. 2012). This phenotype was
studies (Baldwin et al. 1992; Firriolo et al. 1995; Smith et al. judged by JECFA as indicative of a mild, chronic inflamma-
1996; Scotter et al. 2003; Griffis et al. 2010; McKee et al. 2012; tory reaction and a mild hepatocyte injury (JECFA 2012).
Cravedi et al. 2017). Granulomas were generally confined to The induction of inflammatory granuloma in the liver of F-
the highest dietary concentrations and mostly seen with low- 344 rats by low-viscosity mineral oils and (n-alkane-rich) par-
viscosity mineral oils (N10A, N15H, P15H, N26A, N69H, N70A, affin waxes is considered an adverse effect. Yet, the relevance
N70H) and paraffin waxes (LMPW, IMPW) (Figure 12). There to humans has been repeatedly questioned because of the
was no evidence for treatment-related granulomatous difference in morphology between the epithelioid cell granu-
changes from subchronic and chronic studies (Smith et al. lomas seen in F-344 rats and the lipogranulomas observed in
1996; Shoda et al. 1997; Scotter et al. 2003; Trimmer et al. humans in association with the ingestion of MOSH (Miller
2004) with medium- and high-viscosity white mineral oils et al. 1996; Smith et al. 1996; Fleming et al. 1998; Carlton
(P70A/H, P70H, P100H) and microcrystalline waxes (HMPW). et al. 2001; Trimmer et al. 2004; Griffis et al. 2010; Adenuga
Compared to female F-344 rats, male F-344 rats exhibited et al. 2017; Fleming and Carrillo 2018). In its opinion on low-
less severe granulomatous lesions (Baldwin et al. 1992; Smith and medium-viscosity mineral oils in 2012, JECFA noted that
et al. 1996). Such treatment-related changes were not the available data were not sufficient to conclude on the
observed in other rat strains (Shubik et al. 1962; Firriolo et al. relevance for humans of the effects seen in F-344 rats (JECFA
1995; Smith et al. 1995; Griffis et al. 2010) and beagle dogs 2012). Also, the CONTAM Panel in its 2012 opinion on min-
at all (Smith et al. 1995). eral oil hydrocarbons in food stated that information was
Histological examination under polarized light of fresh-fro- insufficient to consider the liver (micro)granulomas in the F-
zen liver sections from female F-344 rats being exposed to 344 rat as specific to this strain of rat (EFSA 2012). A recent
(n-alkane-rich) LMPW revealed the presence of birefringent analysis in terms of a mode of action/human relevance
material within hepatocytes, Kupffer cells (resident macro- framework (MoA/HRF) came to the conclusion that the MoA
phages), and some granulomas, being indicative of crystalloid underlying the formation of MOSH-induced epithelioid granu-
structures (Smith et al. 1996; Scotter et al. 2003). Further, lomas in the F-344 rat model is not relevant for humans
electron microscopy showed Kupffer cells to contain crystal- (Adenuga et al. 2017).
loid structures (Griffis et al. 2010) and large, irregularly
shaped vacuoles, being suggestive of the presence of phago-
cytozed foreign material (Hoglen et al. 1998). These observa- Histopathological lesions associated with the deposition
tions would be consistent with the hypothesized of MOSH in human tissues
crystallization of n-alkanes above C25 in liver of female F-344 MOSH-associated lesions in human tissues occur as oil drop-
rats (Barp et al. 2017b). An increased density of lymphoid cell lets. In microscopic images of paraffin-embedded tissue sec-
clusters in livers of female F-344 rats exposed to light-liquid tions, they appear as clear spaces, demarcating previous sites
paraffin oil (S-C25), paraffin wax-containing liquid paraffin oil of putative MOSH deposits that had been dissolved out dur-
(L-C25W), and LMPW was observed as an additional change ing histological processing (Carlton et al. 2001). Mainly
(Hoglen et al. 1998; Cravedi et al. 2017). affected are the abdominal lymph nodes, spleen, and liver.
The specific mechanism of the development of these epi- The deposits appear as focal collections of extra- and intra-
thelioid cell granulomas in the liver of F-344 rats is not well cellular vacuoles of markedly varying size (Stryker 1941;
understood (Fleming et al. 1998; Hoglen et al. 1998; Carlton Boitnott and Margolis 1966b; Rose and Liber 1966; Liber and
et al. 2001; Griffis et al. 2010; Adenuga et al. 2017). In prin- Rose 1967; Kelsall and Blackwell 1969; Boitnott and Margolis
ciple, granuloma formation is regarded as a cellular attempt 1970; Nochomovitz et al. 1975; Blewitt et al. 1977; Klatskin
to engulf an offending agent that is difficult to eradicate, 1977; Dincsoy et al. 1982; Cruickshank 1984; Cruickshank
such as a persistent microbe (e.g. M. tuberculosis) or a self- and Thomas 1984; Wanless and Geddie 1985; Fleming et al.
antigen in immune granulomas (Kumar et al. 2018). 1998; Carlton et al. 2001; Fleming and Carrillo 2018).
762 R. PIROW ET AL.

The intracellular vacuoles occur in lipophages (vacuolated 1966b; Rose and Liber 1966; Liber and Rose 1967; Boitnott
macrophages) and in multinucleated giant cells, which are and Margolis 1970; Nochomovitz et al. 1975; Blewitt et al.
occasionally present (Stryker 1941; Boitnott and Margolis 1977; Dincsoy et al. 1982). MS identified complex mixtures of
1966b; Kelsall and Blackwell 1969; Boitnott and Margolis predominantly branched and cyclic compounds, a compos-
1970; Fleming et al. 1998; Carlton et al. 2001; Fleming and ition typical of mineral oils (Boitnott and Margolis 1966b,
Carrillo 2018). Other associated inflammatory cells, such as 1970; Barp et al. 2014). GC–FID analysis of saturated hydro-
lymphocytes and plasma cells, are present in varying carbons isolated from histologically positive spleens revealed
amounts (Stryker 1941; Rose and Liber 1966; Kelsall and a largely unresolved hump ranging from C21 to C28 (Rose and
Blackwell 1969; Nochomovitz et al. 1975; Blewitt et al. 1977; Liber 1966; Liber and Rose 1967). A clear positive correlation
Klatskin 1977; Dincsoy et al. 1982; Cruickshank and Thomas was observed between the incidence and severity of histo-
1984; Wanless and Geddie 1985; Fleming et al. 1998; Fleming logical lesions and the MOSH concentration in the respective
and Carrillo 2018). Associated scarring fibrosis is rare and, if tissues (Figure 13) (Boitnott and Margolis 1966b; Liber and
present, usually of mild degree (Blewitt et al. 1977; Rose 1967; Boitnott and Margolis 1970; Cruickshank 1984).
Cruickshank and Thomas 1984; Fleming et al. 1998; Fleming Affected lymph nodes show lesions restricted to the cor-
and Carrillo 2018). All the above-mentioned components tex and medullary cords (Stryker 1941; Kelsall and Blackwell
form an aggregate, which is commonly called 1969), but also a scattered distribution of lesions throughout
“lipogranuloma” (Klatskin 1977; Dincsoy et al. 1982; the node has been described (Carlton et al. 2001). The lymph
Cruickshank 1984; Cruickshank and Thomas 1984; Wanless nodes mainly affected are those associated with the gastro-
and Geddie 1985; Fleming et al. 1998; Bellamy and Burt 2018; intestinal tract and the liver (Boitnott and Margolis 1966b;
Fleming and Carrillo 2018). Kelsall and Blackwell 1969; Nochomovitz et al. 1975;
The characteristic morphology and its correlation with Cruickshank and Thomas 1984). Hepatic lymph nodes receive
deposits of mineral oil was first discovered in postmortem tis- lymph fluid formed in the perisinusoidal spaces of Disse and
sues by Stryker (1941). Studies published later used speci- drained into the lymphatic vessels surrounding the portal tri-
mens from autopsies and biopsies performed between as ads (Ohtani and Ohtani 2008; Moore et al. 2014). Lymph
early as 1880 and 1980. Lipid histochemistry involving differ- nodes from non-abdominal sites (e.g. neck and axilla) were
ential staining of fresh-frozen tissue sections was applied in found to show only an infrequent and scanty involvement
an attempt to distinguish MOSH droplets from deposits of (Kelsall and Blackwell 1969; Cruickshank 1984). In hepatic
biological lipids (Stryker 1941; Boitnott and Margolis 1966a, lymph nodes obtained from 49 autopsies, oil droplets were
1966b; Liber and Rose 1967; Boitnott and Margolis 1970; virtually absent at estimated MOSH concentrations of less
Nochomovitz et al. 1975; Blewitt et al. 1977; Dincsoy et al. than 100 ppm but invariably present at levels greater than
1982). Chemical methods comprised the extraction of tissue 250 ppm (Figure 13) (Boitnott and Margolis 1966b). A high
lipids followed by identification and, sometimes, quantifica- incidence of hepatic lymph node lesions of 55–88% was
tion of saturated hydrocarbons using, e.g. molecular sieves, noted in autopsies (N ¼ 61–300) performed in North America
column and thin-layer chromatography, gravimetry, infrared and Australia in the 1960s and early 1970s (Boitnott and
spectroscopy, GC–FID, and MS (Boitnott and Margolis 1966a, Margolis 1966b; Kelsall and Blackwell 1969; Cruickshank and

Figure 13. Histological grading of oil droplet lesions in human autopsy material of lymph nodes, liver, and spleen. Left: Stacked bar chart showing the proportion
of histological grades in relation to the estimated MOSH content in hepatic lymph nodes. The MOSH content, originally reported as percent of the total lipid content
(Boitnott and Margolis 1966b), was estimated by assuming the total lipid content to be 5% of the tissue wet weight (Boitnott and Margolis 1970). Values within the
bars indicate the number of cases. Right: Violin and density scatter plot showing the distribution of MOSH concentration values in tissue conditional on the histo-
logical grading of the lesions in spleen and hepatic portal triads. Sample sizes (n) are indicated. Data were compiled from Boitnott and Margolis (1966b, 1970).
 CRITICAL REVIEWS IN TOXICOLOGY 763

Thomas 1984). For the mesenteric lymph nodes, somewhat lipogranulomas might even persist in subjects with resolved
lower incidences were reported (Stryker 1941; Kelsall and fatty liver disease (Zhu et al. 2010). Lipid histochemistry with
Blackwell 1969; Cruickshank and Thomas 1984). differential staining and chemical analysis of tissue lipid
Splenic lesions are generally located in or close to lymph- extracts are therefore crucial, but have not always been used
oid follicles and in the periarteriolar lymphoid sheaths to support the identification of MOSH-related lesions.
(Stryker 1941; Hudson and Robertson 1966; Liber and Rose In 42 autopsy livers, oil droplets were absent in the portal
1967; Boitnott and Margolis 1970; Nochomovitz et al. 1975; tracts at MOSH concentrations less than 380 ppm but invari-
Cruickshank 1984; Carlton et al. 2001). The pathology is also ably present at levels greater than 800 ppm (Figure 13)
described as “follicular lipidosis” (Rose and Liber 1966; Liber (Boitnott and Margolis 1970). MOSH concentration in liver
and Rose 1967; Cruickshank 1984; Cruickshank and Thomas was positively correlated with that in spleen (r ¼ 0.76, N ¼ 23)
1984). In 26 autopsy spleens, oil droplets were absent at (Boitnott and Margolis 1970). In North America, a high inci-
MOSH concentrations less than 200 ppm but invariably pre- dence (45 and 48%) of hepatic lesions was reported for aut-
sent at levels greater than 500 ppm (Figure 13) (Boitnott and opsy material (N ¼ 100 and 465) from the early to mid-1970s
Margolis 1970). A similar correlation was obtained in another (Cruickshank and Thomas 1984; Wanless and Geddie 1985); a
autopsy study: the six histologically negative spleens had three-fold lower incidence (16%) was reported for autopsy
MOSH concentrations of 60–590 ppm whereas seven positives material (N ¼ 25) from the 1940s (Stryker 1941), which is con-
had levels of 270 ppm and 1120–2660 ppm, respectively sistent with the historical trend observed for the splenic
(Rose and Liber 1966; Liber and Rose 1967). In North lesions. A strong association was found between the pres-
America, a high incidence of 46–76% was reported for aut- ence of lipogranulomas in liver and spleen of the same cases
opsy material (N ¼ 100–465) from the 1960s and 1970s (Liber (Wanless and Geddie 1985). In 824 consecutive liver biopsies
and Rose 1967; Cruickshank and Thomas 1984; Wanless and performed in North America in 1978–1980, a considerably
Geddie 1985); three-fold lower incidences (18–24%) were lower incidence (4.6%) of lipogranulomas in non-fatty livers
observed for the 1940s (Stryker 1941; Cruickshank 1984). This was reported (Dincsoy et al. 1982). A review of 240 biopsies
historical trend can be traced back further: the incidence was carried out at the same center in the early 1950s showed an
zero in 1882–1937 (N ¼ 115) compared to 33% in 1938–1956 incidence of 1.7% (Dincsoy et al. 1982), again indicating a his-
(N ¼ 70) (Liber and Rose 1967). Epidemiological analyses of torical trend.
autopsy material from the 1970s identified a higher incidence The only recent study is that of Barp et al. (2014). Liver
and severity of splenic lesions in men than in women and other tissues were collected from 37 subjects during aut-
(Cruickshank 1984; Wanless and Geddie 1985). A histologic opsy performed in Austria in 2013. MOSH concentrations
study of spleens from autopsies (N ¼ 50 per center) per- were in the range of 14–901 ppm for liver and 6–1400 ppm
formed in 74 centers of 41 countries showed a marked geo- for spleen (Barp et al. 2014). The LC–GC–FID chromatograms
graphic variation: The incidences were 50% in North America for both tissues showed MOSH humps that were completely
and Australasia, 30% in Great Britain, and 0–11% in continen- devoid of skinny peaks indicative of n-alkanes (Barp et al.
tal Europe countries (Cruickshank 1984). The study authors 2014). In a later publication Barp and others indicated that
explained this variation by the ingestion of MOSH from sour- “virtually no granulomas were observed” in both tissues
ces related to the packaging and protective coating of food- (Barp et al. 2017b). The absence of MOSH-associable lesions
stuffs in the more affluent societies (Cruickshank 1984). in these autopsy patients could result from a considerably
MOSH-related hepatic lesions tend to occur in the portal lower dietary exposure in post-2000 Europe compared to
fields rather than in the parenchyma. They are usually found pre-2000 North America. In the U.S., the average exposure to
in the pericentral region, i.e. located around the central vein mineral oils and waxes from food-use applications was esti-
of the liver lobule (Stryker 1941; Boitnott and Margolis 1970; mated to be approximately 2 mg/kg bw per day in the 1960s
Nochomovitz et al. 1975; Blewitt et al. 1977; Dincsoy et al. (Boitnott and Margolis 1966b) and 0.875 mg/kg bw per day
1982; Cruickshank and Thomas 1984; Wanless and Geddie in 2002 (Heimbach et al. 2002). These intakes are 1–2 orders
1985; Trivalle et al. 1991; Fleming et al. 1998; Carlton et al. of magnitude higher than the mean background exposure to
2001; Bellamy and Burt 2018; Fleming and Carrillo 2018). This MOSH which was estimated by EFSA using European food
differential zonal involvement is, however, not consistent contamination data, most of which were collected in
across studies (Dincsoy et al. 1982; Wanless and Geddie 1997–2010 (EFSA 2012).
1985). Reversibility of MOSH retention as well as a different In conclusion, MOSH-related lipogranulomas in human liv-
persistence potential between severe and mild lesions (e.g. ers are morphologically quite different from inflammatory
regression of smaller oil droplets) following reduction in epithelioid granulomas seen in F-344 rats following the oral
exposure might be additional explanatory factors to those exposure to low-viscosity mineral oils and (n-alkane-rich) par-
already discussed elsewhere (Wanless and Geddie 1985). affin waxes (Miller et al. 1996; Smith et al. 1996; Fleming
What complicates the identification of MOSH-related lesions et al. 1998; Carlton et al. 2001; Fleming and Carrillo 2018).
is that morphologically similar changes may result from the The lesions in human livers and other affected tissues are
accumulation of biological lipids, mainly triglycerides, in stea- generally regarded as incidental findings with no clinical sig-
totic livers of patients with, e.g. fatty liver disease or hepatitis nificance (Stryker 1941; Klatskin 1977; Dincsoy et al. 1982;
C infection (Delladetsima et al. 1987; Denk et al. 1994; Zhu Cruickshank and Thomas 1984; Wanless and Geddie 1985;
et al. 2010; Coash et al. 2012; Bellamy and Burt 2018; de Fleming et al. 1998; Carlton et al. 2001; Bellamy and Burt
Mello and Ferreira Alves 2018). Some fat-related 2018; Fleming and Carrillo 2018). The EFSA CONTAM Panel in
764 R. PIROW ET AL.

its 2012 opinion noted that “the clinical significance of the and Barrowman 1987; Barrowman et al. 1989), but also for
lipogranulomas is not known, but their presence had not di-isopropylated naphthalene (Kojima et al. 1978).
been associated with inflammatory responses, and not Upon dermal exposure, PAHs are well absorbed and
reported to be associated with clinical abnormalities” (EFSA become systemically available. When reaching the viable epi-
2012). The Panel concluded that “whilst dietary exposure to dermis, PAHs can already be converted into a variety of
mineral oil hydrocarbons could result in the formation of metabolites such as phenols (hydroxy derivatives), dihydro-
lipogranulomas in the liver and other tissues of humans, the diols (diols), quinones (diones), and catechols (Pannatier et al.
current incidence is very low and that these do not appear 1978; Ng et al. 1991, 1992; Jacques et al. 2010; Brinkmann
to have any adverse consequences”. et al. 2013). These derivatives can be further converted into
While a general agreement has been reached on the non- glucuronide and sulfate conjugates (Pannatier et al. 1978; Ng
adversity of MOSH-related lipogranulomas in humans, this is et al. 1992; Jacques et al. 2010). The extent of percutaneous
not the case for the human relevance of the epithelioid cell absorption of PAHs is negatively related to their molecular
granulomas seen in the F-344 rat model (EFSA 2012; JECFA size and hydrophobicity (Vanrooij et al. 1995; Roy et al. 1998;
2012; Adenuga et al. 2017). To solve this issue, the question Gute et al. 1999; Sartorelli et al. 1999; Moss et al. 2002; Beriro
has been raised whether there could be an atypical response et al. 2016). Dermal absorption is also influenced by extrinsic
to MOSH in humans resulting in granulomas of similar factors such as PAH concentration in the vehicle, the amount
morphology to those in F-344 rats (Fleming et al. 1998; of formulation applied per skin area, the viscosity of the
Fleming and Carrillo 2018). This led to the question whether vehicle, the stratum corneum/vehicle partition coefficient
some/all of the idiopathic (unexplained) granulomas in (Dankovic et al. 1989; Sartorelli et al. 1999), and the anatom-
human biopsies could be atypical reactions to MOSH. There ical site of application (Vanrooij et al. 1993). In vitro and
is no evidence that this is the case. But even if it were, a in vivo experiments with different mineral oil products spiked
worst-case scenario suggests that the implied numbers of with radiolabelled benzo[a]pyrene (B[a]P) showed a reduced
cases across the EU are low and a progression to significant percutaneous absorption of B[a]P with increasing viscosity of
liver disease is unlikely (Fleming and Carrillo 2018). the mineral oil product (Roy et al. 1996; Potter et al. 1999).

MOAH Carcinogenicity of PAHs and MOAH

Several PAHs with four to six aromatic rings, including
Gastrointestinal and dermal absorption of MOAH methylated derivatives, are carcinogenic in animal studies
and PAHs (JECFA 2006; EFSA 2008; IARC 2010). Mono- and polymethyla-
Data on the gastrointestinal and dermal absorption of complex tions can convert non-carcinogenic PAHs (e.g. pyrene, anthra-
mixtures of highly alkylated MOAH are virtually non-existent. cene) into genotoxic carcinogens, depending on the
Given that MOAH are structurally and physico-chemically methylation position (LaVoie et al. 1985; Rice et al. 1987;
related to PAHs and MOSH, and given that MOAH occur asso- Surh et al. 1990). Conversely, a higher degree of alkylation
ciated with MOSH in petroleum products, it is likely that they abrogates carcinogenicity of certain PAHs if the alkylated
are absorbed to comparable degrees. Upon absorption MOAH derivatives become directed to non-toxifying metabolic path-
are likely being metabolized (oxidized) as they could not be ways (! side-chain oxidation instead of ring oxidation).
detected in human tissues that are burdened by high levels of Isopropylation of naphthalene is an example (Honda et al.
MOSH (Barp et al. 2014). The absorption of MOSH has already 1990; Ho €ke and Zellerhoff 1998). Bulky ring substitutions can
been reviewed in the previous sections. This section comple- also eliminate carcinogenicity by preventing the intercalation
ments the picture with information on PAHs. of activated derivatives into DNA. Apart from alkylation, par-
Following oral ingestion, PAHs (including simple methy- tial hydrogenation can also modulate the tumorigenic activity
lated compounds) are absorbed in the gastrointestinal tract of PAHs (Lijinsky et al. 1965; Lijinsky et al. 1970; Lijinsky and
via the lymphatic system and portal vein, then metabolized, Garcia 1972). In general, however, it is not predictable how
and finally excreted (Laher et al. 1984; Laher and Barrowman alkylation and partial hydrogenation affect the carcinogenic
1987; Lindstrom et al. 1987; Laher and Barrowman 1988; potential of parent PAHs.
Laurent et al. 2002; Cavret et al. 2003; Ramesh et al. 2004; For mineral oils, products with a high concentration of
Harris et al. 2013; Harris et al. 2016). PAHs with two or three polycyclic aromatic compounds (PAC) with 3–7 rings (parent
aromatic rings can be absorbed more rapidly and extensively PAHs, short-alkylated MOAH, compounds containing heteroa-
than those with five or six rings (Rahman et al. 1986; Laurent toms), such as unrefined and poorly refined base oils, were
et al. 2002; Cavret et al. 2003). The transfer into circulation carcinogenic in mouse skin painting assays (Roy et al. 1988;
occurs concurrently with dietary lipids (Stavric and Klassen McKee, Daughtrey, et al. 1989; Blackburn et al. 1996;
1994; Harris et al. 2016). Co-administration of long-chain diet- Mackerer et al. 2003). The aromatics concentration was deter-
ary lipids and bile salts significantly facilitates the absorption mined by target analytics for specific parent PAHs (McKee,
of the larger, more lipophilic PAHs (Laher et al. 1983; Daughtrey, et al. 1989; Concawe 1994) and by solvent extrac-
Rahman et al. 1986; Barrowman et al. 1989). Bioavailability tion with dimethyl sulfoxide (DMSO) followed by GC separ-
data exist for some simple substituted compounds such as ation according to the ring number using two to seven ring
mono- and di-methylated naphthalene and di-methylated unsubstituted PAHs as internal standards (Roy et al. 1988;
benzanthracene (Laher et al. 1983; Rahman et al. 1986; Laher Mackerer et al. 2003; Gray et al. 2013). The latter technique is
 CRITICAL REVIEWS IN TOXICOLOGY 765

used by the Mobil PAC Method for predicting dermal car- 50 lg/kg is specified for B[a]P. MOAH (total aromatics) are
cinogenicity (see below). not addressed in these purity specifications but are still pre-
The mouse skin painting bioassay is used as an indicator sent in these products. Based on available data, the ANS
assay for carcinogenicity. It is performed in two different Panel concluded that there is no concern for genotoxicity of
experimental designs, as a chronic or as an initiation/promo- these products. In addition, no carcinogenic effect was
tion (I/P) bioassay. Mineral oils (C15–C50) were mostly tested observed in chronic dietary studies with HVMO and MVMO in
in chronic bioassays (see below), with the exception of one F-344 rats (Shoda et al. 1997; Trimmer et al. 2004), and with
highly solvent-refined mineral oil stream, which was tested in paraffin and microcrystalline waxes in SD rats (Shubik
both kinds of protocols (Skisak et al. 1994). Substances of et al. 1962).
other petroleum-derived categories with lower carbon num- There is sufficient evidence from occupational epidemio-
ber ranges were tested as well, including kerosenes (C9–C16) logical studies that dermal exposure to untreated and mildly
such as jet fuel, gas oils (C9–C30) such as diesel fuel, and treated mineral oils causes skin cancer in humans (IARC
hydrocarbon solvents (C5–C20) (Clark et al. 2013; McKee and 2012). The current gold standard for determining the dermal
White 2014; McKee et al. 2015). These substance mixtures carcinogenicity of base oils is the chronic mouse skin-paint-
represent, together with mineral oils, relevant sources of ing bioassay (Chasey and McKee 1993; Concawe 1994;
food contamination (see Chapter “Evaluation of mineral oil Blackburn et al. 1996; Mackerer et al. 2003), which was
hydrocarbons in food by the EFSA CONTAM Panel”) so that designed to mimic the adverse effects seen in humans. For
they are briefly discussed below with respect to their carcino- routine use, the petroleum industry has developed quicker
genic properties. alternatives based on in vitro bioassays and chemical proce-
Some gas oils contain aromatic constituents with more dures for predicting dermal carcinogenicity. These are the
than three rings in toxicologically relevant concentrations modified Ames test (Mackerer et al. 2003), the Mobil PAC
which induce skin tumors in mice via genotoxicity (Clark Method (Roy et al. 1988; Roy et al. 1995; Mackerer et al.
et al. 2013; McKee and White 2014). In addition, kerosenes, 2003), and the IP 346 method (Institute of Petroleum 2004).
gas oils, and hydrocarbon solvents can produce skin tumors. Of regulatory relevance in the EU is the IP 346, which is a
These tumors appear to be caused by a promotional process gravimetric method that determines the weight percentage
secondary to repeated dermal irritation as they are not muta- of DMSO extractables (e.g. PAC) in the oil (Chasey and McKee
genic and thus unable to act as cancer initiating agents 1993; Mackerer et al. 2003). The two-step solvent extraction
(McKee, Plutnick, et al. 1989; Nessel et al. 1998; Nessel et al. protocol enables a selective enrichment of fused aromatic
1999). This skin irritation-based promotion effect seems to ring systems with three to seven rings. This is due to the
apply to alkylbenzene mixtures such as C10–C15 (predomin- affinity of the sulfur atom of DMSO to the p-electron-rich aro-
antly C12) branched-chain alkyl benzene (i.e. dodecylbenzene) matic ring systems (Natusch and Tomkins 1978). The extrac-
as well (Iversen 1989; Assogne 1990; Ingram and Grasso tion efficiency decreases with decreasing size and increasing
1991; European Chemicals Bureau 1999). It also seems to extent of alkylation of the aromatic ring systems,
apply to a jute batching oil variety (JBO-P) (Mehrotra et al. respectively.
1988), a straight-run unrefined broad cut from the atmos- According to Concawe, a division of the European
pheric crude distillation unit that includes constituents of Petroleum Refiners Association, the IP 346 method specifically
both kerosene and base oil (Agarwal et al. 1985; Agarwal applies to unused lubricating base oils with at least 95% of
et al. 1986; Chaudhuri 2011). This JBO-P additionally carries components with a boiling point beyond 300  C (Concawe
cancer initiating activity due to the presence of a fraction of 2016). The method has been used in the EU for the classifica-
PAHs with more than three rings (Agarwal et al. 1988; tion of base oils as carcinogens. Under the CLP Regulation
Mehrotra et al. 1988). (EC) No 1272/2008, the members of three categories of pet-
The following paragraphs are focused on mineral oils and roleum substances (i.e. other lubricant base oils, foots oils,
waxes. The carcinogenic potential of mineral oils and waxes treated distillate aromatic extracts) (Concawe 2001) are con-
has been assessed in the food safety area for the oral expos- sidered carcinogenic (category 1B) unless it can be shown
ure route and in the area of industrial chemicals for the der- that the substance contains less than 3% (w/w) DMSO
mal exposure route. extractable material.
The Panel on Food Additives and Nutrient Sources added The applicability of the IP 346 for predicting the dermal
to Food (ANS Panel) of the European Food Safety Authority carcinogenicity of certain base oils is based on the positive
(EFSA) evaluated the safety of certain mineral hydrocarbon correlation between the DMSO extractable material and the
products for their use as food additives, including certain skin cancer incidence observed in more than 100 mouse
medium viscosity white mineral oils (MVMO) (EFSA 2013b) skin-painting studies performed by the petroleum industry in
and high viscosity white mineral oils (HVMO) (EFSA 2009), as the 1970s and 1980s (Concawe 1994). The data base describ-
well as a microcrystalline wax (E 905) (EFSA 2013a). The phys- ing this relationship includes samples of substances from five
ical specifications of these products are given in Table 3. petroleum substance categories (Concawe 2016): highly
Purity specifications were set in order to limit the residual refined base oils (HRBO, the white oils), other lubricant base
amounts of PAHs in these products. For MVMO and HVMO, oils (OLBO), unrefined/acid-treated oils, treated distillate aro-
the purity specifications stipulate that the UV absorbance of matic extracts, and untreated distillate aromatic extracts
a DMSO extract of the white mineral oil is not higher than (Figure 14). The data base also includes blends thereof. HRBO
that of a reference. For microcrystalline wax, a limit level of and OLBO are commonly known as “mineral oils”. Test
766 R. PIROW ET AL.

additional negative controls (Doak et al. 1985; Blackburn

et al. 1986; McKee, Scala, et al. 1990).
The dermal carcinogenicity studies were conducted with
two mouse strains of different sex, female CF1 mice and
male C3H mice. Dose-response profiles of these mice demon-
strate a somewhat different responsiveness (Figure 14). The
data for C3H mice showed a pronounced scattering around
the steep part of the fitted dose response curve (Figure
14(B)). This suggests that additional factors (e.g. PAH profile,
different treatment regimens) influence the tumorigenic
response of samples with elevated levels of DMSO extract-
able material. Unfortunately, incomplete information on test
conditions and on physical and chemical data prevents a
statistically based analysis of additional predictors.
In the respective studies an oil sample was judged (poten-
tially) carcinogenic if at least two out of 50 test animals
(4%) developed skin tumors of benign or malignant nature
(Blackburn et al. 1986; Concawe 1994). The background
tumor rate in negative controls treated with highly refined
mineral oil was well below 1%. An analysis of negative con-
trols of three carcinogenesis programs revealed that follow-
ing treatment with highly refined mineral oil two out of 610
C3H mice (¼0.3%) developed benign papillomas and none
developed carcinomas (McKee, Nicolich, et al. 1990). The
same order of magnitude was found when the relationship
between skin cancer incidence and DMSO extractable con-
tent was modeled. For CF1 and C3H mice this results in com-
parable background tumor rates of 0.19 and 0.12%,
respectively (Figure 14). Similarly, rates of 0.2% and <0.5%
(none out of 200) were reported for shaved and untreated
controls (Doak et al. 1983; Blackburn et al. 1986). Toluene-
Figure 14. Correlation between skin cancer incidence (%) in CF1 (A) and C3H treated vehicle controls showed tumor rates of 0.6% (2 out
(B) mice and the DMSO extractable content (%) of dermally applied mineral oil of 350 animals) (Blackburn et al. 1986).
samples. Symbol types indicate the different petroleum substance categories
and blends thereof. Data were taken from Concawe (1994) and were fitted by a Compared to these background rates the selected carcino-
three-parameter log-logistic model with the upper asymptote fixed to 100% genic threshold of 4% might appear high. However, given
(Ritz et al. 2015) (solid curves). The dashed horizontal lines and associated num- the typical sample size of 50 animals per group this value is
bers indicate the lower asymptotes of the fits, providing estimates of the back-
ground tumor level in CF1 and C3H mice. The threshold for carcinogenicity statistically well justified as the following example illustrates.
classification at 3% (w/w) DMSO extractable material is indicated by a dot- If 5 out of 50 animals (i.e. 10%) were to develop a tumor in
ted line. the treatment group and none of the animals in the control
group, the one-sided Fisher’s exact test results in a p-value of
0.028 and thus well below the common significance level of
0.05. Hence a threshold of 4% (i.e. 2 of 50 animals), instead
materials were applied undiluted in a defined volume to the
of 10%, can be deemed conservative. Alternatively one could
shaved backs of the animals 1–3 times per week (Doak et al.
refer to the tumor rate of the historical controls as a set
1983; Blackburn et al. 1984; Doak et al. 1985; Blackburn et al.
background. With a set background of 0.2% and a sampling
1986; Roy et al. 1988; McKee, Daughtrey, et al. 1989; McKee,
size of 50 animals, 4% is then the minimum tumor rate which
Nicolich, et al. 1990; Chasey and McKee 1993; Concawe
can be statistically resolved by a one-sided exact binomial
1994). In some cases, test materials were diluted in highly
test and a significance level of 0.05. Discussions of this value
refined mineral oil or toluene for reasons of high toxicity or by Concawe with the EU Technical Progress Committee
high viscosity (Blackburn et al. 1986; McKee, Daughtrey, et al. Classification and Labeling working group and the IARC,
1989; McKee, Nicolich, et al. 1990). Only some studies respectively, found this 4% classification threshold “to be
reported the size of the dermal treatment area (McKee, valid, although possibly severe” (Concawe 1994).
Daughtrey, et al. 1989; Chasey and McKee 1993). White min- To determine the threshold for the DMSO extractable con-
eral oil and toluene were reported to be used as negative/ tent that gives the most accurate discrimination between
vehicle controls, and B[a]P (in toluene or acetone) and cata- potentially carcinogenic and non-carcinogenic base oils,
lytically cracked clarified oil as positive controls (Doak et al. Concawe applied a binary classification analysis to a data set
1985; Blackburn et al. 1986; McKee, Daughtrey, et al. 1989; of 104 samples using decision thresholds (or cutoffs) of 1, 2,
McKee, Nicolich, et al. 1990; McKee, Scala, et al. 1990). and 3% (w/w) of DMSO extract (Concawe 1994). The data set
Shaved but untreated animals were reported to be used as included samples of substances from five petroleum
 CRITICAL REVIEWS IN TOXICOLOGY 767

substance categories and blends thereof (Figure 14). The cut- mouse skin painting bioassay. The IP 346 is used as a quasi-
off of 3% (w/w) DMSO extractables yielded the highest accur- analytical tool to predict the carcinogenicity of mineral oils.
acy (¼94%). There were three false negative predictions and Since its introduction, it developed into a key indicator
three false positives which resulted in a sensitivity of 92% method applied in carcinogenicity classification of certain
and a specificity of 96%. In 2016, Concawe reviewed the data petroleum stream categories under the CLP regulation. It is
and included 29 new study results on substances within the also used as a qualifier for refining efficiency in the manufac-
chemical scope of the IP 346 (Concawe 2016). The re-analysis turing of mineral oils and waxes. For highly refined white
of the enlarged data set of 133 samples yielded a virtually mineral oils and waxes, there are no indications from animal
identical sensitivity of 93% and a slightly lower specificity studies for and no epidemiological evidence of
of 91%. carcinogenicity.
For mineral oil, it is particularly the performance character-
istics of the 92 tested base oils in the HRBO and OLBO cate-
gories which are relevant (Concawe 2016). There were three Evaluation of the inhalation toxicity of
false negative predictions and seven false positive ones. The pharmaceutical white oil by the german
false negatives had a tumor incidence of 5–10% and MAK commission
1.77–2.82% (w/w) of DMSO extractable content (Figure 14(B)).
The Permanent Senate Commission for the Investigation of
The sensitivity was 86% and the specificity 90%. The sensitiv-
ity describes the ability of the IP 346 to positively identify Health Hazards of Chemical Compounds in the Work Area
base oils that are also tested carcinogenic in the mouse skin (MAK Commission) proposes, inter alia, maximum workplace
test. The alignment of the two tests is crucial for the applic- concentrations (MAK values) for volatile chemicals and dusts
ability of the IP 346 for safety testing. Of equivalent import- in the air. These proposals are published in the annual list of
ance, at least for consumer safety, is the negative predictive MAK values. In 2015, the MAK Commission published its
value (NPV). The NPV describes the probability that a base oil evaluation of pharmaceutical white oil and proposed a MAK
being predicted to be non-carcinogenic by the IP 346 also is value for the inhalation exposure of workers to aerosols of
non-carcinogenic in the mouse skin test. The NPV can be cal- such oils (MAK 2015).
culated from sensitivity, specificity, and prevalence, the latter White mineral oil (CAS no. 8042-47-5) is defined as a
being the proportion of base oils that are (or would be) car- “highly refined petroleum mineral oil consisting of a complex
cinogenic in mice. The prevalence among the 92 base oils in combination of saturated hydrocarbons (carbon numbers pre-
the aforementioned data set was 21%, which translates into dominantly in the range of C15–C50) obtained from the inten-
a NPV of 95% (Concawe 2016). It should be noted that for sive treatment of a petroleum fraction with sulfuric acid and
commercial products the NPV would rather expected to be oleum, or by hydrogenation, or by a combination of hydro-
higher, as the prevalence amongst these oils is likely to be genation and acid treatment” (ECHA 2017c). Additional wash-
lower than 21%. The reason is that a large part of the ing and treating steps may be included in the processing
Concawe database originates from one company’s samples, operation. Compared to technical white oil, which may con-
most of which were prepared with laboratory pilot units with tain traces of potentially carcinogenic aromatic hydrocarbons,
the tumorigenic oils being specifically refined by experimen- pharmaceutical white (mineral) oil is more thoroughly refined
tal conditions that are, according to Chasey and McKee and has to comply with the European Pharmacopeia (see
(1993), not representative of those used in commer- Chapter “Mineral oils in medicinal products”). Pharmaceutical
cial refineries. white oil is for example used as a laxative, as a base for
In addition to the assessment of carcinogenicity under the ointments, creams, hair care products, and baby oils.
CLP regulation, the IP 346 is used as a qualifier for refining
Furthermore, it serves as lubricant for machines used in the
efficiency. In case of mineral white oils and waxes used in
pharmaceutical, cosmetics, and food packaging industry.
food, pharmaceutical or cosmetic applications, the IP 346 is
Inhaled mineral oil deposited in the lungs is taken up by
applied to the solvent-extracted vacuum distillate (also called
macrophages and, presumably because of incomplete phago-
waxy raffinate) from which these substances originate. Only if
cytosis and/or biotransformation, can cause inflammation and
the feedstock passes the IP 346 test, it is subjected to further
microgranulomas through to fibrotic responses. This path-
processing such as dewaxing, de-oiling, hydro-treatment, acid
ology is called exogenous lipid pneumonia. For humans,
and clay treatment (Figure 1). According to industry repre-
sentatives, these processing steps are quite efficient and there are reports on exogenous lipid pneumonia after inhal-
result in a pronounced reduction of PAC and MOAH concen- ation or accidental aspiration of mineral oil products (Cullen
trations, DMSO extractable contents, and mutagenicity et al. 1981; Spickard and Hirschmann 1994; Bandla et al.
indexes (Jouanneau 2017; Sothmann and Su €dkamp 2017). 1999). Quantitative exposure data are lacking though. For the
The achievable reduction of MOAH depends on whether it is derivation of a MAK value, the MAK Commission followed
a white oil, a hard paraffin wax, or a microcrystalline wax. the same approach as the Scientific Committee on
Representative data for commercial products from different Occupational Exposure Limits (SCOEL 2011). Since inhalation
oil companies are not available. studies with pharmaceutical white oil were not available, four
In summary, mineral oil products with a high proportion animal inhalation studies with highly refined mineral oils as
of three to seven aromatic ring constituents, such as unre- well as with white oils of not exactly known composition and
fined and poorly refined base oils, are carcinogenic in the PAH content were used for deriving the MAK value.
768 R. PIROW ET AL.

The MAK Commission evaluated a 4-week study with contaminated batches of sunflower oil being imported into
hydro-treated and acid-washed white oil (60% paraffinic, “no the EU. The term “mineral oil hydrocarbons” was used by the
aromatics”) (Dalbey et al. 1991) and a 13-week study with CONTAM Panel in a broader sense, as it comprised not only
three highly refined mineral oils (cutting oil, gear oil, engine the entry into and contamination of food from mineral oil
oil) (Dalbey 2001). Both studies were conducted with SD rats. products (e.g. white oils, lubricants) but also the contamin-
Two chronic (6–26 months) studies with a white mineral oil ation from products belonging to other categories of petrol-
(25–30 carbon atoms, 95% naphthenic with 1–6 rings) eum-derived substances such as diesel fuel and hydrocarbon
(Wagner et al. 1964) and with a fiber-yarn preparation (70% solvents. The focus was particularly on hydrocarbons within
paraffinic mineral oil, 30% synthetic fiber adjuvants) plus the carbon number range of C10 to C50. Hydrocarbons with
1000 ppm acetone (fiber solvent) in the test atmosphere carbon numbers outside this range were not considered rele-
were also evaluated (Stula and Kwon 1978). These studies vant because of the high volatility of the smaller and the low
used SD rats, mongrel dogs, CF and CAF/JAX mice, rabbits, absorption of the larger compounds.
Syrian hamsters, and gerbils. The animals were exposed to a MOH occur in food as a result of intended uses of food-
respirable mineral oil aerosol with a mass median aero- grade products (e.g. as food additives, additives used in
dynamic diameter (MMAD) of approximately 1–2 mm. food-contact materials; see Table 2) and due to contamin-
Rats and dogs responded most sensitively in these studies. ation. Food becomes contaminated via the environment,
In the chronic studies at an aerosol concentration of 5 mg/ food processing, and migration from food packaging materi-
m3, these species showed oil-filled macrophages in the lung als such as paperboard (see Chapter “Activities of surveiil-
which, in the absence of any other toxicity, was considered lance and regulatory authorities on MOH in food and food
as a physiological reaction rather than an adverse effect, so contact materials”) and jute bags. Depending on the source,
that this concentration was regarded as the chronic NOAEC the composition of the contaminating MOH mixtures varies.
(No Observed Adverse Effect Concentration). Subacute and The CONTAM Panel based its exposure assessment on a
subchronic exposures of rats to higher aerosol concentrations set of 1455 data on MOH in food, which were provided by
resulted in the following effects: occurrence of oil-filled mac- four European countries. Most of the data were from
rophages (foam cells) in the alveoli, macrophage accumula- 1997–2000 and from 2008–2010. Sufficient data were avail-
tion in the alveoli and tracheobronchial lymph nodes, able only for the MOSH fraction. The MOAH fraction was spe-
cifically determined in few samples only, and it was
neutrophil infiltration in the alveoli, thickening of alveolar
estimated to be approximately in the range of 15–35% of the
walls, epithelial hyperplasia, increase of pulmonary hydroxy-
total MOH in the different food groups. The highest average
proline (collagen marker for fibrotic changes), and increased
background concentrations for MOSH were identified in con-
lung wet weight. The chronic exposure of rats and dogs to
fectionery (46 ppm), vegetable oils (41–45 ppm) and canned
100 mg/m3 resulted in an increase in alkaline phosphatase
fish (40 ppm).
(indication of inflammatory processes in the lung) and in the
Bread and rice were cases for which the MOSH concentra-
occurrence of microgranulomas and lipogranulomas in the
tion values followed a bimodal distribution. This was
lung. These granulomatous reactions were considered as
explained by a low background exposure (mean: 4 ppm)
adverse and therefore established a LOAEC (Lowest Observed
and a high extra exposure with mean values of 532 ppm
Adverse Effect Concentration). At the same aerosol concen-
(bread) and 997 ppm (rice). These high values were likely
tration, hamsters, mice, and gerbils only showed some oil-
caused by the use of food additives (E 905, E 905a; see
filled macrophages in the lung. The most sensitive mouse
Table 2) as release agents in bakeries and as spraying agents
strain (CAF/JAX mice) gave no indications of tumor promo-
in rice. These specific uses are not authorized in the EU today.
tion in the lung.
In dry food such as breakfast cereals, packaging material
Based on these results, the MAK Commission derived a based on recycled paper can form a major source of MOSH
MAK value of 5 mg/m3 for the respirable fraction (i.e. alveolar and MOAH. This is due to the transfer of volatile compounds
fraction) of aerosols of pharmaceutical white oil. Given the via the gas phase (evaporation and re-condensation). Data
large margin between the chronic NOAEC (5 mg/m3) and the from a German survey with samples collected in 2010
chronic LOAEC (100 mg/m3) as well as the NOAEC of 50 mg/ (Vollmer et al. 2011) revealed that among the dry food items
m3 in a subchronic study, and further considering that the packaged in recycled paperboard without barrier the highest
effect was attributed to a local overload of the lung, for average MOSH concentrations were detected in cream pud-
which the inter-individual differences were judged to be low, ding mix (32.4 ppm) and semolina (23.9 ppm).
and that there was no evidence for tumor promotion in the Based on the data for MOSH in food and on food con-
lung, no further safety factors were applied. sumption data (from 32 different dietary surveys carried out
in 22 European countries), average and high (95th percentile,
Evaluation of mineral oil hydrocarbons in food by P95) consumptions were estimated for different age groups
the EFSA CONTAM panel of the general population. Background levels of MOSH in
food resulted in chronic exposures between 0.03 (mean) and
In 2012 the EFSA Panel on Contaminants in the Food Chain 0.1 mg/kg bw per day (P95) for adults and the elderly.
(CONTAM) published a scientific opinion on mineral oil Children (3–10 years) had higher exposures of up to 0.17
hydrocarbons (MOH) in food (EFSA 2012). The initiation of (mean) and 0.32 mg/kg bw per day (P95). Specific scenarios
risk assessment was triggered by the detection of highly for the consumption of bread and rice with high MOSH levels
 CRITICAL REVIEWS IN TOXICOLOGY 769

resulted in a chronic exposure of up to 6.4 mg/kg bw per day petroleum-derived substances. Several food-grade mineral
for bread and 1.3 mg/kg bw per day for rice. In addition, oils and waxes, which very likely contained (but had not
MOSH from dry food packed in recycled paperboard without been tested for) MOAH, were not carcinogenic in chronic
barrier forms a relevant contribution to total diet- dietary studies (see Section “Carcinogenicity of PAHs and
ary exposure. MOAH”). No data are available for the dietary exposure to
Information on the toxicity of MOSH derives mainly from non-food grade mineral oils. Dermal and inhalation studies
rodent studies. A subchronic study in SD rats with gavage were therefore used instead. The carcinogenicity of petrol-
administration of de-aromatized hydrocarbon solvents (car- eum-derived materials appeared to be in general due to the
bon number range: C10–C13) resulted in moderate liver cell presence of PAC containing three or more aromatic rings.
hypertrophy (Adenuga et al. 2014) which, in the absence of The CONTAM Panel concluded that many MOAH with three
any pathological alterations, was considered by the CONTAM or more aromatic rings and little or no alkylation can be bio-
Panel to be an adaptive response and not an adverse effect. activated to genotoxic carcinogens. C10–C15 branched-chain
Subchronic studies with dietary exposure of F-344 rats to alkyl benzene, which was taken as a representative of highly
highly refined mineral oils and waxes showed that MOSH in alkylated MOAH, has a skin irritation-related promotion effect
the carbon number range C16–C40 can accumulate in tissue (see Section “Carcinogenicity of PAHs and MOAH”). Some
and may cause microgranulomas in the MLNs and the liver simple MOAH such as naphthalene (C10) are carcinogenic by
of this rat strain, in particular in females (see Sections “MOSH a non-genotoxic mode of action, involving cytotoxicity and a
levels in animal tissues” and “Effects associated with the proliferative response.
accumulation of MOSH in animal tissues”). MOSH-related lipo- The data situation for MOAH did not permit a hazard char-
granulomas had been observed in human liver, spleen and acterization for carcinogenicity. Given that MOAH in food ori-
lymph nodes, without clear association with any adverse ginate from different petroleum-derived substances and
effect (see Section “Histopathological lesions associated with sources, and given the lack of information on the carcinogen-
the deposition of MOSH in human tissues”). icity of non-food grade mineral oils by the oral route, the
The CONTAM Panel’s hazard characterization of MOSH CONTAM Panel considered the MOAH fraction as potentially
identified inflammatory liver microgranulomas in F-344 rats genotoxic and carcinogenic. Given further that the MOAH
as the critical adverse effect. Since none of the highly refined fraction can be up to 30–35% of the MOH present in food,
mineral oils and waxes tested in animal studies were the CONTAM Panel considered the dietary exposure to
regarded as representative of the human dietary exposure in MOAH of potential concern.
terms of carbon number distribution, the CONTAM Panel In summary, the CONTAM Panel considered the dietary
selected NOAEL values for the different tested grades as ref- exposure to MOSH (background exposure and exposures
erence points and applied a margin of exposure (MOE) associated with the use of white oils as release agents in
approach. For the background exposure scenario, the lowest bread and for spraying of grains) and MOAH to be of poten-
NOAEL of 19 mg/kg bw per day for the most potent grades tial concern.
(i.e. low and intermediate melting point waxes) (Smith et al. The CONTAM Panel recommended that future monitoring
1996) was selected. For the chronic high exposure scenarios should distinguish between MOSH and MOAH, and between
reflecting the consumption of highly contaminated bread subclasses of MOSH based on carbon numbers and chemical
and rice, the CONTAM Panel selected the NOAEL of 45 mg/kg structures. Moreover, migration from recycled paperboard
bw per day for a hydro-treated white oil (HTWO, similar to used for food packaging should be prevented by the inclu-
P70H) (Baldwin et al. 1992), because white oils, but not sion of a functional barrier into the packaging assembly.
waxes, were used as release and spraying agents in Regarding toxicology, the relevance to humans of the liver
these cases. microgranulomas seen in F-344 rats should be investi-
Risk characterization for MOSH under the background gated further.
exposure scenario yielded MOEs of 100–680 for average con- In order to fill data gaps in risk assessment, EFSA funded a
sumers. For high consumers under the same scenario, the project on bioaccumulation and toxicity of mineral oil hydro-
MOEs were lowest in toddlers and other children (MOE range: carbons in female F-344 rats. The results of this project were
59–110). For the chronic high exposure scenario involving published recently by Cravedi et al. (2017) and others (Barp
the exposure to high MOSH levels in bread, the MOEs were et al. 2017a, 2017b) (see Sections “Distribution, metabolism,
16–65 for average consumers and 7–32 for high consumers. and excretion of MOSH” and “MOSH in animal tissues”). This
For highly contaminated rice, the MOEs were 35–1900 for project focused, inter alia, on the accumulation of the different
average consumers and 12–200 for high consumers. The MOSH substructures and in particular on the impact of n-alka-
MOEs obtained in the high exposure scenarios (especially for nes on granuloma formation. It included a study arm with sub-
bread high in release agents) were in most/many cases chronic exposure to a diet containing a MOSH mixture
below 100, in some cases below 10, which were considered representative of the whole MOSH range to which humans are
to be of particular concern. exposed via the diet. This is important to mention since none
Concerning the carcinogenicity of MOAH, information was of the petroleum products tested in previous animal studies
derived from animal studies with PAHs (including methylated were regarded by the CONTAM Panel as representative of the
derivatives), C10–C15 (predominantly C12) branched-chain alkyl human background exposure via food. To nevertheless enable
benzene (i.e. dodecylbenzene), a partially hydrogenated aro- risk characterization under the background exposure scenario,
matic hydrocarbon (tetralin), mineral oils, and other the CONTAM Panel in its 2012 opinion had chosen a
770 R. PIROW ET AL.

pragmatic conservative approach by using the lowest NOAEL barrier function, whereas bags made of polyolefins turned
of those studies as reference point for the MOE calculation. out to be less effective for long-term storage.
The results of EFSA-funded project offer an opportunity for On March 15, 2017, the German Federal Ministry of Food
revisiting the reference point selection. An evaluation of the and Agriculture published a new draft version of the 22nd
study results by the CONTAM Panel is pending. ordinance amending the Consumer Goods Ordinance (the
“Mineral oil Ordinance”). Since for MOAH a carcinogenic
potential cannot be excluded, the transfer into food should
Activities of surveillance and regulatory authorities be reduced as far as possible by the introduction of func-
on MOH in food and food contact materials tional barriers if transfer is not prevented by other conditions.
In 2009, the Official Food Control Authority of the Swiss According to the draft ordinance the functionality of the bar-
Canton of Zu €rich reported on high contents of MOH in rier is defined by a specific detection limit for the transfer of
paperboard food packaging (BfR 2010a; Lorenzini et al. 2010). MOAH into food of 0.5 mg/kg food (BMEL 2017).
Their presence was attributed to MOH from offset printing In 2017, the Commission Recommendation (EU) 2017/84
inks and recycled fibers. It was found that hydrocarbons up on the monitoring of mineral oil hydrocarbons in food and in
to about C24 are sufficiently volatile for relevant migration materials and articles intended to come into contact with
into dry food. To minimize the transfer of MOH from recycled food was issued. The monitoring is aimed to a better under-
carton-board packaging into food, the German Federal standing of the relative presence of MOSH and MOAH in
Institute for Risk Assessment (BfR) in 2010 proposed the use food commodities. It should focus on the food categories
of fresh fiber cartons and internal bags with barrier effect as which have been addressed in the EFSA Opinion (EFSA 2012)
well as a reduction of MOH entering the paper recycling pro- as main contributors to dietary exposure and should also
cess (BfR 2010c). From today’s view, the former management cover food contact materials used for those products. It was
option has come into effect. originally planned to conduct the monitoring in 2017 and
To improve the data situation for the evaluation of MOH 2018, but this was hampered by the fact that the guidance
mixtures, the German Federal Ministry of Food, Agriculture developed by the EU Reference Laboratory for Food Contact
and Consumer Protection funded a project entitled “Degree Materials was not available until February 2019 (JRC 2019). It
of migration of undesirable substances from packaging mate- is to be expected that the results of the monitoring are used
rials made of recycled paper into food” (Vollmer et al. 2011). by the European Commission for a decision on regulatory
119 samples of dry foods intended for long-term storage at measures on MOH under the food contaminants law.
ambient temperature were collected from the German mar-
ket in 2010 (Vollmer et al. 2011; Harling et al. 2012). The sam-
Mineral oil hydrocarbons in cosmetic products
ples were analyzed for MOH in the packaging materials
(paperboard, internal bag) and the food at three time points Highly refined mineral oils and waxes (e.g. Paraffinum
in 2010 and 2011 (Harling et al. 2012). The maximum concen- liquidum and petrolatums) are found as ingredients in many
tration in food was 100 mg/kg for MOSH and 16 mg/kg for products available to consumers including cosmetics (Table
MOAH. Concentrations in the packed dry foods increased 2). Annex II of the Regulation (EC) No 1223/2009 on cosmetic
with increasing storage duration. products prohibits substances of other petroleum categories
The above-mentioned project had a special focus on the (Concawe 2001) including other lubricant base oils (OLBO),
volatile MOH fraction from C16 to C24 since transfer via the foots oils, slack waxes, petrolatums, treated distillate aromatic
gas phase is regarded as main route for the contamination of extracts, and other gas oils. The ban does not apply to e.g.
dry food by recycled paper and board. This range covers the petrolatums and slack waxes if the full refining history is
lower part of the carbon-number range of low-viscosity min- known and if it can be shown that the substance from which
eral oils of the JECFA class III (Table 3). For this class of oils, it is produced is not a carcinogen. The ban does also not
JECFA had specified a temporary acceptable daily intake apply to e.g. OLBO if they contain less than 3% (w/w) DMSO
(ADI) of 0–0.01 mg/kg bw per day (JECFA 2002), which was extractable material. Although mineral oils and waxes as
later withdrawn (JECFA 2012). In the project this reference ingredients of cosmetics in the European market place are
value was used to derive a specific migration limit (SML) of subject to regulation (Regulation [EC] No 1223/2009) requir-
0.6 mg/kg food for packaging materials (Vollmer et al. 2011). ing industry to demonstrate lack of carcinogenicity, recent
In 58% of the samples (excluding those packed in imperme- findings of MOAH in products have caused general concern
able internal bags of polyethylene terephthalate (PET) or alu- about potential risk from PAC that might remain entrained
minium), the transfer of MOSH exceeded the SML at the within refined mineral oils.
respective best-before date or after 16 months of storage Mineral oils, waxes and petrolatum as currently used in
(Harling et al. 2012). The MOH transferred into food com- cosmetic products are complex combinations of hydrocar-
monly contained 10–20% MOAH. In 58% of the samples the bons obtained through chemical refinement including distilla-
transfer of MOAH exceeded 1 mg/kg food. In products pack- tion, extraction, crystallization and hydrogenation from
aged in recycled cardboard without an internal bag acting as petroleum fractions (Figure 1). The carbon chain length in
a functional barrier, the extent of migration after prolonged these mixtures ranges from C18 to > C90 (Petry et al. 2017).
storage was 70–80% of the MOH originally contained in the Mineral oils serve diverse functions in cosmetics e.g. as
packaging. Internal bags made of PET provided a good vehicles, viscosity regulators and moisturizers, and are
 CRITICAL REVIEWS IN TOXICOLOGY 771

consequently found in various product groups (e.g. skin care formation of inflammatory lesions or granulomas in the liver
products, sunscreen, lipsticks, Vaseline and baby oil). or any other adverse effects.
Depending on the product type, the mineral oil content in Market analysis of mineral oil qualities in lipstick has
cosmetics may vary from low to nearly 100%. An example for shown that consumers may be exposed to non-compliant
the latter is petrolatum that was patented under the brand qualities. A more detailed analysis of MOSH revealed that a
name Vaseline by Robert Chesebrough in 1872 and has been high percentage of the products contained more than 5% of
marketed ever since. Petrolatums are blended from high hydrocarbons below C25 indicating that mineral oils of a
quality white oils and petroleum waxes (Health lower molecular mass range have been used. Female F-344
Canada 2016). rats exposed to low viscosity oils in their daily diet for
Compositional testing of dermally applied cosmetic prod- 90 days exhibited inflammatory liver granulomas which are
ucts that contain mineral oil indicated MOAH concentrations regarded as adverse by EFSA and JECFA (see Section “Effects
of up to 4.5%. Vaseline appeared to contain 1.7–5.0% MOAH; associated with the accumulation of MOSH in animal tissues”
although only four samples have been analyzed (BfR 2018; for details). Mineral oil-induced histological changes can also
references therein). Elevated MOAH levels in cosmetics occur in the human liver, noteworthily, with a different
appear to be correlated with the presence of petrolatum in a morphology designated as lipogranuloma. The induced effect
formulation (BfR, unpublished data). As part of petrolatum, has been considered clinically unimportant in humans
refined microcrystalline waxes are characterized by a seem- because there is no evidence of a concurrent inflammatory
ingly high MOAH content. This is due to residual one or two response (for details see Section “Histopathological lesions
aromatic and partially hydrogenated ring systems bearing associated with the deposition of MOSH in human tissues”).
long alkyl chains. These high molecular weight structures The amount of MOSH per day (up to 0.7 mg/kg bw per day)
contribute significantly to the weight percentage of MOAH. that a consumer accidentally ingests using a lipstick contrib-
According to industry, food-grade mineral microcrystalline utes roughly 10% to the ADI (BfR 2018).
waxes may contain up to 7.0% MOAH but nevertheless com- In summary, mineral oil-containing cosmetic products do
ply with medicinal quality standards in terms of PAC content not pose a health risk to the consumer when the ingredients
(Jouanneau 2017; Sothmann and Su €dkamp 2017). Medicinal
are of food and medicinal grade quality. Regarding MOAH
white oils (e.g. Paraffinum liquidum) typically retain less than occurring in cosmetic products it should also be kept in
0.1% of MOAH (Jouanneau 2017; Sothmann and mind that currently this fraction cannot be specifically corre-
Su€dkamp 2017).
lated either with biological activity nor adverse health effects
Testing of a subset of MOAH-containing samples of cos-
due to a lack of appropriate assays.
metic products indicated compliance with international purity
standards (Ph. Eur.). This finding is in line with a statement
published recently by industry representatives to only use Mineral oils in dermatology
mineral oils and waxes of pharmaceutical grade in European
cosmetic products (Petry et al. 2017). It should be noted that From the dermatological point of view, the pharmacological
this negotiated agreement has not been officially endorsed effect of mineral oil-based products such as petrolatum
yet by Cosmetics Europe (the European trade association for (see Chapter “Mineral oils in medicinal products”) is of pri-
the cosmetics and personal care industry) and in any case is mary importance for the health assessment of mineral oils in
not legally binding. cosmetic products. In the US, petrolatum is approved for
There are multiple lines of evidence that the dermal infection prophylaxis after dermatosurgery and for the main-
absorption of highly refined mineral oil and wax is restricted tenance therapy of atopic dermatitis (AD). There is mounting
to the stratum corneum (i.e. the outmost layer of the epider- evidence that PAHs play a critical role in mediating the
mis) indicating a negligible risk of bioavailability (see Section beneficial effect (see below). PAHs occur not only in mineral
“Dermal absorption of MOSH” for details). Long-term dermal oil-based products, they are highly enriched in coal tar also.
exposure studies with highly refined mineral oils and waxes Coal tar-based products are also used to treat AD and other
failed to produce any local or systemic toxicity including chronic skin diseases such as psoriasis. The following sum-
tumor formation (Shubik et al. 1962; Nash et al. 1996; mary of selected studies on petrolatum and coal tar consid-
Concawe 2016). ers the dermatological viewpoint.
Mineral oil-containing lip care may lead to an oral expos- Molecular responses induced by white petrolatum USP
ure of consumers. To ensure a high level of safety for this (United States Pharmacopeia) in the skin were studied in
product group, Cosmetics Europe has issued recommenda- patients with moderate AD and healthy subjects by using dif-
tions with regard to the quality standard of the respective ferent methods performed on control skin, petrolatum-
oils and waxes (Cosmetics Europe 2004, 2018). To comply occluded skin (48-h occlusion using a Finn chamber), and
with those recommendations, mineral oils must be of food- skin occluded by the Finn chamber only (Czarnowicki et al.
grade purity and fulfill the physico-chemical properties of 2016). Petrolatum-occluded skin showed an enhanced expres-
medium and high viscosity oils and microcrystalline waxes sion of antimicrobial peptides (AMPs), which are regulated by
for which ADI levels by EFSA (EFSA 2009, 2013b, 2013a) and the interleukin IL-17/IL-22 signaling pathway. Control experi-
JECFA (JECFA 2002, 2012) have been established. It should ments showed that the induction of AMPs was not attribut-
be stressed that in chronic feeding studies in female F-344 able to the occlusive effect itself. The application of
rats these highly refined mineral oils did not cause the petrolatum also induced the expression of key barrier
772 R. PIROW ET AL.

differentiation markers (filaggrin and loricrin), increased stra- repeated exposure of the scalp to coal tar-containing sham-
tum corneum thickness, and significantly reduced T-cell infil- poo resulted in the metabolic activation of hair follicle
trates in the setting of ‘‘normal-appearing’’ or non-lesional enzymes, which manifested themselves in vitro in DNA-bind-
AD skin, which is known to harbor barrier and immune ing experiments by an enhanced B[a]P-DNA adduct
defects. Among the non-occluded controls, non-lesional AD formation.
skin compared with non-AD skin displayed a disturbed kera- A US retrospective study on the incidence rates of skin
tinization (parakeratosis) and a reduced expression of key cancer was conducted in patients with psoriasis that had
barrier differentiation markers (filaggrin and loricrin). received an initial Goeckerman therapy (combination of
Petrolatum-occluded AD skin demonstrated a restoration of crude coal tar and artificial ultraviolet radiation) and of which
normal keratinization (orthokeratosis) and an enhanced many continued to use coal tar regularly on an outpatient
expression of loricin und filaggrin. It is unclear how petrol- basis (Pittelkow et al. 1981). The 25-year follow-up analysis of
atum induces the molecular and structural changes in the the initial patient population revealed that 19 out of 260
epidermis. A possible mechanism could involve the activation patients reported the incident of non-melanoma skin cancer
of the arylhydrocarbon receptor (AhR) in Th17 (T-helper 17) (NMSC), and one patient reported the development of a
cells by PAHs, which activates the interleukin IL-17/IL-22 sig- malignant melanoma. Expected incidence rates of NMSC for
naling pathway and thereby induces the AMPs in the study population were calculated from annual age-spe-
the epidermis. cific incidence rates for four US areas, which were available
The pharmacological effect is presumably linked to the from the 3rd National Cancer Survey. In a study group of 260
presence of PAHs which are AhR ligands. Certain PAHs, which patients the number of subjects expected to develop skin
bind to or activate the AhR, are regarded as model carcino- cancer was 49 for Dallas, 16 for Iowa, 19 for Minneapolis and
gens in coal tar, which has a dermato-pharmacologically simi- 23 for San Francisco. This suggests that the incidence of skin
lar effect to petrolatum. The role of the AhR in epidermal cancer is not appreciably increased above the
effects induced by coal tar treatment was studied in vitro in expected incidence.
human keratinocytes (from AD patients and healthy volun- The possible percutaneous absorption of mutagens or
teers) (van den Bogaard et al. 2013). Immunofluorescence pro-mutagens (i.e. substances metabolically converted to
microscopy showed a nuclear translocation of the AhR fol- mutagens in the body or by the UV radiation applied) from
lowing treatment with 2% medicinal coal tar (pix lithanthra- crude coal tar in subjects receiving a Goeckerman therapy
cis). The expression of AhR-responsive genes encoding for was investigated in volunteers and psoriatic patients by
proteins such as CYP1A1 and CYP1B1 was increased. The measuring the mutagenicity of urine using the Ames test
expression of genes encoding for the epidermal differenti- (Salmonella typhimurium strain TA98 plus S9 mix) (Wheeler
ation proteins filaggrin, hornerin, and involucrin, which are all et al. 1981). Two healthy volunteers and a psoriatic patient
pivotal for skin barrier function (and all are functionally (all non-smokers) received a single application of 5% crude
related to AD), was also increased. AhR knockdown by siRNA coal tar (USP) in petrolatum (30 g) to the trunk skin for 6 h.
completely abrogated this effect. These data support a regu- This was followed by ultraviolet light treatment. The muta-
latory role of the AhR signaling pathway in the coal tar-medi- genic activity in urine samples was measured in a time win-
ated induction of epidermal differentiation genes. This dow from two hours before to 30 h after the start of the
hypothesis was confirmed in a follow-up study with AhR-defi- experiment. As a negative control, the two healthy volunteers
cient and AhR agonist-treated keratinocytes (van den had been treated with petrolatum and ultraviolet light at an
Bogaard et al. 2015). Histological and immunohistochemical earlier time point, using the same experimental regimen. The
investigations in human organotypic skin models revealed an mutagenic activity in the urine of coal-tar-treated subjects
accelerated epidermal differentiation following coal tar treat- increased after the start of treatment, reached its maximum
ment (van den Bogaard et al. 2013). It was also shown that after 6–8 h, then declined and approached the individual
the AhR activated the Nrf2 antioxidative stress pathway in background level after a total of 30 h. No mutagenicity was
keratinocytes. Coal tar induced the nuclear translocation of detected in the urine after petrolatum treatment. Given the
Nrf2 and the subsequent induction of its target gene, the small number of patients, however, only very limited conclu-
NAD(P)H quinone oxidoreductase 1 (NQO1). Upon AhR sions can be drawn from this study.
knockdown, induction of NQO1 expression was inhibited. Whether the dermatological exposure to coal tar oint-
AhR-mediated Nrf2 activation due to coal tar treatment could ments, which leads to a urinary excretion of (carcinogenic)
thus inhibit (Th2-induced) oxidative stress and inflammatory PAH metabolites, is associated with an increased risk of blad-
processes resulting in a favorable effect on the treatment of der cancer was addressed in a population-based case-control
psoriasis and AD. study performed in the Netherlands (Roelofzen et al. 2015).
The dermal metabolism and effects of the model carcino- About 1387 cases diagnosed with bladder cancer between
gen B[a]P was studied in human hair follicles (Merk et al. 1995 and 2006 and 5182 population controls were included.
1987). Human hair was plucked from the scalp of healthy vol- Information on the use of coal tar and the history of skin dis-
unteers before and after shampooing their hair daily for ease was obtained through questionnaires. Logistic regres-
4 days with crude coal tar-containing shampoo. Hair follicles sion analyses were performed to estimate the risk of bladder
were shown to convert B[a]P to several metabolic products cancer after coal tar treatment, adjusted for age, gender,
including 7,8-dihydroxy-B[a]P (B[a]P-7,8-diol), a major precur- smoking status, duration of smoking, and intensity of smok-
sor of the ultimate carcinogenic metabolite of B[a]P. The ing. The use of coal tar ointments was approximately equal
 CRITICAL REVIEWS IN TOXICOLOGY 773

among cases and controls (3.8% vs. 3.0%, respectively). cyclic alkanes, which is important for the manufacturing of
Dermatological application of coal tar was not significantly petrolatum, as it imparts a low bleeding tendency (i.e. the
associated with bladder cancer (adjusted odds ratio: 1.37, tendency for the oil component to separate from the wax
95% CI: 0.93–2.01). An inverse association between skin dis- component). In the USP and the NF, the corresponding qual-
ease and bladder cancer was observed (adjusted odds ratio: ities are termed paraffin and microcrystalline wax.
0.74, 95% CI: 0.61–0.90). The results of this study provide no Paraffins are characterized in the Ph. Eur. by physical
evidence of bladder cancer due to dermatological coal tar parameters, including viscosity, drop point, and melting
treatment. Limitations of the study are the low number of point, depending on what is applicable to the respective
patients and the relatively short duration of dermatological product. The test for impurities includes a proof of absence
coal tar exposure in comparison to occupational exposures. of PAHs by measuring the UV absorbance (260–420 nm) of a
In the context of dermal mineral oil exposure, AhR hence DMSO extract of a paraffin sample dissolved in hexane, using
is a janus-faced protein: On the one hand it induces enzymes a naphthalene solution as reference. The limit values (in
which are involved in the formation of mutagenic/carcino- naphthalene equivalents) are 0.5 ppm for liquid and light
genic PAH metabolites, while on the other hand it mediates liquid paraffins, <70 ppm for hard paraffin, <300 ppm for
protective effects involved in the treatment of skin diseases. white soft paraffin, and <450 ppm for yellow soft paraffin.
The composition of coal tar with respect to PAHs deviates Mineral oils are used predominantly as excipients in top-
from that of petrolatum and mineral oils in cosmetic prod- ical pharmaceutical formulations such as ointment bases
ucts, which is due to the different source/origin and differen- (emollients, solvents), emulsions (microencapsulation of many
ces in the manufacturing process. In addition, medicinal drugs), ophthalmic ointments, and otic preparations. They are
products, as opposed to consumer products, are subject to a rarely used as excipient in capsules and tablets (solvents and
balancing of the therapeutic benefits and the risk of lubricants). In Germany, the use of liquid paraffin as an active
adverse effects. substance in mild laxatives is currently under reevaluation by
the German Federal Institute for Drugs and Medical
Devices (BfArM).
Mineral oils in medicinal products
The legal framework for medicinal products for human
Mineral oils and waxes have been used in medicinal products use in Europe is set by the Directive 2001/83/EC. Annex I
for decades. The European Pharmacopeia (Ph. Eur.) 8.0 distin- contains the analytical, pharmacotoxicological and clinical
guishes between liquid, semi-liquid, and solid paraffins standards and protocols with respect to the testing of medi-
(Table 2). cinal products. The standard requirements for an authoriza-
Liquid paraffin (Paraffinum liquidum) and light liquid paraf- tion dossier include quality and nonclinical assessment. The
fin (P. perliquidum) are defined in the respective Ph. Eur. nonclinical assessment follows the requirements described in
Monographs as a purified mixture of liquid saturated hydro- Module 2 and Module 4 of Annex I. The toxicity testing is
carbons obtained from petroleum. In the United States focused on active substance(s) but is often performed with
Pharmacopeia (USP) and the National Formulary (NF), the cor- final medicinal products (e.g. dermal and ophthalmic prod-
responding qualities are termed mineral oil (USP) and light ucts). For excipients such as medicinal paraffins, no pharma-
mineral oil (NF). cokinetic data are required for the authorization of medicinal
Semi-liquid paraffin is a blend of solid and liquid qualities, products. Limited “indirect” toxicity data on excipients can
comprising paraffin wax, microcrystalline wax, and a white oil potentially be derived from studies with the final formulation
(Barry and Grace 1971; van Heugten et al. 2017). Semi-liquid (active pharmaceutical ingredient and excipients), in particu-
paraffin is also called petrolatum, Vaseline, or petroleum jelly. lar from the comparison of the vehicle control with the
The Ph. Eur. categorizes semi-liquid paraffin further into untreated control group (e.g. irritant potential, skin sensitiza-
white soft paraffin (Vaselinum album) and yellow soft paraffin tion). The quality assessment of substances follows the
(V. flavum). In contrast to liquid paraffin, which is based on requirements specified in Module 3. For excipients such as
medicinal white oil, semi-liquid paraffin can be blended from medicinal paraffins, the Ph. Eur. Monographs are applicable.
technical white oil. This explains why the attribute The applicant’s confirmation of compliance with the
“saturated” is missing in the definition in the respective Monograph is mostly acceptable for excipients.
Monographs and why the limit values for impurities of PAHs The Ph. Eur. provides substance identification criteria and
are higher (see below). Yellow soft paraffin derives its color impurity requirements, which are key elements for the quality
from base materials that are not as thoroughly purified as and safety of the overall drug product. For example, the
those used for white soft paraffin. In the USP, the corre- Monograph on liquid paraffin (P. liquidum) specifies an identi-
sponding qualities are termed white petrolatum fication based on infrared absorption spectrophotometry
and petrolatum. (comparison with a Ph. Eur. reference spectrum of liquid par-
The solid paraffins are differentiated in the Ph. Eur. into affin), a viscosity measurement, and a phenolphthalein test.
hard paraffin (P. solidum), also called paraffin wax, and micro- The test on impurities includes, inter alia, a proof of absence
crystalline wax, for which currently only a draft Monograph of PAHs by measuring the UV absorbance of a DMSO extract
exists. Both products are defined as saturated hydrocarbons. using a naphthalene solution as reference. An identification
Hard paraffin mainly contains straight-chain alkanes. of individual aromatic hydrocarbons is not requested and no
Microcrystalline wax has a high content of branched and specific limit has been implemented.
774 R. PIROW ET AL.

Finished medicinal products containing light liquid paraf- standards, indexes and existing codes, and/or analytical fin-
fin for oral use include soft capsules, where the active ingre- gerprints. More specifically, the guidance requires that for a
dients are dissolved or suspended in the oil, and hard UVCB substance, “[ … ] all known constituents and all constit-
capsules, where the oil is used as plasticizer in film coatings. uents present at concentrations 10% should be specified by
The mass of light liquid paraffin per dosage form is in the at least an English-language IUPAC name and preferably a
range of 0.35–73.59 mg for products on the German market. CAS number; the typical concentrations and concentrations
Medicinal products containing liquid paraffin for oral use ranges of the known constituents should be given as well.
include, for example, laxatives where the oil is the active sub- [ … ] Unknown constituents should be identified as far as
stance. The amount of liquid paraffin per dosage for products possible by a generic description of their chemical nature.
can be up to 10 g for a laxative. It should be noted that such Additives should be completely specified in a similar way to
medical preparations are to be used only in the short term. that described for well-defined substances”. Furthermore, and
Medicinal products containing white soft paraffin are widely of great importance in the case of mineral oil-based substan-
applied for topical treatments including therapeutic treat- ces, “[ … ] constituents that are relevant for the classification
ments (see Chapter “Mineral oils in dermatology”). They are [ … ] of the substance shall always be identified by the same
not approved for oral use. Yellow soft paraffin is rarely used identifiers, independently from their concentration”. However,
for dermatological preparations. Finally, hard paraffins serve the ECHA guidance does not specify the nature or extent of
as embedding or encapsulating material for active substances analytical investigations which registrants need to perform in
in oral dosage forms with retarded release. The amount of order to ascertain or exclude the presence of constituents in
hard paraffin per dosage form is in the range of 0.1–100 mg amounts relevant for classification (ECHA 2017b).
for products on the German market. In addition to the ECHA guidance, other sector-specific
guidance exists, such as the OECD guidance documents for
characterizing oleochemical substances or hydrocarbon sol-
Mineral-oil based substances under REACH and CLP vents for assessment purposes, which are less strict regarding
the specification of relevant individual constituents, but more
Regulation (EC) 1907/2006 (REACH) provides the legal basis
specific with respect to naming conventions and analytical
for the registration, evaluation, authorization, and restriction
verification of composition (OECD 2014, 2016). With respect
of chemicals in the EU. It is interconnected in multiple ways
to the latter, it is of particular importance for hazard assess-
with Regulation (EC) 1272/2008 (CLP) on the classification,
ment that specified compositions not only cover all poten-
labeling, and packaging of hazardous chemical substances
tially relevant constituents (as far as known) but also the
and mixtures, which translates the UN GHS, the so-called
inter-batch variability in PetCo substances from the same
Globally Harmonized System which has been developed for
refinery and process. To this end both OECD guidance docu-
these purposes at the UN level, into European Law.
ments endorse the concept of “five-batch analysis”: “The typ-
ical, minimum and maximum concentration value for a given
Identification of mineral oil-based substances constituent should be included in the substance specification.
[ … ]. This information can be established by analyzing a
Under the REACH and CLP regulations mineral oil-based sub- number of separate batches (typically five) over a period of
stances (as a subgroup of substances derived from petroleum time. The average value of this multiple batch analysis should
or coal streams, so-called “PetCo substances”) belong to the be used to set the mean (typical value). The minimum and
group of UVCBs, i.e. “substances of unknown or variable maximum values should be no more than 3 standard devia-
composition, complex reaction products, or biological materi- tions from the mean/typical value” (OECD 2014).
als”. More specifically, often both the “U” and the “V” apply As a basis for their approach to the environmental risk
to mineral oil-based substances, as it is impossible to specify assessment of petroleum-based substances (PETRORISK
the exact content of each constituent over the whole carbon approach), Concawe has introduced the so-called
number and substance class range covered by a given “hydrocarbon block method” (HCBM) (King et al. 1996;
stream and because refineries will use crude oil from varying Comber et al. 2014). Briefly, a matrix of 3-carbon number
sources for production, resulting in a potentially significant ranges (e.g. C15–C17) vs. substance class (e.g. n-P ¼ n-paraffins
variation of composition between batches. or DiAr ¼ di-aromatics) is constructed. Composition is then
Requirements for the identification and naming of UVCB specified by giving the content of each individual fraction
substances under REACH and CLP are detailed in the corre- characterized by a specific combination of 3-carbon number
sponding guidance provided by the European Chemicals ranges and substance class (e.g. C12–C14 DiAr) as determined
Agency (ECHA). While acknowledging that “[ … ] UVCB sub- by GC
GC analysis. Rorije and coworkers (Rorije et al. 2012)
stances either cannot be uniquely specified with the IUPAC from the Dutch National Institute for Public Health and the
name of the constituents, as not all the constituents can be Environment (RIVM) in reply to a service request by ECHA
identified; or they may be generically specified but with a have identified shortcomings of the HCBM as practically
lack of specificity due to variability of the exact composition”, implemented (and even more so, of the PETRORISK approach
the ECHA guidance nevertheless requests registrants under itself, for which they saw “[ … ] a serious possibility that the
REACH to give the chemical composition and the identity of tool PETRORISK [ … ] will lead to an underestimation of the
the constituents as far as known, e.g. at least in a more gen- (environmental) risk related to the production and use of pet-
eric way, on the basis of well-known reference samples or roleum products”) and recommended improvements.
 CRITICAL REVIEWS IN TOXICOLOGY 775

However, while certain practical aspects may need further the total tonnage level registrants must fulfill a set of stand-
discussion, at least the theoretical concept of the HCBM ard information requirements (mostly in vitro or in vivo tests
matrix as such can be a helpful starting point for characteriz- in animals, cf. REACH Annexes VII-X) which can be adapted
ing the composition of mineral oil-based UVCB substances in under certain circumstances (REACH Annex XI). In addition, if
a transparent and comparable way, as a pre-requisite for risk certain conditions are fulfilled, they must submit a so-called
assessment based on the presence/absence of certain hydro- “Chemical Safety Assessment (CSA)” in order to demonstrate
carbon fractions or constituents (cf. Section “SVHC Roadmap that potential risks due to hazardous properties of the regis-
2020 and PetCo group”). tered substance are adequately controlled. For the registration
of most, if not all petroleum-derived substances, the REACH
standard information requirements have been adapted in favor
Classification for hazardous properties
of category/grouping approaches, i.e. individually registered
While both the CLP (through hazard classes and categories) substances are allocated to groups/categories (e.g. based on
and REACH (through chemical safety assessment) regulations comparable production processes) and their risk assessment is
essentially address all relevant toxicological endpoints in the based on a matrix of physico-chemical and (eco)toxicological
area of human health, they place particular focus on carcino- properties, and biological fate data which are read across
genic, mutagenic, and reprotoxic (CMR) properties. According between group members in order to fill data gaps. While this
to Article 36 (1) of the CLP Regulation, substances with these approach is sensible as such, in particular from an animal wel-
properties require “harmonized classification” (CLH), i.e. these fare and resource perspective, it is clear that it can only pro-
classifications are officially identified by the European duce reliable results if sufficient mutual similarity in
Commission based on an evaluation by the Risk Assessment composition between all materials registered under each indi-
Committee (RAC) of the European Chemicals Agency (ECHA). vidual registration, between category members, and between
By subsequent publication in Annex VI of the CLP regulation the substances used for testing can be established
such classifications then become legally binding in the EU. with confidence.
Classification not only applies to the main constituent(s) Under Title VI, REACH also foresees processes by means of
of well-defined substances. CLP Art. 11 states that “[ … ] which registrants, ECHA or EU Member State Competent
where a substance contains another substance, itself classi- Authorities (MSCAs) can scrutinize registration dossiers for
fied as hazardous, whether in the form of an identified potential data gaps and propose (registrants) or request
impurity, additive or individual constituent, this shall be (authorities) the generation of further data. Again, a sufficient
taken into account for the purposes of classification, if the specification of the composition is key, in particular to estab-
concentration of the identified impurity, additive or individual lish the representativeness of test materials used in the stud-
constituent is equal to, or greater than, the applicable cutoff ies submitted for the registered composition.
value [ … ]”. For CMR properties these cutoffs are given in
the form of endpoint-specific “Generic Concentration Limits”
Restriction
(GCLs, e.g. 0.1% (w/w) in the case of carcinogens of CLP cat-
egory 1) or as “Specific Concentration Limits” based on indi- MSCAs or ECHA (on request by the European Commission)
vidual substance data (SCLs, e.g. 0.01% (w/w) for the can initiate the restriction of one or more uses of a substance
carcinogenic PAH benzo[a]pyrene). under REACH Title VIII, if they are able to demonstrate that
In line with the CLP guidance issued by ECHA, CLH of this use/these uses pose an unacceptable risk to human
UVCB mineral oil-based substances for CMR properties should health. This process normally involves scrutiny by RAC and
be done preferably based on the presence of one or more SEAC (ECHA’s Committee for socio-economic analysis) and –
relevant constituents/additives/impurities (e.g. B[a]P) above aside from the burden of proof – places a heavy workload on
the applicable concentration limits whereas for other human the dossier submitter. In the case of a substance with CMR
health endpoints, CLH is in general obtained based on data properties which may be used by consumers (on its own, in
for the substance as a whole. It is self-evident that for con- mixtures, or articles), a simplified route according to REACH
stituent-based classification an adequate and comprehensive Art. 68 (2) is available, which e.g. has been used for the
specification of composition is a key pre-requisite. restriction of PAHs in consumer products proposed by the
The official identification of CMR properties by CLH in German MSCA in 2010 (for details of the risk assessment for
itself already constitutes a regulatory measure which may consumers performed by BfR in this context, cf. BfR (2010b)).
have severe consequences in sector-specific downstream While in general restriction is a powerful and versatile regula-
legislation. It also constitutes a fundamental pre-requisite for tory tool, in particular for the regulation of substances in
any other regulatory risk management measures under articles, it is often difficult to prove unacceptable risk, most
REACH, such as identification of “Substances of Very High of all for consumers, where reliable data on exposure (includ-
Concern” (SVHC), authorization, or restriction. ing migration of hazardous substances from articles) are
often not available. An advantage is given by the fact that
for petroleum-derived substances, restriction can focus on
Registration and evaluation under REACH
relevant hazardous constituents, since the origin of e.g. car-
Under REACH, substances must be registered at a market vol- cinogenic polycyclic aromatic compounds (PAC, i.e. including
ume per registrant of 1 ton or more per year. Depending on also those polycyclic aromatic substances containing
776 R. PIROW ET AL.

heteroatoms) in articles cannot be traced back to the use of obliges ECHA to consider “whether the use of that substance
a specific petroleum-based substance in the produc- in articles poses an unacceptable risk to human health or the
tion process. environment that is not adequately controlled”. In cases
where such a risk is established, ECHA then must prepare a
restriction dossier (cf. previous section).
SVHC identification and authorization
In 2006, as a follow-up to the 2002 Johannesburg sustainabil-
SVHC roadmap 2020 and PetCo group
ity summit, the policy goal “to achieve, by 2020, that chemi-
cals are used and produced in ways that lead to the To address the SAICM 2020 goal, the European Commission
minimization of significant adverse effects on human health in 2012 devised the so-called “SVHC roadmap 2020” with the
and the environment, using transparent science-based risk aim to have all “relevant currently known SVHCs” included in
assessment procedures and science-based risk management the Candidate List by 2020 (European Commission 2013).
procedures, taking into account the precautionary approach” This goal not only included the aim to determine the rele-
was expressed at UN level in the form of the Strategic vance of SVHCs that are known today, but also to identify
Approach to International Chemicals Management (SAICM, cf. new potential SVHCs that will come out of REACH registra-
www.saicm.org, UN (2003)). This has been translated under tion and evaluation. Since 2013, comprehensive screening
REACH into the concept of so-called “Substances of Very activities by ECHA and the MSCAs have taken place and sev-
High Concern” (SVHCs). SVHCs are officially identified and eral working groups on EU level have been working on the
regulated under REACH Title VII (authorization) with the aim implementation of this roadmap. The progress of this work is
“[ … ] to ensure the good functioning of the internal market tracked on the website of the European Chemicals Agency
while assuring that the risks from substances of very high (https://echa.europa.eu/de/svhc-roadmap-to-2020-implemen-
concern are properly controlled and that these substances tation, last accessed 2019-06-20).
are progressively replaced by suitable alternative substances Depending on the source material and the production
or technologies where these are economically and technically process, petroleum- and coal-derived (UVCB) substances may
viable” (REACH Art. 55). contain constituents with SVHC properties. Since they form a
Inter alia, CMR properties of CLP category 1 can trigger large group of substances with a number of specific prob-
official identification of a substance as SVHC (by the lems, the roadmap activities related to regulatory risk man-
European Commission based on a vote of the MSC, ECHA’s agement of petroleum and coal stream (PetCo) substances
Member State Committee), in which case it is put first on a are coordinated since 2015 by the so-called PetCo Working
so-called Candidate List (CL) for authorization. In a second Group (WG), which consists of representatives of ECHA, the
step, based on certain prioritization criteria as well as on European Commission, EU Member States, and industry
available capacity of the authorities, it is transferred to stakeholders; for more details on the composition and remit
REACH Annex XIV, the list of substances requiring authoriza- of this group, cf. https://echa.europa.eu/de/petco-working-
tion. CL entries refer to single substances or to closed lists of group (last accessed 2019-06-20). In August 2017, the PetCo
substances. As a consequence, one of the problems encoun- WG has published a general approach for how to further
tered in practice is presented by the fact that petroleum- handle the roadmap work on these substances (ECHA 2017a).
based UVCB substances can either be put on the CL one by Briefly, this approach foresees a first prioritization for further
one (which is not efficient, given the large number of these work of substances with consumer or professional uses
substances) or as groups defined in such a way that a defini- (widespread uses, highest priority) over those with uses only
tive (closed) list of affected substances can be identified at industrial sites (medium priority), or used only as fuel or
(with the chance of missing relevant similar substances). It is intermediates (lowest priority). Next the prioritized substan-
not possible to include open (insufficiently determined) ces will be further categorized as substances of known haz-
lists of substances, e.g. “all substances containing >0.01% ard (regulatory action possible, if needed), of no known
benzo[a]pyrene” (substance-in-substance approach), both hazard (no regulatory action needed for the time being), and
because there could be insufficient certainty for registrants of potential hazard (further investigation of hazardous prop-
about whether their substance is affected and because the erties needed).
total number of affected substances would not be known. As On-going discussions in the PetCo WG inter alia circle
a result of the latter, the authorities’ capacities for e.g. further around the question which type of composition information
processing later applications for authorization (AfAs) could be is needed to reliably identify constituents relevant for classifi-
overtaxed, blocking other important regulatory activities in cation and labeling. For human health hazard assessment,
the field of chemical safety. A further disadvantage of the the HCBM approach might serve as a basis, if solutions for
authorization procedure is the fact that it can regulate certain problems (e.g. insufficient granularity of the blocks,
the use of substances in the production of articles, but not partial ambiguity of the blocks, lack of a standardized
the articles themselves, hence the import of articles manufac- GC
GC method) can be found. As part of the PetCo work,
tured outside the EU cannot be regulated via an Annex XIV ECHA has compiled a library of potential PetCo constituents.
entry directly. However, after the so-called “sunset date”, i.e. Constituents characterized as potentially hazardous based on
the date from which a substance in Annex XIV may no longer experimental data or using in silico modeling could then be
be placed on the market unless authorized, REACH Art. 69(2) mapped to specific hydrocarbon blocks. In the future, this
 CRITICAL REVIEWS IN TOXICOLOGY 777

might provide registrants with a means of easily identifying mesenteric lymph nodes (MLNs), which suggests an exposure
which “constituents relevant for classification and labeling” route that is primarily oral. Intestinally absorbed MOSH follow
they should address (e.g. by further targeted chemical ana- the route of transport of lipids and are primarily taken up by
lysis and/or by risk assessment) based on a reliable GC
GC the liver. Biotransformation of MOSH in liver is slow. Human
analysis of their registered material. For the latter a 5-batch- (and animal) data indicate a lifelong accumulation in adipose
analysis of representative samples could increase the reliabil- tissue and MLNs but not in liver and spleen. Although conta-
ity of the assessment. minated food is the main culprit, mineral oil-containing cos-
Practical experience from screening projects has shown metics are also contributing to the body burden of
that many PetCo registration dossiers suffer from insufficient ingested MOSH.
substance identification and, as a result, insufficient justifica- MOSH in human liver are complex mixtures consisting pre-
tion of the substance categories used for risk assessment. On dominantly of highly branched and cyclic compounds.
top of compliance issues observed also in REACH registra- Autopsy studies from the mid-1960s showed livers to contain
tions of mono- or multi-constituent substances (UBA 2015), oil droplets, so-called lipogranuloma; their occurrence and
insufficient or absent specification of composition, insufficient extent correlated with the MOSH content in human liver tis-
justification of category formation, huge variation of compos- sue. In F-344 rats, dietary exposure to certain highly refined
itional parameters – where specified – that are relevant for oils and waxes caused inflammatory epithelioid cell granulo-
classification and labeling (e.g. under the same registration mas in liver. These adverse lesions are morphologically dis-
ID, substances with DMSO extractables ranging from <1 to tinct from the MOSH-induced non-inflammatory
>8% are found), absence of key data for CMR properties lipogranuloma in human livers. The incidence and severity of
with missing/insufficient justification, and unclear identity of rat liver granulomas correlated with the tissue MOSH content.
test material used in toxicological studies, are all deficiencies In histologically positive rat livers, n-alkanes made up a
frequently encountered in PetCo registration dossiers. prominent part of the MOSH fraction. The MOSH content in
The need to improve the justification of the categories rat liver correlated with the viscosity and n-alkane content of
formed so far for the PetCo substances has already been the administered substances. Paraffin waxes (n-alkane-rich)
acknowledged in general by industry. To that end, Concawe induced the strongest response, followed by low-viscosity
inter alia has initiated the so-called Cat-App program. mineral oils. To assume that the causative agents for granu-
According to Concawe, “the concept is to develop a frame- loma formation in F-344 rat liver by low-viscosity mineral oils
work based on chemical-biological read-across, a novel direc- are the n-alkanes is conceivable, but this remains speculative
tion in regulatory decision making. The approach is to without further supportive data.
integrate innovations in (i) in vitro testing, (ii) high-through- Since the mid-1960s, exposure to contaminated food has
put genomics, and (iii) integrative data analyses and visualiza- continuously decreased and, consequently, so has the MOSH
tion into a workflow for read-across assessment of UVCBs in level in human liver. Virtually no granulomas were found in
regulatory programs“. First results were published on the autopsy patients in a more recent study. The relevance of
Concawe website in September 2018 (https://www.concawe. hepatic lesions in F-344 rats for humans is still unclear, but
eu/publication/cat-app-project-2018-results-brochure, last doubts regarding the suitability of this rat model are war-
accessed 2019-06-20) and in the peer-reviewed literature ranted. Present-day human data, ideally derived from sub-
(Kamelia et al. 2017; Kamelia et al. 2018; Kamelia et al. 2019a; jects with a higher exposure to contamined food due to
Kamelia et al. 2019b). The project is interesting from a scien- eating habits, are needed to clarify this issue. Since MOSH
tific perspective while the regulatory acceptability of its have the intrinsic property to induce tissue lesions, to minim-
results will need further discussion. Anyway, it is noted that – ize their contamination level in food is mandatory. For the
provided the project will be successful – the subsequent revi- intended use of highly refined mineral oils and waxes in lip
sion of PetCo categories as a result cannot be expected to care products, the recommendations of Cosmetics Europe
be completed by the end of this decade, i.e. in time for the should be followed.
2020 goal. Recent analytical findings of MOAH in cosmetic products
have caused concern about potential dermal cancer risk aris-
ing from MOAH constituents. For highly refined mineral oils
Conclusions
and waxes, which are used inter alia as cosmetic ingredients,
The objective of this review was to evaluate the potential animal studies support the safe use of these substances.
health risks arising from oral and dermal exposures of con- Additionally, the safety of these cosmetic products is docu-
sumers to mineral oil in food and non-food products such as mented by their prevalent and long-term use, with a simul-
cosmetics. Mineral oils are complex substances. Analytically, taneous lack of clinical and epidemiological evidence of
two fractions can be distinguished: mineral oil saturated adverse health effects. Regarding consumer exposure to less
hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons refined and characterized mineral oil, however, data on safety
(MOAH). The carcinogenic potential of individual constituents is incomplete. This applies, for example, to contamination of
of the MOAH fraction and the bioaccumulation potential of food by unrefined mineral oils. Given that MOAH in food ori-
MOSH represent two of the main toxicological concerns. ginate from different sources, and given the lack of informa-
Autopsy and biopsy studies showed the presence of tion on the carcinogenic potential of non-food grade mineral
MOSH in human tissue. Highest MOSH levels occurred in the oils, the MOAH impurities in food should be minimized.
778 R. PIROW ET AL.

Acknowledgement Bakker GCM. 2011. Absorption and kinetics of P15H oil after single oral
administration of P15H oil in human volunteers. TNO Quality of Life
This review emanated from two expert meetings and a consumer protec- Report V9501.
tion forum held in Berlin at the BfR in between 2015 and 2017. The Bandla HP, Davis SH, Hopkins NE. 1999. Lipoid pneumonia: a silent com-
meetings brought together experts from German federal and state plication of mineral oil aspiration. Pediatrics. 103(2):E19.
authorities, from European authorities as well as from academia and Barp L, Biedermann M, Grob K, Blas YEF, Nygaard UC, Alexander J,
from industry. The authors would like to thank the following experts/ Cravedi JP. 2017a. Accumulation of mineral oil saturated hydrocarbons
speakers for their valuable contributions/presentations: R€
udiger Bartsch, (MOSH) in female Fischer 344 rats: comparison with human data and
Jan van Benthem, Maurus Biedermann, Marco Binaglia, Juan Carlos consequences for risk assessment. Sci Total Environ. 575:1263–1278.
Carillo, Eddo Hoekstra, Ute Galle-Hoffmann, J€ urgen Lademann, Hans Barp L, Biedermann M, Grob K, Blas YEF, Nygaard UC, Alexander J,
Merk, Jochen Norwig, Kristof Seubert, Michal Skowron, Dierk Sothmann, Cravedi JP. 2017b. Mineral oil saturated hydrocarbons (MOSH) in
Klaus S€udkamp, Chrystele Tissier. The authors would like to thank Koni female Fischer 344 rats; accumulation of wax components; implica-
Grob and Nicole Concin for providing the raw data on MOSH levels in tions for risk assessment. Sci Total Environ. 583:319–333.
human tissues. Barp L, Kornauth C, Wuerger T, Rudas M, Biedermann M, Reiner A,
Concin N, Grob K. 2014. Mineral oil in human tissues, Part I: concen-
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All authors are or have been employed by the German Federal Institute
for Risk Assessment (BfR). Two coauthors (B€arbel Vieth and Karla Pfaff) absorption and metabolism of hydrocarbons. Prog Lipid Res. 28(3):
have retired recently, but were working for the BfR for the entire drafting 189–203.
period. The manuscript resulted from the authors’ risk assessment activ- Barry BW, Grace AJ. 1971. Structural, rheological and textural properties
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Bartsch N, Heidler J, Vieth B, Hutzler C, Luch A. 2016. Skin permeation of
there are no conflicts of interest associated with this academic
publication. polycyclic aromatic hydrocarbons: a solvent-based in vitro approach
to assess dermal exposures against benzo[a]pyrene and dibenzopyr-
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van den Bogaard EH, Bergboer JG, Vonk-Bergers M, van Vlijmen-Willems tion of MOSH.
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Health. 38(4):355–368. vaseline$ OR "petroleum jell" OR "microcrystalline wax" OR
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2 Choe et al. (2015a) included
high-density lipoproteins in rat. Lipids. 19(6):423–435. 3 Choe et al. (2014) included
Wagner WD, Wright PG, Stokinger HE. 1964. Inhalation toxicology of oil 4 Ghadially et al. (1992) included
mists. I. Chronic effects of white mineral oil. Am Ind Hyg Assoc J. 5 Nash et al. (1996) included, secondary literature
25(2):158–168. 6 Mao-Qiang et al. (1995) included
Walborg EF, Jr., DiGiovanni J, Conti CJ, et al. 1998. Short-term biomarkers 7 Patzelt et al. (2012) included
of tumor promotion in mouse skin treated with petroleum middle dis- 8 Rossmiller and Hoekstra (1966) included
9 Stamatas et al. (2008) included
tillates. Toxicol Sci. 45(2):137–145.
10 Strakosch (1943) included
Wanless IR, Geddie WR. 1985. Mineral oil lipogranulomata in liver and 11 Suzuki et al. (1978) included
spleen. A study of 465 autopsies. Arch Pathol Lab Med. 109(3): 12 Concawe (2010) excluded, test substances
283–286. not representative of MOSH,
Wang T, Si HZ, Chen PP, Zhang KJ, Yao XJ. 2008. QSAR models for the secondary literature
dermal penetration of polycyclic aromatic hydrocarbons based on 13 Jakasa et al. (2015) excluded, test substances
Gene Expression Programming. QSAR Combinat Sci. 27(7):913–921. not representative of MOSH
Wislocki PG, Fiorentini KM, Fu PP, et al. 1982. Tumor-initiating ability of 14 Singh et al. (2002) excluded, test substances
not representative of MOSH
the twelve monomethylbenz[a]anthracenes. Carcinogenesis. 3(2):
215–217.
Wheeler LA, Saperstein MD, Lowe NJ. 1981. Mutagenicity of urine from
psoriatic patients undergoing treatment with coal tar and ultraviolet
light. J Invest Dermatol. 77(2):181–185.
Appendix Table 2. References retrieved from literature search in Web
Willard-Mack CL. 2006. Normal structure, function, and histology of
of Science.
lymph nodes. Toxicol Pathol. 34(5):409–424.
Withey JR, Law FC, Endrenyi L. 1993. Percutaneous uptake, distribution, No Reference Comment
and excretion of pyrene in rats. J Toxicol Environ Health. 40(4): 1 Brown et al. (1995) already included
601–612. 2 Choe et al. (2015a) already included
Zesch A, Bauer E. 1985. Quantitative Aspekte zur perkutanen Aufnahme 3 Choe et al. (2017) included
4 Choe et al. (2014) already included
von Wollwachsalkoholen (Cetylalkohol) und Paraffinen (Octadecan) 5 Ghadially et al. (1992) already included
aus verschiedenen Salbengrundlagen [Quantitative aspects of the per- 6 Intarakumhaeng et al. (2018) included
cutaneous uptake of wool wax alcohols (cetyl alcohol) and paraffins 7 Patzelt et al. (2012) already included
(octadecane) from different ointment bases]. Dermatosen. 33(1):15–20. 8 Petry et al. (2017) already included, secondary literature
German. 9 Stamatas et al. (2008) already included
Zhu H, Bodenheimer HC, Jr., Clain DJ, Min AD, Theise ND. 2010. Hepatic 10 Choe et al. (2015b) excluded, no quantitative information
lipogranulomas in patients with chronic liver disease: association with 11 Concin et al. (2011) excluded, no quantitative information
12 Patzelt et al. (2011) excluded, no quantitative information
hepatitis C and fatty liver disease. World J Gastroenterol. 16(40):
5065–5069.
786 R. PIROW ET AL.

Appendix Table 3. Additionally included reference, identified from the refer- Appendix Table 4. Continued.
ence list of a retrieved article. No Reference Comment
No Reference Comment 21 Kolattukudy and Hankin (1966) included
1 Zesch and Bauer (1985) included 22 Kusunose et al. (1969) included
23 Le Bon et al. (1988) included
24 McCarthy (1964) included
25 Miller et al. (1996) included, second literature
This search in Web of Science returned 355 records, of which 343 26 Mitchell and Hübscher (1968) included
were excluded after examination of the title and/or abstract. Of the 27 Nochomovitz et al. (1975) included
remaining 12 full-text records, three references were excluded due to 28 Noti et al. (2003) included
the lack of quantitative information on dermal absorption. The remaining 29 Olson et al. (1986) included
nine hits comprised the review of Petry et al. (2017) and six references, 30 Perdu-Durand and Tulliez (1985) included
which were already identified for inclusion from Petry et al. (2017), and 31 Pokrovskii et al. (1969) included
32 Popovic et al. (1973) included
two new references for inclusion. An additional article (in German) was
33 Savary and Constantin (1967) included
identified for inclusion from the reference list of a retrieved article. To 34 Scotter et al. (2003) included
sum up, 13 unique articles (11 experimental papers, 2 review papers) on 35 Shubik et al. (1962) included
dermal absorption of MOSH were finally included. 36 Smith et al. (1996) included
37 Trimmer et al. (2004) included
38 Trivalle et al. (1991) included
Appendix 2: Literature search for ADME, tissue 39 Tulliez J (1986) included, secondary literature
40 Tulliez and Bories (1975a) included
levels, and histopathological effects of MOSH upon 41 Tulliez and Peleran (1977b) included
oral exposure 42 Tulliez and Bories (1975b) included
43 Tulliez and Bories (1978) included
The starting point for the literature search was the comprehensive scien- 44 Tulliez and Bories (1979) included
tific opinion of EFSA (2012) on mineral oil hydrocarbons in food. 53 refer- 45 Wanless and Geddie (1985) included
ences on the absorption (upon oral exposure), distribution, metabolism, 46 Zhu et al. (2010) included
and excretion (ADME) of MOSH, including individual saturated hydrocar- 47 Bakker (2011) excluded, inaccessible study report,
results also published
bons as model compounds for MOSH, as well as on the tissue levels and
in Boogaard et al. (2012)
associated histopathological effects of MOSH (in animals and humans) 48 Baxter et al. (1967) excluded, test compounds not
were identified from this opinion. Of the 53 references that were representative of MOSH
screened for inclusion, seven were excluded because of inaccessible 49 Bird et al. (1990) excluded, inaccessible abstract
study information, low information content (abstract), unnatural (intra- 50 Cnubben and van Stee (2011) excluded, inaccessible study report,
peritoneal) route of administration, or ingestion of vegetable food con- results also published in
taining hydrocarbons of plant origin. The remaining 46 references were Boogaard et al. (2012)
51 Janz and Shacter (1995) excluded, intraperitoneal
found suitable for inclusion.
administration
A systematic literature search was subsequently performed in the 52 Salvayre et al. (1988) excluded, ingestion vegetable
Science Citation Index Expanded database of Web of Science for the food containing hydrocarbons of
time span from 1945 to 2018. The search was conducted with the plant origin
advanced search option using the following set of queries: 53 Tulliez, Bories, Boudene, et al. (1975) excluded, ingestion vegetable
food containing hydrocarbons of
plant origin
Set Query
#3 #1 AND #2
#2 TS¼(spleen$ OR splenic OR "lymph node$" OR hepati Appendix Table 5. References retrieved from literature search in Web
OR liver$ OR tissue$) of Science.
#1 TS¼(MOSH OR "saturated hydrocarbon$" OR No Reference Comment
"mineral hydrocarbon$" OR "mineral oil$")
1 Adenuga et al. (2017) included
2 Baldwin et al. (1992) included
3 Barp et al. (2017b) included
Appendix Table 4. References retrieved from the EFSA (2012) opinion. 4 Barp et al. (2017a) included
5 Barp et al. (2014) included
No Reference Comment 6 Biedermann et al. (2015) included
1 Albro and Fishbein (1970a) included 7 Blewitt et al. (1977) already included
2 Albro and Thomas (1974) included 8 Boitnott and Margolis (1966a) included
3 Baldwin et al. (1992) included 9 Boitnott and Margolis (1966b) included
4 Blewitt et al. (1977) included 10 Boitnott and Margolis (1970) already included
5 Boitnott and Margolis (1970) included 11 Boogaard et al. (2012) included
6 Carlton et al. (2001) included, secondary literature 12 Carlton et al. (2001) already included, secondary literature
7 Concin et al. (2008) included 13 Concin et al. (2008) already included
8 Concin et al. (2011) included 14 Concin et al. (2011) already included
9 Cravedi et al. (2011) included 15 Cruickshank (1984) already included
10 Cravedi and Perdu (2012) included 16 Cruickshank and included
11 Cruickshank (1984) included Thomas (1984)
12 Delladetsima et al. (1987) included 17 Dincsoy et al. (1982) already included
13 Dill et al. (2003) included 18 Firriolo et al. (1995) already included
14 Dincsoy et al. (1982) included 19 Fleming and Carrillo (2018) included
15 Elliott et al. (1966) included 20 Fleming et al. (1998) already included
16 Firriolo et al. (1995) included 21 Griffis et al. (2010) already included
17 Fleming et al. (1998) included 22 Halladay et al. (2002) already included
18 Griffis et al. (2010) included 23 Hoglen et al. (1998) included
19 Halladay et al. (2002) included 24 Hudson and Robertson (1966) included
20 Ichihara et al. (1981) included 25 Liber and Rose (1967) included
(continued) (continued)
 CRITICAL REVIEWS IN TOXICOLOGY 787

Appendix Table 5. Continued. Appendix 3: Literature search for gastrointestinal

No Reference Comment absorption of MOAH and PAHs
26 McKee et al. (2012) included
27 Miller et al. (1996) already included, second literature The starting point for the literature search was the comprehensive scien-
28 Rose and Liber (1966) included tific opinion of EFSA (2012) on mineral oil hydrocarbons in food. Six
29 Scotter et al. (2003) already included references on the gastrointestinal absorption of PAHs (including simple
30 Shoda et al. (1997) included methylated compounds) were identified from this opinion. All six refer-
31 Smith et al. (1995) included ences turned out to be suitable for inclusion.
32 Smith et al. (1996) already included
A systematic literature search was subsequently performed in the
33 Trimmer et al. (2004) already included
34 Trivalle et al. (1991) already included Science Citation Index Expanded database of Web of Science for the
35 Wanless and Geddie (1985) already included time span from 1945 to 2018. The search was conducted with the
36 Zhu et al. (2010) already included advanced search option using the following set of queries:
37 Boitnott and Margolis (1966c) excluded, precedent abstract publication
of Boitnott and Margolis (1966b)
38 Dincsoy and Weesner (1981) excluded, precedent abstract publication
of Dincsoy et al. (1982) Set Query
39 Hard (2000) excluded, second literature, different study #3 #1 AND #2
focus (toxic oil syndrome) #2 TS¼("intestinal absorption")
40 Gazzarrini and Nagy (1966) excluded, single-case study with #1 TS¼(MOAH OR "aromatic hydrocarbon$" OR
no information on the "mineral hydrocarbon$" OR "mineral oil$" OR "polycyclic")
target tissues in focus
41 Jampolis et al. (1953) excluded, second literature,
focus on mineral oil aspiration
42 Nagy et al. (1969) excluded, non-target species, This search in Web of Science returned 42 records, of which 36 were
saturated hydrocarbons excluded after examination of the title and/or abstract. Of the remaining
of mainly plant origin 6 records, five were included, and all of these represented newly identi-
43 Vachha et al. (1967) excluded, no information on
fied references. Two additional articles were identified from the reference
MOSH levels in tissues
list of a retrieved paper. Also additionally included was a relevant
book chapter.

Appendix Table 7. References retrieved from the EFSA (2012) opinion.

Appendix Table 6. Additionally included references which were identified
from the reference list of retrieved articles, from the search for authors con- No Reference Comment
stantly contributing to the field (“Tulliez J”, “Popovic M”), and among the 1 Barrowman et al. (1989) included, secondary literature
EFSA Supporting Publications. 2 Laher and Barrowman (1987) included
No Reference Comment 3 Laher and Barrowman (1988) included
4 Laher et al. (1983) included
1 Albro and Fishbein (1970b) included
5 Laher et al. (1984) included
2 Cravedi et al. (2017) included
6 Rahman et al. (1986) included
3 Hansen (1968) included
4 Popovic (1970) included
5 Popovic et al. (1982) included
6 Skipski et al. (1967) included
7 Stryker (1941) included
8 Tulliez, Bories, and Peleran (1975) included Appendix Table 8. References retrieved from literature search in Web
9 Tulliez and Peleran (1977a) included of Science.
10 Tulliez et al. (1981) included No Reference Comment
11 Vost and Maclean (1984) included 1 Cavret et al. (2003) included
2 Harris et al. (2013) included, secondary literature
3 Laurent et al. (2002) included
4 Ramesh et al. (2004) included, secondary literature
5 Stavric and Klassen (1994) included
This search in Web of Science returned 425 records, of which 382 6 Rees et al. (1969) excluded, inaccessible abstract
were excluded after examination of the title and/or abstract. Of the
remaining 43 records, seven references were excluded because of being
precedent abstract publications of later published (and already included)
full-papers, or non-usable information (single-case study with no infor- Appendix Table 9. Additionally included references which were identified
from the reference list of a retrieved article. Additionally included was a rele-
mation on the target tissues in focus, non-target species with presence
vant book chapter.
in tissue of saturated hydrocarbons of mainly plant origin, different study
focus, pulmonary effects caused mineral oil aspiration, topic related to No Reference Comment
the “toxic oil syndrome”). The remaining 36 hits comprised 18 references, 1 Lindstrom et al. (1987) included
which were already identified for inclusion from the EFSA (2012) opinion. 2 Kojima et al. (1978) included
3 Harris et al. (2016) included, secondary literature
The systematic literature search identified 18 new references, among
which the paper of Boogaard et al. (2012) containing the results of the
two inaccessible study reports cited by EFSA, and the paper of Smith
et al. (1995) relating to the precedent abstract publication cited by EFSA. Appendix 4: Literature search for dermal absorption
11 additional articles related to the ADME of MOSH were identified for of MOAH and PAHs
inclusion from reference lists of retrieved papers, from the search for
authors constantly contributing to the field (“Tulliez J”, “Popovic M”), A systematic literature search was performed in the Science Citation
and among the EFSA Supporting Publications. To sum up, 75 unique Index Expanded database of Web of Science for the time span from
articles on the ADME, tissue levels, and histopathological effects of 1945 to 2018. The search was conducted with the advanced search
MOSH upon oral exposure were finally included. option using the following set of queries:
788 R. PIROW ET AL.

This search in Web of Science returned 638 records, of which 614

Set Query
were excluded after examination of the title and/or abstract. Of the
#4 #1 AND #2 AND #3 remaining 24 records, six were included, and all of these represented
#3 TS¼(absorption OR penetration OR permeation OR permeability)
newly identified references. The remaining 18 references were not
#2 TS¼(skin OR dermal OR transdermal OR cutaneous
OR transcutaneous OR percutaneous) included for the sake of conciseness. Additional references with extra
#1 TS¼(MOAH OR "aromatic hydrocarbon$" information on structure-carcinogenicity relationships for alkylated and
OR "mineral hydrocarbon$" OR "mineral oil$" OR polycyclic) partially hydrogenated PAHs as well as with relevant special information
on the mouse skin painting assay and related assays (Mobil PAC
Method), and on major categories of petroleum-derived substances were
included. Also included were relevent publications from CONCAWE,
This search in Web of Science returned 316 records, of which 302 EFSA, European Union, Institute of Petroleum, IARC, and JECFA as well as
were excluded after examination of the title and/or abstract. Of the two references to relevant expert workshop presentations.
remaining 14 records, 10 references with relevant information on the
dermal absorption of PAHs were included. The remaining four references
were not included for the sake of conciseness. Five additional references Appendix Table 12. References retrieved from the EFSA (2012) opinion.
with extra information on the skin metabolism of PAHs were included.
No Reference Comment
1 Agarwal et al. (1986) included
Appendix Table 10. References retrieved from literature search in Web of Science. 2 Agarwal et al. (1988) included
No Reference Comment 3 Doak et al. (1983) included
1 Beriro et al. (2016) included, secondary literature 4 Iversen (1989) included
2 Dankovic et al. (1989) included 5 LaVoie et al. (1985) included
3 Gute et al. (1999) included 6 McKee, Daughtrey, included
4 Moss et al. (2002) included et al. (1989)
5 Potter et al. (1999) included 7 Shoda et al. (1997) included
6 Roy et al. (1996) included 8 Shubik et al. (1962) included
7 Roy et al. (1998) included 9 Trimmer et al. (2004) included
8 Sartorelli et al. (1999) included 10 Bingham (1988) not included for the sake of conciseness,
9 Vanrooij et al. (1993) included secondary literature
10 Vanrooij et al. (1995) included 11 Bingham and Barkley (1979) not included for the sake of conciseness
11 Devillers (2000) not included for the sake of conciseness 12 Fischer et al. (1951) not included for the sake of conciseness
12 Payan et al. (2008) not included for the sake of conciseness 13 Horton et al. (1957) not included for the sake of conciseness
13 Wang et al. (2008) not included for the sake of conciseness 14 LaVoie et al. (1982) not included for the sake of conciseness
14 Withey et al. (1993) not included for the sake of conciseness 15 LaVoie et al. (1981) not included for the sake of conciseness
16 Mehrotra and Saxena (1979) not included for the sake of conciseness
17 Murata et al. (1993) not included for the sake of conciseness
18 Murata et al. (1997) not included for the sake of conciseness
19 Shubik and Saffiotti (1955) not included for the sake of conciseness
Appendix Table 11. Additionally included references with extra information 20 Smith et al. (1951) not included for the sake of conciseness
on skin metabolism. 21 Wislocki et al. (1982) not included for the sake of conciseness
No Reference Comment
1 Brinkmann et al. (2013) included
2 Jacques et al. (2010) included
3 Ng et al. (1991) included
4 Ng et al. (1992) included Appendix Table 13. References retrieved from literature search in Web
5 Pannatier et al. (1978) included, secondary literature of Science.
No Reference Comment
1 Blackburn et al. (1996) included
2 Chasey and McKee (1993) included
Appendix 5: Literature search for (dermal) 3 Ingram and Grasso (1991) included, secondary literature
carcinogenicity of PAHs and MOAH 4 Nessel et al. (1999) included
5 Nessel et al. (1998) included
The starting point for the literature search was the comprehensive scientific 6 Skisak et al. (1994) included
opinion of EFSA (2012) on mineral oil hydrocarbons in food. 21 references 7 Amin et al. (1992) not included for the sake of conciseness
on the carcinogenicity (with focus on dermal carcinogenicity) of PAHs 8 Amin et al. (1993) not included for the sake of conciseness
(including alkylated compounds) and mineral oils were identified from this 9 Amoruso et al. (2003) not included for the sake of conciseness
10 Blanding et al. (1951) not included for the sake of conciseness
opinion. 12 references were not considered for the sake of conciseness.
11 Broddle et al. (1996) not included for the sake of conciseness
A systematic literature search was subsequently performed in the 12 Cavalieri et al. (1991) not included for the sake of conciseness
Science Citation Index Expanded database of Web of Science for the 13 Freeman et al. (1993) not included for the sake of conciseness
time span from 1945 to 2018. The search was conducted with the 14 Gerhart et al. (1988) not included for the sake of conciseness
advanced search option using the following set of queries: 15 Gradiski et al. (1983) not included for the sake of conciseness
16 Habs et al. (1980) not included for the sake of conciseness
17 Higginbotham et al. (1993) not included for the sake of conciseness
Set Query 18 Ingram et al. (1994) not included for the sake of conciseness
#5 #1 AND #2 AND #3 AND #4 19 LaVoie, Cai, et al. (1993) not included for the sake of conciseness
#4 TS¼(mouse OR mice OR bioassay) 20 LaVoie, He, et al. (1993) not included for the sake of conciseness
#3 TS¼(tumo$r OR carcinogen OR cancer) 21 Lewis (1983) not included for the sake of conciseness
#2 TS¼(skin OR dermal OR cutaneous) 22 Nessel (1999) not included for the sake of conciseness
#1 TS¼(MOAH OR "aromatic hydrocarbon$" OR "mineral hydrocarbon$" OR 23 Roy et al. (1996) not included for the sake of conciseness
"mineral oil$" OR polycyclic OR polynuclear OR petroleum) 24 Walborg et al. (1998) not included for the sake of conciseness
 CRITICAL REVIEWS IN TOXICOLOGY 789

Appendix Table 14. Additionally included references.

No Reference Comment
1 Agarwal et al. (1985) included
2 Assogne (1990) included
3 Blackburn et al. (1986) included
4 Blackburn et al. (1984) included
5 Chaudhuri (2011) included, secondary literature
6 Clark et al. (2013) included, secondary literature
7 Concawe (1994) included, secondary literature
8 Concawe (2001) included, secondary literature
9 Concawe (2016) included, secondary literature
10 Doak et al. (1985) included
11 EFSA (2008) included, secondary literature
12 EFSA (2009) included, secondary literature
13 EFSA (2013a) included, secondary literature
14 EFSA (2013b) included, secondary literature
15 European Chemicals Bureau (1999) included, secondary literature
16 Gray et al. (2013) included, secondary literature
17 H€oke and Zellerhoff (1998) included, secondary literature
18 Honda et al. (1990) included
19 IARC (2010) included, secondary literature
20 IARC (2012) included, secondary literature
21 Institute of Petroleum (2004) included
22 Jouanneau (2017) included
23 JECFA (2006) included, secondary literature
24 Lijinsky and Garcia (1972) included
25 Lijinsky et al. (1970) included
26 Lijinsky et al. (1965) included
27 Mackerer et al. (2003) included, secondary literature
28 McKee et al. (2015) included, secondary literature
29 McKee, Nicolich, et al. (1990) included
30 McKee, Plutnick, et al. (1989) included
31 McKee, Scala, et al. (1990) included
32 McKee and White (2014) included, secondary literature
33 Mehrotra et al. (1988) included
34 Natusch and Tomkins (1978) included
35 Rice et al. (1987) included
36 Roy et al. (1995) included
37 Roy et al. (1988) included
38 Sothmann and S€ udkamp (2017) included
39 Surh et al. (1990) included