Toxicological and ecotoxicological properties of gas-to-liquid (GTL) products. 1. Mammalian toxicology

Abstract

Gas-to-liquid (GTL) products are synthetic hydrocarbons produced from natural gas using a Fischer–Tropsch process. This process yields a synthetic crude oil that consists of saturated hydrocarbons, primarily linear alkanes, with increasing amounts of branched (methyl-groups) alkanes as the chains get longer. In addition, small amounts of cycloalkanes (branched cyclopentanes and cyclohexanes) may be formed as the polymerization reaction prolongs. This synthetic crude can subsequently be refined to a range of products very similar to petroleum refining. However, in contrast to their petroleum-derived analogs, GTL products are essentially free of unsaturated or aromatic constituents and also no sulfur-, oxygen-, or nitrogen-containing constituents are present.

From a regulatory perspective, GTL products are new substances which require extensive testing to assess their hazardous properties. As a consequence, a wide range of GTL products, covering the entire portfolio of GTL products, have been tested over the past few years in a wide variety of toxicological studies, including reproductive and prenatal development toxicity studies. This review provides an overview of the hazardous properties of the various GTL products.

In general, the data collected on GTL products provide strong proof that they exert minimal health effects. In addition, these data provide supporting evidence for what is known on the mechanisms of mammalian toxicology of their petroleum-derived analogs. In the few cases where adverse effects were found for the GTL substances, these were usually less severe than the adverse effects observed with their petroleum-derived analogs.

Keywords:

• Base oil
• diesel
• Fischer–Tropsch
• fuel
• gas-to-liquid
• gas oil
• hazardous properties
• naphtha
• paraffinic hydrocarbon
• regulatory
• solvents
• toxicity

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Erratum

Introduction

Gas-to-liquid (GTL) products are synthetic hydrocarbons produced from natural gas as a feedstock. The basic chemistry of the GTL process was developed in 1925 in Mülheim an der Ruhr (Germany) at the Kaiser Wilhelm Institut für Kohlenforschung by Franz Fischer and Hans Tropsch and is known, after the inventors’ names, as the ‘Fischer–Tropsch process’. In essence, this process involves the synthesis of higher hydrocarbons from a simple carbon source, via so-called synthesis gas (or syn-gas, a mixture of carbon monoxide and hydrogen), using a catalyst. For GTL products, the main carbon source is methane, usually from so-called stranded gas, but it can also be methane obtained from biomass digestion or from coal gasification. The methane is converted, by a process called steam-reforming, to syn-gas, according to the following reaction formula: H₂O + CH₄ → CO +3 H₂. The syn-gas produced in this process is subsequently catalytically converted to a range of saturated hydrocarbons, according to the following reaction formula: (2n + 1) H₂ + n CO → C_nH_(2n + 2) + n H₂O. The saturated hydrocarbons formed are primarily linear alkanes, with increasing amounts of branched (methyl-groups) alkanes if the chains get longer. In addition, small amounts of cycloalkanes (branched cyclopentanes and cyclohexanes) may be formed as the reaction prolongs. Essentially, the process yields a synthetic crude oil that is made up of a wide variety of alkanes but that is essentially free of unsaturated or aromatic constituents. In addition, in contrast to petroleum crudes, no sulfur-, oxygen-, or nitrogen-containing constituents are present in GTL synthetic crude. This synthetic crude can subsequently be refined to a range of products. The main GTL products are listed in Table 1. In addition, several GTL specialty products, including GTL solvents, have been developed with specific (physico-chemical) properties and specific applications (Tables 2 and 3). Essentially these GTL specialty products are fractions (cuts) of the GTL products listed in Table 1 but with a smaller range of carbon chain lengths. They are specially distilled to obtain products with very specific physico-chemical properties (such as viscosity and boiling point). A typical example are the GTL solvents (Table 3), which represent narrow cuts with partially overlapping carbon chain lengths and which have been designed to provide highly specific solvency properties.

Table 1. Main GTL products.

Product (CAS name)	Common name	CAS RN	CLP classification for health hazards	GHS classification for health hazards
Naphtha (Fischer–Tropsch), light, C4–C10 branched and linear	GTL naphtha	848301-65-5	Aspiration Cat. 1 – H304 Skin irritation Cat. 2 – H315 STOT-SE Cat. 3 – H336 (CNS-depression) STOT-RE Cat. 2 – H373^a Reproductive toxicity Cat. 2 – H361f^b	Aspiration Cat. 1 – H304; Skin irritation Cat. 2 – H315 STOT-SE Cat. 3 – H336 (CNS-depression)
Kerosine (Fischer–Tropsch), full-range, C8–C16 branched and linear	GTL kerosine	848301-66-6	Aspiration Cat. 1 – H304	Aspiration Cat. 1 – H304
Distillates (Fischer–Tropsch), C8–C26 branched and linear	GTL gas oil (GTL diesel, GTL fuel)	848301-67-7	Aspiration Cat. 1 – H304	Aspiration Cat. 1 – H304
Distillates (Fischer–Tropsch), heavy, C18–C50 branched, cyclic and linear	GTL base oil	848301-69-9	Aspiration Cat. 1 – H304^c	Aspiration Cat. 1 – H304^c
Residual oils (Fischer–Tropsch), base oil production, C40–C70 branched, cyclic and linear aliphatic hydrocarbon fraction	GTL residual oil	848301-71-3	Not classified	Not classified
Paraffin waxes (Fischer–Tropsch), light, C15–C27 branched and linear	GTL light waxy raffinate	848301-90-6	Not classified	Not classified
Paraffin waxes (Fischer–Tropsch), full-range, C15–C50 branched and linear	GTL wax (Waxy raffinate)	848301-87-1	Not classified	Not classified

^a EU classification based on CLP harmonized classification of n-hexane for peripheral neuropathy, which has a specific concentration limit of ≥5%.

^b EU classification, based on CLP harmonized classification of n-hexane for fertility (f) effects category 2, which has a classification cut off of >3%.

^c Applicable if viscosity is ≤20.5 mm²/s @ 40 °C.

Table 2. GTL specialty products.

Product (CAS name)	Common name	CAS RN	GHS and CLP classification for health hazards
GTL light kerosine C8–12	SMDS kerosine	1046149-76-1	Aspiration Cat. 1 – H304
GTL n-paraffins C13–17	Heavy detergent feedstock (HDF), Sarapar 147	90622-45-0	Not classified
GTL linear paraffins C18–25	Sarawax 30 (SX30)	8002-74-2	Not classified
GTL linear paraffins C19–36	Sarawax 50 (SX50)	8002-74-2	Not classified
GTL linear paraffins C25–48	Sarawax 70 (SX70)	8002-74-2	Not classified
GTL linear paraffins C38–90	Sarawax 100 (SX100)	8002-74-2	Not classified

Table 3. GTL solvents.

Chemical name^a	GTL solvent name	CAS RN	GHS and CLP classification for health hazards
Hydrocarbons, C8–C11, n-alkanes, isoalkanes, <2% aromatics	GS160	1437281-13-4	Aspiration hazard Cat.1 – H304; Skin irritation Cat. 2 – H315; STOT SE Cat.3 – H336
Hydrocarbons, C9–C12, n-alkanes, isoalkanes, <2% aromatics	GS170	1437281-04-3	Aspiration hazard Cat.1 – H304; Skin irritation Cat. 2 – H315; STOT SE Cat.3 – H336
Hydrocarbons, C10–C13, n-alkanes, isoalkanes, <2% aromatics	GS190	185857-36-7	Aspiration hazard Cat.1 – H304
Hydrocarbons, C9–C13, n-alkanes, isoalkanes, <2% aromatics	GS210	1437280-84-6	Aspiration hazard Cat.1 – H304
Hydrocarbons, C12–C15, n-alkanes, isoalkanes, <2% aromatics	GS215	1437281-03-2	Aspiration hazard Cat.1 – H304
Hydrocarbons, C14–C16, n-alkanes, isoalkanes, <2% aromatics	GS250	1174918-46-7	Aspiration hazard Cat.1 – H304
Hydrocarbons, C15–C19, n-alkanes, isoalkanes, <2% aromatics	GS270	1437281-01-0	Aspiration hazard Cat.1 – H304
Hydrocarbons, C18–C24, isoalkanes, <2% aromatics	GS310	1437280-85-7	Aspiration hazard Cat.1 – H304

^a The carbon number range presented for GTL Solvents is consistent with the Hydrocarbon Solvents Producers Association (HSPA) naming convention (HSPA 2011) that has been adopted within Europe for REACH to identify hydrocarbon solvent type products, and allows for a comparison of GTL Solvents with existing hydrocarbon solvent products.

Chemistry and testing approach

The chemistry of GTL products is rather straightforward as they comprise primarily alkanes. Up to carbon chain lengths of approximately C₁₂ about 60% of the constituents are linear alkanes (n-alkanes) and about 40% branched alkanes (isoalkanes), mostly monomethylalkanes. With carbon numbers greater than C₁₂, the branching rapidly increases from about equal amounts of n-alkanes and isoalkanes at C₁₄ to about 25% n-alkanes and 75% isoalkanes at C₁₈. When the total carbon number is greater than C₂₅, the branching is virtually complete and the percentage of n-alkanes gets very low. When the carbon numbers get even higher, cyclic structures (5- and 6-ring cycloalkanes) may also be formed. The percentage of cycloalkanes in GTL base oil (C₁₈–C₅₀) is usually <5%, whereas it may increase to 35–40% for GTL residual oil (C₄₀–C₇₀). The branching is almost exclusively monomethylalkanes for the lighter products (GTL naphtha and GTL kerosene) but with increasing carbon numbers, the branching increases and GTL gas oil and GTL base oil contain about 25% monomethylalkanes, 50% dimethylalkanes, and 25% trimethylalkanes. Tertiary branching is virtually absent.

Although the GTL products have to a large extent the same properties and applications as conventional, petroleum-derived products, they are expected to be less hazardous since they are practically devoid of unsaturated and aromatic constituents. However, as they are not derived from crude oil, they are considered to be new substances according to most chemicals regulations. As a consequence, the GTL products have been extensively tested for their intrinsic hazardous properties, including both mammalian and ecological toxicity, as required by several regulatory schemes. As these products have global uses but without necessarily the same application in different regions, the actual testing may vary for the various products based on specific regional applications and regulatory demands. Overall, this has led to a wide range of toxicity testing giving a broad overview of the hazardous properties of the various GTL products. The carbon chain lengths of the various GTL products discussed are depicted in Figure 1. This review presents the mammalian toxicology data on GTL products and compares these to similar data obtained with conventional, petroleum-derived products. All studies reported here were performed under Good Laboratory Practice (GLP), according to adopted OECD testing guidelines for animal testing in certified laboratories. The ecotoxicological data on GTL products will be reviewed in a separate paper.

Figure 1. Schematic overview of carbon chain lengths in the various GTL products.

Toxicity testing

Acute toxicity

Testing results for GTL products indicate that acute exposure poses no significant hazard. Low-viscosity GTL products are considered to present a risk of aspiration if ingested and, as a consequence, are classified as aspiration hazards. Inhalation exposure of high concentrations of aerosols of low-viscosity GTL substances induced lung lesions which are considered to be secondary to aspiration, due to coalescence of the aerosol droplets, since the histopathology of these lesions was fully consistent with those observed following aspiration and no adverse effects were noted in organs other than the lungs.

Acute oral toxicity

All the GTL products listed in Table 1, covering the entire hydrocarbon range from C₄ to C_70, have been tested for acute oral toxicity in the Sprague–Dawley CD rat in a series of oral gavage studies according to OECD testing guideline 420 (acute oral toxicity – fixed dose procedure). For all these GTL products, the LD₅₀ was greater than 5000 mg/kg body weight, with no gross signs of any toxicity, indicating minimal overall oral toxicity Sanders (2006a, 2006b, 2006c, 2007a, 2007c, 2007e, 2007f). In addition, two GTL waxes, Sarawax 30 (SX30) and Sarawax 100 (SX100), were tested in an acute oral toxicity limit dose test in Sprague–Dawley rats by oral gavage according to OECD testing guideline 401 (acute oral toxicity) and found to be not toxic at the limit dose of 5000 mg/kg without any treatment-related changes in behavior or condition and without any findings in necropsy (Robbins 1993a, 1993b).

Acute dermal toxicity

SMDS kerosine, a light cut of GTL kerosine (linear and branched alkanes covering the carbon range from C₈ to C₁₂), GTL gas oil (linear and branched alkanes covering the carbon range from C₈ to C₁₆), GTL base oil (linear, branched and cyclic alkanes, covering the carbon range from C₁₈ to C₅₀), and SX30 (only linear alkanes, covering the carbon range from C₁₈ to C₂₅), were tested for acute dermal toxicity according to OECD testing guideline 402 (acute dermal toxicity) in Wistar rats (SMDS kerosine) and Sprague–Dawley CD rats (GTL gas oil, GTL base oil, and SX30). No signs of local or systemic toxicity were seen in the rats treated with SMDS kerosine. In some female rats treated with GTL gas oil, GTL base oil and SX30 light irritation (erythema or desquamation of the skin) were seen, but no signs of systemic toxicity. Overall, all GTL products tested for acute dermal toxicity were found to be not systemically toxic with LD₅₀ values greater than 2000 mg/kg (Robbins 1993c; Rees 1997a; Sanders 2014a, 2014b).

Acute inhalation toxicity

GTL gas oil was tested in nose-only acute inhalation toxicity studies in rats according to OECD testing guideline 436 (acute inhalation toxicity – acute toxic class method). Since GTL products with carbon chain lengths greater than ∼ C₁₂ will have low vapor pressures, they can only be tested as aerosols. Initially, the substance was tested in RccHan:Wist rats at an aerosol concentration of 5.61 ± 0.23 mg/l (4 h exposure) (Griffiths 2013). However, at this concentration, the substance was found to be toxic to the lungs as some of the treated animals showed dark patches in the lungs or abnormally dark lungs. One out of three male rats and one out of three female rats did not survive the exposure. The surviving animals, in contrast, totally recovered in 8–9 d after cessation of exposure (see Table 4). Detailed histopathology of the animals that died following exposure showed pulmonary congestion and edema, the presence of alveolar macrophages, acute alveolar inflammation, and acute perivascular inflammation of the lungs with no effects on the trachea, which is fully consistent with toxicity following aspiration of fluid from coalesced aerosol. The nose-only acute inhalation toxicity study with GTL gas oil was subsequently repeated at a lower concentration (5.11 ± 0.09 mg/l, 4-h exposure) and, at this concentration, all males survived and one out of three female rats died. All surviving animals fully recovered in 7–8 d after cessation of exposure.

Table 4. Acute inhalation toxicity of low-viscosity GTL products.

Product	Viscosity (mm^2/s @ 40 °C)	Concentration (mg/l, 4h)	MMAD^a (μm)	Inhalable fraction (%)	Deaths (# animals died/total #)	Recovery (days) of survivors
GTL base oil 4	18	5.12 ± 0.18	1.22	88.6	0/6	9-10
GTL base oil 3	10	5.09 ± 0.07	1.92	77.3	1/3 ♂, 3/3 ♀	8 (♂)
GTL base oil 3	10	1.05 ± 0.04	1.64	89.4	1/3 ♂, 1/3 ♀	8 (♂), 13 (♀)
GTL solvent GS310	5.9	4.98 ± 0.17	3.17	60.9	2/3 ♂, 1/3 ♀	8
GTL solvent GS310	5.9	1.14 ± 0.12	2.45	78.1	0/3 ♂, 1/3 ♀	6-7
GTL gas oil	3.4	5.61 ± 0.23	2.78	65.7	1/3 ♂, 1/3 ♀	8-9
GTL gas oil	3.4	5.11 ± 0.09	2.16	77.5	0/3 ♂, 1/3 ♀	7-8

^a Mass median aerodynamic diameter.

Two low-viscosity GTL base oil fractions were also tested in a nose-only inhalation acute toxicity test according to OECD testing guideline 436 in RccHan:Wist rats (Griffiths 2014a, 2014b, 2014c). The lowest-viscosity oil, GTL base oil 3 (a cut of GTL base oil, C₁₈–C₃₀, with a viscosity of ∼10 mm²/s at 40 °C and ∼3 mm²/s at 100 °C) was tested at an aerosol concentration of 5.09 ± 0.07 mg/l (4-h exposure). At this concentration, one of the three males and all three female rats in the study died, showing that an aerosol of this material has to be considered to be acutely toxic with a LC₅₀ value below 5 mg/l (4 h), according to the current UN GHS guidelines for Classification and labeling (6th edition, revised 2015). Animals that died during the study showed no clinical signs of toxicity or any gross pathology apart from abnormally dark lungs, again compatible with aspiration into the lungs of the low-viscosity material. The surviving males fully recovered in 8 d. The study was repeated at a concentration of 1.05 ± 0.06 mg/l (4-h exposure). At this concentration, one out of three male rats and one out of three female rats died, indicating that the LC₅₀ value lies between 1.05 and 5.09 mg/L (4-h exposure) (Griffiths 2014b). All surviving animals recovered fully. The surviving male rats appeared normal at day 8 but the surviving females recovered more slowly and appeared normal only at day 13. Detailed histopathology of the lungs indicated that the lesions observed in the lungs, such as congestion, hemorrhage, edema, and alveolar and perivascular inflammation, are consistent with hydrocarbon aspiration-induced inflammation, i.e. a chemical pneumonitis (Griffiths 2014c). When GTL base oil 4 (a cut of GTL base oil, C₂₁–C₅₀, with a viscosity of ∼18 mm²/s at 40 °C and ∼4 mm²/s at 100 °C) was tested at 5.12 ± 0.18 mg/L (4 h), no deaths occurred at all (see Table 4). Some common abnormalities occurred during the study, such as increased respiratory rate, hunched posture, pilo-erection, and wet fur. All three male and two of the female rats exhibited minimal body weight losses on the first day post-exposure. All animals started to show increasing body weights during the recovery period. All animals recovered to appear normal from days 9 to 10 after exposure (Griffiths 2014a).

GTL solvent GS310 (branched alkanes, covering the carbon range from C₁₈ to C₂₄), which has a kinematic viscosity of 5.9 mm²/s at 40 °C, was also tested for acute inhalation toxicity according to OECD testing guideline 436 in RccHan:Wist rats. At an aerosol inhalation concentration of 4.98 ± 0.17 mg/L (4 h) two out of three male rats and one out of three females rats died. All animals exhibited increased respiratory rates during the study and the day after. The surviving animals recovered in 8 d. The study was repeated at a lower concentration of 1.14 ± 0.12 mg/L (4 h). At this concentration all males survived and one out of three females died. All survivors recovered to normal within 6–7 d (Griffiths 2014d). Like GTL base oil 3, aerosols of GTL solvent GS310 have to be considered to be acutely toxic by inhalation with a LC₅₀ value below 5 mg/l (4 h), following the GHS guidelines.

Primary irritation

Some GTL products may induce slight irritation to the skin or the eyes, both in vivo and in vitro. However, the irritating effect is transient and never sufficient to trigger classification under GHS with exception of the GTL solvents GS160 and GS170 (with carbon chain lengths of C₈–C₁₁ and C₉–C₁₂, respectively) which are classifiable as skin irritants.

Dermal irritation

The following 12 materials covering the carbon range of C₈–C₇₀ were tested for primary skin irritation in the New Zealand white rabbit according to OECD testing guideline 404 (acute dermal irritation/corrosion): GTL solvent GS160 (linear and branched alkanes, covering the hydrocarbon range from C₈ to C₁₁), SMDS-kerosine, GTL solvent GS170 (linear and branched alkanes, covering the hydrocarbon range from C₉ to C₁₂), GTL solvent GS215 (linear and branched alkanes, covering the hydrocarbon range from C₁₂ to C₁₅), Sarapar 147 (linear alkanes, covering the hydrocarbon range from C₁₃ to C₁₇), GTL solvent GS310, GTL wax, the Sarawaxes SX30, SX50, and SX70) (n-alkanes, covering the hydrocarbon range from C₁₅ to C₅₀, from C₁₈ to C₂₅, from C₁₉ to C₃₆, and from C₂₅ to C₄₈, respectively), the GTL base oil, and the GTL residual oil (branched, linear and cyclic alkanes, covering the hydrocarbon range from C₄₀ to C₇₀).

The GTL solvents GS160 and GS170 were irritating to the skin with 24–72 h mean erythema scores of 2.2 and 2.1, respectively, and a 24–72 h mean edema score of 2.0 for both solvents (Latour 2014b, 2014c). SMDS kerosine was found to be slightly irritating to the skin, with a primary irritation index of 1.1 (Rees 1997c). SX30 also showed mild transient irritant properties to the skin, with a primary irritation index of 1.2 over the assessment period and 24, 48, and 72 h scores for erythema and edema of 0.89 and 0.67, respectively (Robbins 1993d), which does not lead to classification under GHS. The heavier Sarawaxes showed very slight, transient erythema which resolved completely within 24 h (Van Huygevoort 2003a, 2003b).

The data across the spectrum of carbon chain lengths strongly suggest that the dermal irritant properties are associated with linear and branched alkanes up to a chain length of about C₁₂. The data on GTL solvents GS160 and GS170 (primary irritation scores ≥2.0) seem to confirm this: GTL solvent GS160 was indeed found to be an irritant and because its carbon number range covers the high end of GTL naphtha (linear and branched alkanes C₄–C₁₀), the later should be also considered a skin irritant. Similarly, GTL solvent GS170 (covering the hydrocarbon range from C₉ to C₁₂) was found to be irritant. SMDS kerosine (covering the hydrocarbon range from C₈ to C₁₂) showed some irritation of the skin (primary irritation score 1.1), but not sufficient for GHS classification.

In contrast, GTL solvent GS215 (which covers C₁₂–C₁₅, but contains less than 20% of hydrocarbons with chain lengths C₁₂ or shorter) did not demonstrate persistent irritation (primary irritation score of <1.0): some erythema and edema were noted in the 24 h following application, but the effects disappeared completely within 72 h (Latour 2014d). The GTL products with longer chain lengths (GTL solvent GS310, Sarapar 147, GTL base oil, GTL wax, SX30, SX50, SX70, and GTL residual oil) were found to be non-irritants under the testing conditions (primary irritation scores of 0.0) (Robbins 1993d, Van Huygevoort 2003a, Van Huygevoort 2003b, Baskaran 2004a, Sanders 2007d, 2008c, 2008f, Latour 2014e).

In addition, all eight GTL solvents listed in Table 3 were tested in an in vitro test system using a three-dimensional human skin epidermal model (Episkin-SM^®). The use of this model, in parallel to in vivo studies, had the purpose of improving read across between GTL substances and in particular for GTL hydrocarbon solvents. In these studies, the substances were tested by topical application for 15 min on the skin model according to OECD testing guidelines 439 (in vitro skin irritation: reconstructed human epidermis test method). It was shown that GTL products that were irritants in the rabbit and which consist predominantly of alkanes with 12 or fewer carbon atoms, were also irritant (GS160) or borderline non-irritant (GS170) under the test conditions (Westerink 2014a, 2014b) whereas the solvents with primarily longer carbon chain lengths than C₁₂, such as GTL solvents GS190, GS210, GS215, GS250, GS270, and GS310 were not (Westerink 2014c, 2014d, 2014e, Westerink 2014f, 2014 g, 2014 h).

Ocular irritation

A range of GTL products was also tested for primary eye irritation in the New Zealand white rabbit according to OECD testing guideline 405 (Acute eye irritation/corrosion): GTL solvents GS 160, GS170, GS215, GS310, SMDS kerosine, Sarapar 147, GTL wax (linear and branched alkanes, covering the carbon range C₁₅–C₅₀), GTL base oil, SX30, and GTL residual oil. Some of the GTL solvents and the SX30 induced transient conjunctiva redness in the rabbits, but like SMDS kerosine and Sarapar 147 they were found to have a maximum score for ocular lesions of 0.0 and were considered non-irritants (Robbins 1993e, Rees 1997b, Baskaran 2004b, Latour 2014f, 2014g, 2014h, Latour 2014i). GTL wax, GTL base oil, and GTL residual oil produced a maximum group mean score of 5.3, 6.0, and 6.0, respectively, and were classified as a minimal irritant (Class 3 on a 1–8 scale) to the rabbit eye according to a modified Kay and Calandra classification system (Kay & Calandra 1962) but insufficient to classify under GHS (Sanders 2008a, 2008b, 2008g). The entire range of GTL solvents was also tested in vitro according to OECD guideline 437 (bovine corneal opacity and permeability test method for identifying (i) chemicals inducing serious eye damage and (ii) chemicals not requiring classification for eye irritation or serious eye damage, BCOP test). The in vitro results were aligned with the in vivo results as none of the eight GTL solvents triggered classification for eye irritation in the BCOP test (Westerink 2014i, 2014j, 2014k, 2014l, 2014m, 2014n, 2014o, 2014p).

Sensitization

GTL products are considered to be not sensitizing. Some GTL substances tested positive in the mouse local lymph node assay but not in the Magnusson–Kligman guinea pig maximization test. Overall, the data indicate that GTL substances are not sensitizing. Currently available alternative skin sensitization tests do not seem suitable for GTL substances.

Several GTL products were tested for skin sensitization. The test materials do not easily dissolve in solvents compatible with the test system and a variety of solvents was used to obtain optimal dissolution. Initially, the GTL light waxy raffinate (linear and branched alkanes, covering the carbon range from C₁₅ to C₂₇) and a light paraffin wax, SX30, were tested in the local lymph node assay in the CBA/Ca mouse according to OECD testing guideline 429 (Skin Sensitization – Local Lymph Node Assay). Surprisingly, the materials, which both consist almost exclusively of linear alkanes, gave different results: the GTL light waxy raffinate was negative at 25% w/w solution in 2-butanone (stimulation index of 2.57) but positive with stimulation indices of 3.94 and 4.97 when tested in a 50% and a 100% w/w solution in 2-butanone, respectively (Sanders 2007b). In contrast, the light paraffin wax SX30 was negative when tested in a 25%, 50%, and 100% w/w solution in dimethyl formamide with a stimulation index of 1.58, 2.24, and 2.47, respectively (Sanders 2008e). The heavier paraffin wax Sarawax 50 (SX50, linear alkanes, covering the carbon range from C₁₈ to C₃₆) tested negative for skin sensitization with no dermal reaction in any animal when tested at 10% and 50% in propylene glycol in a Magnusson–Kligman maximization test in the albino Dunkin Hartley Guinea pig according to OECD testing guideline 406 (Skin Sensitization) (Rees 1997d). As it was suspected that the weak positive result for the GTL light waxy raffinate might be false, this material was also tested in the same Magnusson–Kligman maximization test setup. In this test system, there was no indication of any sensitization by the GTL light waxy raffinate (Richeux 2007). GTL wax (covering the carbon range from C₁₅ to C₅₀), GTL base oil (covering the carbon range from C₁₈ to C₅₀), and GTL residual oil (covering the carbon range from C₄₀ to C₇₀) also tested negative in the Guinea pig Magnusson–Kligman maximization test (Richeux 2008a, Richeux 2008b, Richeux 2008c). In addition, four GTL solvents (GTL solvent GS160, GS170, GS215, and GS310) and SMDS kerosine, which is with a carbon range from C₈ to C₁₂ rather similar to some GTL solvents, were tested for skin sensitization in the Guinea pig Magnusson–Kligman maximization test according to OECD testing guideline 406. As expected, all the GTL solvents and the SMDS kerosine gave a sensitization rate of 0% confirming the absence of sensitizing potential for GTL products (Rees 1997e, Latour 2014a, Van Huygevoort 2014a, 2014b, 2014c).

Apart from these in vivo skin sensitization tests, all eight GTL hydrocarbon solvents (Table 3) were tested for their potential to cause skin sensitization in vitro, using the Direct Peptide Reactivity Assay (DPRA) (Gerberick et al. 2004,. 2007, Jeong, An et al. 2013). The DPRA is based on the principle that skin sensitization induction follows three key steps: first, penetration into the viable cell layers of skin tissue, second, binding of the hapten to endogenous proteins and, third, activation of the skin Langerhans cells inducing their maturation and migration to the lymph nodes. A sensitizing substance has to have an electrophilic character or must have ability to induce oxidation of the amino acid moieties within the proteins forming a hapten. Thus, the DPRA mimics the second step where the electrophilic potential of substances is evaluated by their reaction with a model peptide containing cysteine. The free SH-group from the cysteine is monitored in the fluorescence assay in a concentration-dependent manner. Since the model peptide is dissolved in water and requires a pH of 7.4, the evaluation of GTL products, which are not water soluble, is a technical challenge when trying to evaluate their reactivity towards the cysteine moiety. Therefore, an array of solvents including DMSO, THF, ethanol, and acetone were assessed for their suitability to bring the alkanes into solution. Generally the lower molecular weight GTL solvents (which have shorter alkane chains and have a much lower content of branched alkanes) were easier to dissolve, but as the carbon chains become longer, the solutions appeared cloudy and turbid, indicative of an (over)saturated solution. As an alternative and in order to ensure solubility of the test material, a non-ionic surfactant was tested as an alternative solvent. The surfactant was identified as alcohol ethoxylate with a carbon chain of C₁₂–C₁₅ and 7 mol of ethoxylation. The test material was dissolved in the surfactant and further diluted with acetonitrile. In general, the results obtained indicated that the GTL solvents, under the test conditions did not show any reactivity towards the peptide, as measured by peptide depletion. If depletion occurred, its level was usually well below the cut-off value of 20% depletion, which is indicative of a lack of sensitization potential. For two cases, GS170 and GS215, a decrease over 20% was observed. However, in those instances, there was no clear dose dependency or there was not further decrease with increasing dose and hence the results could not be unambiguously explained. In addition, the in vivo tests using the Magnusson–Kligman guinea pig maximization assay were negative for both these solvents. The conclusion is that the DPRA assay does not seem to be well suited to test the lipophilic GTL substances due to poor solubility and unstable solutions obtained with alternative solvents.

Repeated-dose toxicity

In general, repeated-dose toxicity studies, both by oral gavage and dietary, with several GTL products showed no systemic toxicity, or selective toxicity to organs relevant to human health. The studies with GTL naphtha, however, showed hydrocarbon-induced male rat nephrotoxicity which was similar to the nephrotoxicity observed following exposure to the conventional petroleum-derived analogs and which is considered to be of no relevance to humans. The studies with GTL waxes showed rat-strain dependent effects, similar to those observed with their conventional petroleum-derived analogs, on liver and mesenteric lymph nodes.

GTL products have been investigated in oral repeated-dose studies in the rat, either by gavage or by dietary intake. GTL naphtha and GTL base oil were tested in 90-d repeated-dose oral gavage toxicity studies in the rat, according to OECD testing Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents). In both studies, an additional group of animals was included to study the recovery over a period of 28 d after the final dosing of the test substance.

For the 90-d oral gavage repeated-dose toxicity studies, GTL naphtha and GTL base oil were chosen to bracket the entire range of GTL products, considering that constituents with carbon chain lengths greater than C₅₀ have negligible bioavailability. Essentially these two products tested cover the carbon range from C₄ to C₁₀ and from C₁₈ to C₅₀, which leaves a gap for the constituents in the carbon range from C₁₀ to C₁₈. To cover this gap, but reduce the number of animals used for toxicity testing of the suite of GTL products, a range of major organs were harvested for the evaluation of histopathological changes in the two-generation reproductive toxicity study in the rat with GTL gas oil according to the OECD guideline 416 (Two-generation Reproduction Toxicity Study) (vide infra). Because these organs are normally assessed in studies according to this OECD testing guideline, additional histopathology does not affect the execution or reliability of this study. GTL gas oil comprises constituents with a carbon range of C₈–C₂₆ and the additional histopathology on the major organs that are usually investigated in a study compliant to the OECD testing guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents) was included to strengthen the read-across case with the two bracketing GTL products

In addition to investigating the systemic toxicity of the heavier (longer carbon chain lengths) materials, GTL residual oil was tested in 28-d repeated dose oral gavage toxicity study in the rat, according to OECD testing Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents). In this study, an additional group of animals was included to investigate the recovery over a period of 14 d following the final dosing of the test material. Finally, a series of 90-d oral dietary repeated-dose toxicity studies with several GTL waxes was performed.

GTL naphtha

In the oral gavage repeated-dose study with GTL naphtha, Sprague–Dawley rats (10 males and 10 females per dose level) were treated for 90 consecutive days with 0, 50, 200, or 750 mg/kg of the test material dissolved in arachis oil. Two recovery groups, each of 10 males and 10 females, were treated with either 750 mg/kg of the test material or the vehicle only for 90 consecutive days and subsequently maintained for a period of 28 d without treatment. All animals were observed throughout the study and blood chemistry and hematological parameters were evaluated at termination. In addition, behavioral assessment and functional tests, including motor activity and sensory reactivity, were conducted during the study. At the end of the study, the animals were sacrificed and organs and tissues selected for detailed histopathological observations. No toxicologically significant effects were identified in clinical observations, behavioral parameters, functional performance tests, or sensory reactivity assessments. Body weight and body weight gain, food and water consumption, ophthalmoscopic evaluations, and hematological parameters were not adversely affected by the test material. As expected, the kidneys of some male rats from the high-dose group (four out of 10) and the middle-dose group (one out of 10) showed adverse effects in the gross pathological evaluation, which did not improve during recovery. Also the mean kidney weights increased statistically significantly with increasing dose and, although there was some improvement, did not turn back to normal within the recovery period (Table 5(a)). The detailed histopathological evaluations showed tubular basophilia with concomitant degeneration and necrosis with accumulation of globular eosinophilic material in the tubular epithelium in the males treated with 750 and 200 mg/kg whereas males treated with 50 mg/kg of the GTL naphtha only showed accumulation of globular eosinophilic material in the tubular epithelium. Additional Mallory's Heidenhain staining was performed on the kidneys to confirm the presence of α₂-microglobulin accumulation in the tubular epithelium. Although the accumulation of globular eosinophilic material had regressed to normal background values in the males treated with 750 mg/kg of the test material during the recovery period, there were no signs of recovery of the tubular degeneration and necrosis. Since these effects are fully consistent with the well-known light-hydrocarbon induced nephropathy, induced by accumulation of α₂-microglobulin, which is peculiar to male rats, they were considered to have no relevance for human hazard assessment (Gibson & Bus 1988). The histopathological examinations also showed, in both male and female rats, centrilobular hepatocyte enlargement, which was fully reversible upon cessation of treatment with NOAELs of 200 and 50 mg/kg body weight for female and male rats, respectively. Hepatocyte enlargement is commonly observed in rats upon administration of xenobiotics and is considered to be adaptive in nature if there are no associated degenerative or inflammatory changes observed as was the case in these studies. In line with this, the increased liver weights observed in the males treated with 200 and 750 mg/kg body weight and in the females treated with 750 mg/kg body weight, returned to normal during the 28 recovery period (Tables 5(a,b)). In addition, the increase of liver weight was less than 10% and the middle-dose level and less than 25% at the highest dose level, which is considered an adaptive rather than an adverse effect (Hall et al. 2012). Therefore, these observations are considered less relevant for human hazard assessment. Since GTL naphtha contains approximately 6% of n-hexane, a substance that is classified as both a neurotoxicant and a reproductive toxicant, it is worth noting that no effects were seen in clinical observations, behavioral parameters, functional performance tests, or sensory reactivity assessments and that also no histopathological effects were observed in the reproductive organs at any dose level (Dunster et al. 2009).

Table 5(a). Group mean terminal body and organ weights (in g) of male rats (n = 10) treated with GTL naphtha by oral gavage for 90 d and after 28 d of recovery.

GTL naphtha	Dose (mg/kg/d)
	Day 91				Day 119 (recovery)
Males	0	50	200	750	0	750
Body weight	581	589	564	558	628	592
SD	43	56	59	57	65	35
Adrenals	0.058	0.0569	0.061	0.053	0.0576	0.0519
SD	0.010	0.0079	0.010	0.014	0.0072	0.0054
Brain	2.15	2.13	2.09	2.08	2.07	2.09
SD	0.07	0.11	0.10	0.10	0.11	0.12
Epididymides	1.51	1.54	1.47	1.51	1.64	1.66
SD	0.13	0.18	0.17	0.19	0.15	0.13
Heart	1.93	1.93	1.94	1.91	1.79	1.74
SD	0.26	0.14	0.26	0.15	0.24	0.14
Kidneys	3.16	3.48*	4.06**	4.37**	3.50	3.97**
SD	0.39	0.25	0.66	0.72	0.39	0.43
Liver	18.1	19.4	19.8**	22.4**	19.3	18.3
SD	1.9	2.2	2.5	3.3	1.9	1.3
Spleen	0.81	0.84	0.89	0.84	0.87	0.90
SD	0.10	0.14	0.20	0.12	0.16	0.12
Testes	3.63	3.64	3.60	3.58	3.84	3.74
SD	0.23	0.22	0.16	0.29	0.25	0.15
Thymus	0.46	0.469	0.49	0.462	0.457	0.412
SD	0.12	0.074	0.15	0.087	0.068	0.078

* p < 0.05; **p < 0.01.

Table 5(b). Group mean terminal body and organ weights (in g) of female rats (n = 10) treated with GTL naphtha by oral gavage for 90 d and after 28 d of recovery.

GTL naphtha	Dose (mg/kg/d)
	Day 91				Day 119 (recovery)
Females	0	50	200	750	0	750
Body weight	304	282	304	282	295	321
SD	22	22	25	22	19	18
Adrenals	0.051	0.0574	0.0520	0.0565	0.057	0.0620
SD	0.013	0.0090	0.071	0.0060	0.015	0.0086
Brain	1.91	1859	1911	1867	1926	1958
SD	0.07	0.041	0.083	0.079	0.077	0.043
Heart	1.18	1.18	1.18	1.12	1.08	1.17
SD	0.16	0.20	0.15	0.17	0.15	0.16
Kidneys	1.79	1.69	1.86	1.82	1.72	1.95*
SD	0.17	0.20	0.20	0.21	0.15	0.12
Liver	10.4	10.0	10.50	10.9*	10.0	10.4
SD	1.2	1.2	0.93	1.7	1.0	1.0
Ovaries	0.114	0.116	0.116	0.095	0.099	0.101
SD	0.032	0.028	0.020	0.020	0.024	0.025
Spleen	0.495	0.465	0.486	0.471	0.499	0.536
SD	0.079	0.057	0.079	0.076	0.058	0.065
Thymus	0.366	0.360	0.387	0.35	0.347	0.304
SD	0.087	0.071	0.082	0.10	0.066	0.064
Uterus	0.64	0.67	0.72	0.73	0.74	0.68
SD	0.15	0.17	0.35	0.24	0.35	0.19

* p < 0.05.

GTL base oil

The study design of the oral gavage repeated-dose study with GTL base oil was very similar to the study with GTL naphtha, but a higher dose level was used for the high dose group (based on a range finding study indicating no toxicity at the limit dose) (Dunster 2009). Sprague–Dawley rats (10 males and 10 females per dose level) were treated for 90 consecutive days with 0, 50, 200, or 1000 mg/kg of the test material dissolved in arachis oil. Two recovery groups, each of 10 males and 10 females, were treated with either 1000 mg/kg of the test material or the vehicle only for 90 consecutive days and subsequently maintained for a period of 28 d without treatment. The same gross pathology and observational, clinical, and functional studies were performed as in the GTL naphtha repeated-dose oral gavage study. No toxicologically significant effects were identified in any of these assessments (Tables 6(a,b)). In the detailed histopathology, however, some distinct observations were made. In the lungs, a higher incidence of higher severity accumulations of alveolar macrophages was observed in the 1000 and 200 mg/kg groups of both sexes. In addition, the cytoplasm of the alveolar macrophages seemed to be more vacuolated than in the low-dose group or controls. These effects did not significantly regress during the recovery period. In the mesenteric lymph nodes, vacuolated histiocytes were observed in the 1000 and 200 mg/kg groups of the females (but not the males). Again, the effect did not seem to regress during the recovery period. An increase in histiocytes within the mesenteric lymph nodes can be seen as a nominal response to poorly absorbed materials (Gopinath et al. 1987). Since contact exposure to the material was via the gut and secondarily through aspiration of small quantities of test material in the lungs, such material would be phagocytosed by histiocytes and macrophages. The cellular breakdown of these inert materials may be slow and the accumulation of the material inside the cells may lead to a “foamy” appearance. This process is neither proliferative nor degenerative but merely a part of a normal body response to high-level exposure to inert materials. As a consequence the observations in the lungs and mesenteric lymph nodes were not considered to be adverse effects. To test this interpretation, a 28-d dietary-repeated dose study was performed in the rat at the request of the USA regulatory authorities. Rats were fed a diet containing GTL base oil 3 (carbon range C₁₈–C₃₀) at concentrations of 0 (control), 750, 3750, and 15 000 ppm, corresponding to the actual mean achieved dose levels of 0, 63, 308, and 1267 mg/kg body weight/day (McRae 2014). In confirmation, no alveolar accumulation of macrophages was observed, nor was any other effect observed in the lungs. Minimal to moderate apoptosis and single cell necrosis were observed in the lamina propria/crypt of the duodenum, jejunum, and ileum of the males of the highest dose group. In addition, slight lymphocytosis was noted in the spleen of three males of the highest dose group. Lymphocytosis was also seen in the mesenteric lymph nodes and Peyer’s patches at a minimal degree in the male low- and middle-dose groups and at a minimal to moderate level in the male high-dose group. Minimal lymphocytosis was also observed in the mesenteric lymph nodes of a single low dose female rat and of Peyer’s patches in four females of the low and of the high-dose female rats, but not in the middle-dose females. The lymphocytosis observed is likely a corpus alienum response in an attempt of the body to clear the inert material.

Table 6(a). Group mean terminal body and organ weights (in g) of male rats (n = 10) treated with GTL base oil by oral gavage for 90 d and after 28 d of recovery.

GTL base oil	Dose (mg/kg/d)
	Day 91				Day 119 (recovery)
Males	0	50	200	1000	0	1000
Body weight	571	581	602	577	603	599
SD	70	57	49	60	58	41
Adrenals	0.0536	0.054	0.0572	0.0567	0.0599	0.0556
SD	0.0086	0.012	0.0097	0.0055	0.0068	0.0049
Brain	2120	2172	2137	2171	2154	2143
SD	0.081	0.088	0.089	0.071	0.085	0.068
Epididymides	1.56	149	1.66	1.50	1.55	1.55
SD	0.14	0.13	0.14	0.18	0.23	0.18
Heart	1.90	1.89	2.14	2.05	2.28	1.94
SD	0.29	0.22	0.25	0.25	0.53	0.28
Kidneys	3.14	3.27	3.41	3.42	3.51	3.48
SD	0.35	0.36	0.37	0.33	0.26	0.25
Liver	17.9	17.6	17.7	17.8	17.7	17.9
SD	2.7	2.9	2.0	1.9	2.8	1.1
Spleen	0.75	0.823	0.891	0.82	0.86	0.97
SD	0.10	0.078	0.098	0.13	0.11	0.11
Testes	3.77	3.78	3.85	3.71	3.61	3.80
SD	0.25	0.29	0.17	0.29	0.32	0.29
Thymus	0.429	0.456	0.460	0.45	0.49	0.47
SD	0.083	0.070	0.078	0.12	0.10	0.11

Table 6(b). Group mean terminal body and organ weights (in g) of female rats (n = 10) treated with GTL base oil by oral gavage for 90 d and after 28 d of recovery.

GTL base oil	Dose (mg/kg/d)
	Day 91				Day 119 (recovery)
Females	0	50	200	1000	0	1000
Body weight	301	325	306	325	334	339
SD	20	27	22	28	26	17
Adrenals	0.056	0.061	0.057	0.062	0.0710	0.0615*
SD	0.013	0.016	0.011	0.016	0.0077	0.0076
Brain	1.90	1.94	1932	1.95	2009	1.97
SD	0.11	0.10	0.066	0.12	0.074	0.10
Heart	1.18	1.15	1.15	1162	1.33	1.34
SD	0.13	0.13	0.13	0.083	0.14	0.10
Kidneys	1.83	1.91	1.84	1.90	2.03	1.89
SD	0.16	0.25	0.17	0.19	0.15	0.16
Liver	9.9	10.6	10.2	10.6	10.59	10.70
SD	1.0	1.1	1.1	1.1	0.93	0.82
Ovaries	0.113	0.124	0.121	0.121	0.1106	0.106
SD	0.024	0.024	0.032	0.025	0.0092	0.018
Spleen	0.546	0.579	0.553	0.578	0.59	0.584
SD	0.079	0.076	0.090	0.077	0.10	0.070
Thymus	0.38	0.360	0.344	0.402	0.334	0.412
SD	0.11	0.078	0.087	0.074	0.082	0.099
Uterus	0.71	0.69	0.78	0.82	0.88	0.83
SD	0.23	0.20	0.26	0.48	0.25	0.23

* p < 0.01.

GTL residual oil

In the oral repeated-dose study with GTL residual oil, Wistar rats (five males and five females per dose level) were treated for 28 consecutive days with 0, 30, 300, and 1000 mg/kg body weight of the test material dissolved in polyethylene glycol 400 (final dosing volume 4.0 ml/kg body weight). Two recovery groups (each five males and five females), were treated with 1000 mg/kg body weight or the vehicle alone for 28 consecutive days and then maintained for a further 14 d without administration of the test material (Dunster 2010). All animals were observed throughout the study for clinical signs, functional observations, body weight change, food and water consumption, blood chemistry, and hematological parameters were evaluated at termination. In addition, behavioral assessment and functional tests, including motor activity and sensory reactivity, were conducted during the study. At the end of the study, the animals were sacrificed and organs and tissues selected for detailed histopathological observations. No toxicologically significant effects were identified in clinical observations, behavioral parameters, functional performance tests, or sensory reactivity assessments. Body weight and body weight gain, food and water consumption, ophthalmoscopic evaluations, and hematological parameters were not adversely affected by the test material. The histopathology showed an increased extramedullary hematopoiesis in the spleen of the females treated with 300 and 1000 mg/kg/d of the GTL residual oil (Dunster 2010). This effect was not seen in the females treated with 30 mg/kg/d or in the males in any dose group. These spleen effects observed in the mid- and high-dose females was fully reversible and were considered adaptive rather than adverse since there was no concomitant effect on any of the hematological parameters (Greaves 2011).

GTL waxes

Historically, repeated dose testing of paraffinic hydrocarbons has been shown to produce strain-specific responses in rats. Several GTL waxes were tested in 90-d dietary repeated-dose toxicity studies in female Fischer 344 and Sprague–Dawley rats (Robbins 1993f). In these studies, three Sarawaxes, SX30, SX50, and SX701, were tested and mixed with the diet at concentrations of 0.002%, 0.02%, 0.2%, and 2%. SX701, and also SX702 (vide infra), are essentially the same as SX70, i.e. waxes predominantly consisting of linear alkanes with chain lengths C₂₅–C₄₈ (see Table 2). However, both the SX701 and SX702 are pre-production materials, synthesized in a pilot plant, and have slightly higher levels of branched (methylated) alkanes than SX70, with SX702 being more branched than SX701. Groups of five female rats were fed these diets for 90 d. In addition, a group of 10 Fischer 344 rats were fed control diet and a group of five Fischer 344 rats were fed a diet containing 5% SX701. Also an additional group of five Fischer 344 rats was fed the 2% diet to provide tissues for non-polar hydrocarbon concentration measurements. Furthermore, groups of five Sprague–Dawley rats were fed the 2% diet of each of the waxes or a control diet to provide a comparison of findings between the two rat strains. The dosing regimen is provided in Table 7(a).

Table 7(a). Comparative study design for the 90-d dietary repeated dose study of SX30, SX50 and SX701 in female Fischer 344 and Sprague–Dawley rats.

Test material	Rat strain	# animals per dose level	Dose level (% in feed)
Control	Fischer 344	10	0
Control	Sprague–Dawley	5	0
SX30	Fischer 344	5	0.002
SX30	Fischer 344	5	0.02
SX30	Fischer 344	5	0.2
SX30	Fischer 344	5 + 5	2.0
SX30	Sprague–Dawley	5	2.0
SX50	Fischer 344	5	0.002
SX50	Fischer 344	5	0.02
SX50	Fischer 344	5	0.2
SX50	Fischer 344	5 + 5	2.0
SX50	Sprague–Dawley	5	2.0
SX701	Fischer 344	5	0.002
SX701	Fischer 344	5	0.02
SX701	Fischer 344	5	0.2
SX701	Fischer 344	5 + 5	2.0
SX701	Sprague–Dawley	5	2.0
SX701	Fischer 344	5	5.0

All animals appeared normal in behavior and no abnormalities in condition, body weight, or food intake were observed in either the Fischer 344 or Sprague–Dawley rats. However, absolute and relative liver weights were increased in the Fischer 344 rats fed 2.0% SX50 and both 2.0 and 5.0% SX701. In the Sprague–Dawley rats only the relative liver weight of the rats fed 2.0% SX30 was increased. With regard to the mesenteric lymph nodes, in the Fischer 344 rats, both absolute and relative weight were increased in rats fed 2.0% SX30, in rats fed 0.2% and 2.0% SX50, and in rats fed 0.02%, 0.2%, 2.0%, and 5.0% SX701. In Sprague–Dawley rats, both absolute and relative weights of not only the mesenteric lymph nodes were increased in animals fed SX30 and SX50 but only the absolute mesenteric lymph node weights in animals fed SX701. Both absolute and relative spleen weights were increased in Fischer 344 rats fed 2.0% SX30, in rats fed 0.2% and 2.0% SX50, and in rats fed 0.2%, 2.0%, and 5.0% SX701. However, no changes in either absolute or relative spleen weights were observed in Sprague–Dawley rats fed any of the three GTL waxes. Detailed histopathology showed treatment-related effects in the liver, mesenteric lymph nodes, and the heart. In the Fischer 344 rats fed SX50 and SX701, microgranulomata were present in the liver at the dose levels of 0.2% and above, while periportal vacuolation was present at dose levels of 2.0% and 5.0% in animals fed SX701. Foci of inflammatory cells were observed in the 0.002% and 0.02% SX701 fed Fischer 344 rats and also in the Sprague–Dawley rats 2.0% SX50 or SX701. No liver lesions were seen in rats, either Fischer 344 or Sprague–Dawley, fed SX30. In the Fischer 344 rats, lesions in the mesenteric lymph nodes were observed which comprised histiocytosis, reactive node, and adenitis. Adenitis was seen in the 2.0% SX30, SX50, and SX701 groups and in the 5.0% SX701 group. Various degrees of histiocytosis were seen in the lower dose groups fed SX50 and SX701. Heart lesions, in the form of thickening, chronic inflammation, and increased basophilia of the mitral valve were seen in Fischer 344 rats fed 2.0% SX50 and 2.0% and 5.0% SX701. Analysis of the organs for non-polar hydrocarbon material showed an accumulation in the livers of the Fischer 344 rats following treatment with SX50 and SX701 and accumulation in the mesenteric lymph nodes following treatment with all three waxes (Robbins 1993f).

Finally, a 90-d dietary repeated dose study was conducted on GTL waxes SX701, SX702, and SX100 (linear alkanes, covering the carbon range C₃₈–C₉₀) in Fischer 344 and Sprague–Dawley rats (Robbins 1993g). Groups of five female Fischer 344 rats were fed a diet containing SX701 at concentrations of 2.0% and 5.0% in the feed and SX702 or SX100 at 0.002%, 0.02%, 0.2%, and2.0% in the feed. The control group consisted of 10 female Fischer 344 rats that received the feed without wax. Additional groups of five female Fischer 344 rats were fed control diet or feed with 5.0% SX701 for 4, 8, or 13 weeks, or control diet or diet with 5.0% SX701 for 13 weeks, followed by control diet for periods of 6 weeks or 12 weeks in a reversal study. Again, groups of five female Sprague–Dawley rats were fed either a control diet or 2.0% SX701 for 13 weeks (Table 7(b)).

Table 7(b). Study design for the 90-d dietary repeated dose study, with recovery periods, of SX701, SX702, and SX100 in female Fischer 344 and Sprague–Dawley rats.

Test material	Rat strain	# animalsper dose level	Dose levels (% in feed)	Exposure duration (weeks)	Recovery duration (weeks)
Control	Fischer 344	5	0	4	0
Control	Fischer 344	5	0	4	6
Control	Fischer 344	5	0	4	12
Control	Fischer 344	5	0	8	0
Control	Fischer 344	5	0	8	6
Control	Fischer 344	5	0	8	12
Control	Fischer 344	5	0	13	0
Control	Fischer 344	5	0	13	6
Control	Fischer 344	5	0	13	12
Control	Fischer 344	10	0	13	0
Control	Sprague–Dawley	5	0	13	0
SX701	Fischer 344	5	2.0	13	0
SX701	Fischer 344	5	5.0	4	0
SX701	Fischer 344	5	5.0	4	6
SX701	Fischer 344	5	5.0	4	12
SX701	Fischer 344	5	5.0	8	0
SX701	Fischer 344	5	5.0	8	6
SX701	Fischer 344	5	5.0	8	12
SX701	Fischer 344	5	5.0	13	0
SX701	Fischer 344	5	5.0	13	6
SX701	Fischer 344	5	5.0	13	12
SX701	Fischer 344	5	5.0	6	24
SX701	Sprague–Dawley	5	2.0	13	0
SX702	Fischer 344	5	0.002	13	0
SX702	Fischer 344	5	0.02	13	0
SX702	Fischer 344	5	0.2	13	0
SX702	Fischer 344	10	2.0	13	0
SX100	Fischer 344	5	0.002	13	0
SX100	Fischer 344	5	0.02	13	0
SX100	Fischer 344	5	0.2	13	0
SX100	Fischer 344	10	2.0	13	0

There were no treatment-related effects on body weight gain or food intake over the course of the studies in any of the exposure groups, with exception of the Fischer 344 rats that were fed 5.0% SX701 for 13 weeks. The animals in this dose group had red staining around the face, occasional staining around the genital area and areas of unkempt fur. These effects appeared intermittently after 10 d of treatment and improved during the reversibility stages. Similar effects were observed in animals from the control and other treatment groups but at lower incidence and severity. In the Fischer 344 rats fed 2.0% SX701 or SX702 for 13 weeks and in the Fischer 344 rats fed 5.0% SX701 for 4, 8, or 13 weeks and for 13 weeks with 6 or 12 week reversal, absolute, and relative liver weights were increased compared to control. The absolute and relative mesenteric lymph node weights were also increased compared with controls in Fischer 344 rats fed SX701 at 2.0% SX701 and Fischer 344 rats fed SX702 at 0.2 and 2.0% for 13 weeks and also in Fischer 344 rats fed SX701 at 5.0% for 4, 8, or 13 weeks or for 13 weeks with 6 or 12 weeks reversal. Also in the Sprague–Dawley rats fed 2.0% SX701, the absolute and the relative mesenteric lymph node weight were higher than in the controls. Absolute and relative spleen weights were higher in Fischer 344 rats fed 2.0% SX701 for 13 weeks and in Fischer 344 rats fed 5.0% SX701 for 4, 8, or 13 weeks or for 13 weeks followed by 6 or 12 weeks reversal. Relative spleen weight only was higher in Fischer 344 rats fed 0.02%, 0.2%, or 2.0% SX702. Liver and spleen weights were not affected by treatment with either SX701, SX702, or SX100 in Sprague–Dawley rats. Treatment-related lesions were observed in the Fischer 344 rats fed SX701 for 4, 6, 8, or 13 weeks and fed for 13 weeks followed with 6 or 12 weeks reversal in the liver, in the mesenteric lymph nodes, and with the exception of the treatment for 4 weeks the mitral valve of the heart. The treatment with SX702 resulted in lesions in the liver and in the mesenteric lymph nodes at all dose levels apart from the lowest level (0.002%). Reversion to the control diet after treatment with SX701 gradually made the lesions less severe. In the mesenteric lymph nodes, a pale eosinophilic homogeneous acellular material, locally extensive, was present. The Fischer 344 rats fed SX100 for 13 weeks showed no treatment-related histopathological changes. In general, effects caused by SX701 were slightly more severe than those caused by SX702 which were in turn more severe than those caused by SX100. The animals partially recovered when the exposure was stopped as treatment-related histopathological changes got less severe during the reversal period (Robbins 1993g).

Reproductive and prenatal developmental toxicity

Based upon test data for GTL naphtha, GTL gas oil, and GTL base oil, the GTL materials are unlikely to harm reproduction or prenatal development. No effects on either male or female reproductive organs were found in 90-d repeated dose studies in rats with GTL naphtha and GTL base oil. No reproductive effects or prenatal harm was found in two-generation and prenatal testing conducted in rats with both GTL gas oil and GTL base oil.

Reproductive toxicity.

The reproductive toxicity of GTL products was investigated in a number of studies in the rat. GTL gas oil and GTL base oil were tested in two-generation reproductive toxicity oral gavage studies in the rat according to OECD testing guideline 416 (Two-generation Reproduction Toxicity Study) (Faiola 2011a, 2011b; Boogaard & Roberts 2015). In addition, the same two GTL substances were also tested in rat prenatal development toxicity studies according to OECD testing guideline 414 (Prenatal Developmental Toxicity Study) (Dunster 2014; Senn 2014).

GTL gas oil.

In the two-generation reproductive toxicity study with GTL gas oil, the Sprague–-Dawley rats were dosed by gavage with GTL gas oil, dissolved in corn oil (dosing volume of 2.0 ml/kg body weight), at dose levels of 0, 50, 200, and 750 mg/kg (Faiola 2011b). The males and females of the F₀ generation (25 males and 25 females per dose group) were 42–46 d old when dosing started and were dosed for at least 70 consecutive days before mating. The test material was administered to the offspring (the F₁ generation, 28 males and 28 females per dose group) as of post-natal day 22. The F₀ and F₁ males received the test material throughout the mating phase until the day prior to euthanasia; the females received the test material throughout mating, gestation, and lactation through the day prior to euthanasia. All animals were observed at least twice daily; clinical observations, body weights, and food consumption were recorded at appropriate intervals for males throughout the study and for females prior to mating and during gestation and lactation. Vaginal smears were performed daily for the determination of estrous cycles beginning 21 d prior to pairing until mating occurred. All F₀ and F₁ females were allowed to deliver and rear their pups until weaning on post-natal day 21. For both generations (F₁ and F₂), on post-natal day 4 litters were arbitrarily culled to 10 pups per litter (five per sex, when possible) to reduce variability due to litter size. Offspring (28/sex/group) from the pairing of the F₀ animals were selected on post-natal day 21 to constitute the F₁ generation. Developmental landmarks (balano-preputial separation and vaginal patency) were evaluated for the selected F₁ rats. Non-selected F₁ pups were necropsied on post-natal day 21, and all F₂ pups were necropsied on post-natal day 21. Selected organs (brain, spleen, and thymus) were weighed from one pup/sex/litter (when possible) from both F₁ and F₂ pups that were necropsied on post-natal day 21. Each F₀ and F₁ parental animal received a complete detailed gross necropsy (the surviving female parental animals were necropsied on lactation day 21), and selected organs were weighed. Spermatogenic endpoints [sperm motility (including progressive motility), morphology, and number] were recorded for F₀ and F₁ males as appropriate, and ovarian primordial follicle counts and corpora lutea counts were recorded for all F₁ females in the control and high-dose groups. Designated tissues from all F₀ and F₁ parental animals were examined microscopically as described in the OECD testing guideline 416. In addition to these designated tissues, a series of additional organs (brains, liver, kidneys, spleen, and thyroid) from both F₀ and F₁ adults also underwent full histopathological examination since a sub-chronic repeated-dose study was not separately performed with GTL gas oil. The fact that in the 90-d repeated-dose studies the test materials were dissolved in another vegetable oil (arachis oil instead of corn oil) was considered inconsequential with regard to histopathology.

Administration of GTL gas oil to male and female rats at dosages up to 750 mg/kg/d had no effect on reproductive performance or gestation length and parturition of both the F₀ and F₁ parental generations (Table 8(a,b)). In addition, there were no test item-related effects on F₁ and F₂ litter parameters, postnatal survival, physical condition/mortality, ano-genital distance, and pup body weights (Table 8(c)). Vaginal patency of F₁ females was unaffected by test item administration. There was a small, but statistically significant, test-article-related increase in the mean age (and adjusted age) of attainment of balano-preputial separation in F₁ males. However, this change was not considered adverse as the age (and adjusted age) of attainment fell within the historical control data range for this parameter. Reproductive performance parameters (mating and fertility indices) for F₁ males given 750 mg/kg/d were not statistically significantly different from the control group and also fell within the historical control data range. Sperm morphology assessments of F₁ males showed a small, but statistically significant, increase in the percentage of abnormal sperm in the males given 750 mg/kg/d (Table 8(b)). This change was also not considered adverse since the percent of abnormal sperm seen fell within the historical control data range for this parameter, reproductive performance parameters (mating and fertility indices) for F₁ males given 750 mg/kg/d were unaffected, and there were no microscopic findings in the testes. The control males from the F₀ generation showed an unexplained low fertility with male mating indices of 69.6%, 80.0%, 95.8%, and 90.9% for controls, 50 mg/kg, 200 mg/kg, and 750 mg/kg dose groups, respectively. To compensate for reduced statistical sensitivity of the first generation litter data, the sizes of the F₁ groups were increased from 25 to 28 rats of each gender. There were no adverse test item-related effects on F₀ and F₁ parental body weights, body weight changes, feed consumption, and food efficiency.

Table 8. Two-generation reproductive toxicity evaluation of GTL gas oil.

(a) Systemic toxicity
GTL gas oil	Dose (mg/kg/d)
F1 adults	0	50	200	750
Pre-mating body weight gain (D 0 – 70) (g)
F0 – males	356	382	363.1	378
SD	11	11	8.8	10
F0 – females	139.6	148.2	151.5	148.7
SD	5.1	5.5	4.5	5.5
Lung weight (g)
F0 – males	2.11	2.318*	2.257	2.917**
SD	0.042	0.054	0.067	0.12
F0 – females	1.595	1.651	1.65	1.817
SD	0.023	0.024	0.037	0.058
Lung histopathology – chronic interstitial alveolar inflammation (incidence of total)
F0 – males	13 of 25	NE	NE	24 of 25
F0 – females	4 of 25	NE	NE	22 of 25
F1 adults
Pre-mating body weight gain (D 0 – 70) (g)
F1 – males	411.8	396.6	430.6	403.5
SD	9.2	6.7	9.3	8.6
F1 – females	128.8	146.8	151.9#	137.4
SD	5.3	5.6	5.9	5.5
Lung weight (g)
F1 – males	2.415	2.498	2.489	3.368**
SD	0.045	0.059	0.056	0.017
F1 – females	1.702	1.756	1.745	1.922**
SD	0.047	0.034	0.032	0.058
Lung histopathology – chronic interstitial alveolar inflammation(incidence of total)
F1 – males	9 of 28	NE	NE	24 of 24
F1 – females	0 of 28	NE	NE	24 of 24

NE: not evaluated. *p < 0.05; **p < 0.01 (Kruskal–Wallis); #p < 0.05 (Dunnett).

(b) Reproductive performance

GTL diesel	Dose (mg/kg/d)
F0 adults – females	0	50	200	750
Ovary weight (pair) (g) – F0	0.1013	0.1082	0.1081	0.104
SD	0.0036	0.0042	0.0036	0.0036
Estrous cycle length (days) – F0	3.9	4.0	4.0	3.8
SD	0.1	0.1	0.1	0.1
GD 0 – 20 body weight gain – F0	142.2	142.3	134.3	132.1
SD	6.1	5.0	6.1	6.7
F1 adults – females
Ovary weight (left) (g) – F1	0.0558	0.0576	0.0564	0.0549
SD	0.0031	0.0024	0.0027	0.0023
Estrous cycle length (days) – F1	4.0	4.3	4.1	4.2
SD	0.1	0.1	0.1	0.1
GD 0 – 20 body weight gain – F1	139.9	144.4	141.9	142.3
SD	6.2	5.2	5.9	7.4
F0 adults – males
Testis weight (left) (g) – F0	1.775	1.842	1.837	1.796
SD	0.066	0.026	0.035	0.031
Sperm production efficiency (10^6/g testis/d) – F0	28.4	NE	NE	30.4
SD	1.5			1.1
Epididymal sperm concentration (10^6/g) – F0	693	742	762	767
SD	33	18	24	18
% Motile sperm – F0	77.1	85.1	81.8	84.6
SD	4.8	1.6	1.3	1.6
% Abnormal sperm – F0	9.50	NE	NE	2.04
SD	5.60			0.11
F1 adults – males
Testis weight (left) (g) – F1	1.917	1.929	1.922	1.954
SD	0.028	0.026	0.036	0.029
Sperm production efficiency (10^6/g testis/d) – F1	25.42	26.22	25.09	23.81
SD	0.61	0.64	0.54	0.75
Epididymal sperm concentration (106/g) – F1	794	803	813	800
SD	17	16	13	24
% Motile sperm – F1	84.4	85.9	86.6	85.8
SD	1.6	1.5	1.2	1.5
% Abnormal sperm – F1	1.13	1.53	1.55	2.29**
SD	0.06	0.09	0.08	0.38

NE: not evaluated.

** p < 0.01, Dunnett-test.

(c) Litter data

GTL gas oil	Dose (mg/kg/d)
F1 pups – females	0	50	200	750
PND 0 weight (g)	6.6	6.7	6.6	6.6
SD	0.2	0.1	0.1	0.2
Adjusted ano-genital distance (mm)	1	1.05	1.04	1.05
SD	0.03	0.03	0.03	0.03
PND 21 weight (g)	58.6	56.8	56.4	55.8
SD	1.2	1.6	1	1.3
Adjusted age at vaginal patency (d)	30.1	30.5	30.6	30.5
SD	0.2	0.2	0.2	0.2
F1 pups – males
PND 0 weight (g)	6.8	7.1	6.9	7.1
SD	0.2	0.2	0.1	0.2
Adjusted ano-genital distance (mm)	2.07	2.17	2.13	2.13
SD	0.05	0.05	0.04	0.05
PND 21 weight (g)	60.9	60.8	59.3	59.7
SD	1.1	1.7	1	1.4
Adjusted age at preputial separation (d)	42	43	43.1	43.3*
SD	0.3	0.3	0.3	0.4
F2 pups – females
PND 0 weight (g)	6.5	6.6	6.4	6.6
SD	0.1	0.1	0.2	0.1
Adjusted ano-genital distance (mm)	0.86	0.84	0.88	0.93
SD	0.02	0.03	0.03	0.03
PND 21 weight (g)	57.3	56	56	56.1
SD	1.4	1.1	1.6	0.9
F2 pups – males
PND 0 weight (g)	6.9	7.1	6.9	7
SD	0.1	0.1	0.1	0.1
Adjusted ano-genital distance (mm)	1.99	1.96	1.88	1.96
SD	0.04	0.04	0.04	0.05
PND 21 weight (g)	61.9	60.1	58.3	59.3
SD	1.3	1.1	1.4	1.3

* p < 0.05 (ANOVA with covariate [body weight]).

Test item-related histopathological lesions were identified in the lungs of both males and females of the F₀ and F₁ generations, consistent with findings noted for GTL naphtha and base oil 90-d repeated-exposure test results: there was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F₀ and F₁ males and females given 750 mg/kg/d (Table 8(a)). This microscopic finding correlated with macroscopic observations in F₀ and F₁ males and F₁ females as well as increased absolute and relative lung weights in F₀ and F₁ males and females. Since this observation of chronic interstitial/alveolus inflammation finding was very similar to what was found in the 90-d repeated-dose study with the same test material, the lungs from the low and mid-dose animals were not evaluated. In the F₁ males, test item-related slight increases in renal tubule degeneration/necrosis and renal tubule hyaline droplets suggestive of hydrocarbon-induced α_2μ-globulin male rat nephropathy were seen in the males given 750 mg/kg/d. Special staining of kidneys from males in the control and high dose group confirmed this lesion to be hydrocarbon-induced α_2μ-globulin male rat nephropathy, an anticipated outcome of this study; therefore, the kidneys of the low and mid dose animals were not evaluated. Another renal effect observed was tubular mineralization in the F₀ males, which may be an artifactual change as it was observed only once amongst F₁ males. As it was observed in the high-dose F₀ males only, it was considered an equivocal test item-related effect.

There were no test item-related effects on F₁ and F₂ pup body weights, microscopic, or macroscopic findings at necropsy (for stillborn, found dead, euthanized, culled, and post-natal day 21 scheduled death pups). Equivocal test item-related, non-adverse decreases in absolute and relative spleen weights were observed in the F₁ and F₂ male and female post-natal day 21 pups in the group given 750 mg/kg/d compared with the control group, but this was not considered a test item-related adverse effect (Faiola 2011b).

GTL base oil.

GTL base oil was also studied in a two-generation reproductive toxicity study in the rat (Faiola 2011a). The set-up and the conduct of this study were almost identical to that for the GTL gas oil, with the test material being dissolved in corn oil and administered by oral gavage (dosing volume 2.0 ml/kg body weight), but the highest dose level was 1000 mg/kg/d, so the dose levels in this study were 0, 50, 250, and 1000 mg/kg/d. Furthermore, the number of animals in each dose group was slightly different: for the F₀ generation, there were either 25 or 26 animals of both genders per dose group, and for the F₁ generation, there were 25 males and 25 females in each dose group. In addition, since a 90-d repeated dose study was already conducted on GTL base oil, no detailed histopathology was performed on tissues that were not required as per the OECD testing 416 guideline.

Administration of GTL base oil to male and female rats at dosages up to 1000 mg/kg/d had no effect on reproductive performance or gestation length and parturition of both the F₀ and F₁ parental generations (Table 9). In addition, there were no test item-related effects on F₁ and F₂ litter parameters, postnatal survival, physical condition/mortality, and pup body weights. Vaginal patency of F₁ females and balanopreputial separation in F₁ males were unaffected by test item administration. Andrology parameters were unaffected by test item administration.

Table 9. Two-generation reproductive toxicity evaluation of GTL base oil.

(a) Systemic toxicity
GTL base oil	Dose (mg/kg/d)
F1 adults	0	50	250	1000
Pre-mating body weight gain (D 0 – 70) (g)
F0 – males	348	350	360	355.2
SD	11	11	11	9.2
F0 – females	128.2	126.6	134.1	133.7
SD	6.6	4.3	7.1	5.4
Lung weight (g)
F0 – males	2.288	2.237	2.380	2.581*
SD	0.065	0.068	0.075	0.077
F0 – females	1.640	1.660	1.724	1.872**
SD	0.029	0.040	0.035	0.075
Lung histopathology – chronic interstitial alveolar inflammation (incidence of total)
F0 – males	3 of 26	NE	NE	25 of 26
F0 – females	2 of 26	NE	NE	20 of 26
F1 adults
Pre-mating body weight gain (D 0 – 70) (g)
F1 – males	460	450	451.6	447
SD	11	11	8.8	9.8
F1 – females	187	183.2	186.1	190.9
SD	5.1	4.2	7.6	6.4
Lung weight (g)
F1 – males	2.4835	2.4473	2.4822	2.6845
SD	0.070	0.070	0.090	0.092
F1 – females	1.700	1.606	1.818	1.860
SD	0.043	0.033	0.070	0.071
Lung histopathology – chronic interstitial alveolar inflammation (incidence of total)
F1 – males	0 of 25	NE	NE	19 of 22
F1 – females	0 of 24	NE	NE	17 of 25

NE: not evaluated.

* p < 0.05; **p < 0.01 (Kruskal–Wallis).

(b) Reproductive performance

GTL base oil	Dose (mg/kg/d)
F0 adults – females	0	50	250	1000
Ovary weight (pair) (g) – F0	0.0614	0.0611	0.0645	0.06
SD	0.0022	0.0028	0.0024	0.002
Estrous cycle length (days) – F0	4.0	4.1	4.3	4.0
SD	0.1	0.1	0.2	0.1
GD 0 – 20 body weight gain – F0	149.3	155	154.6	139.7
SD	5.5	3.5	3.6	5.1
F1 adults – females
Ovary weight (left) (g) – F1	0.0636	0.059	0.0603	0.0615
SD	0.0033	0.0027	0.0028	0.0023
Estrous cycle length (days) – F1	4.1	4.0	4.1	4.1
SD	0.1	0.1	0.1	0.1
GD 0 – 20 body weight gain – F1	140.3	145.6	142.7	157.1
SD	5.6	4.0	7.8	5.2
F0 adults – males
Testis weight (left) (g) – F0	1.841	1.749	1.803	1.801
SD	0.025	0.040	0.030	0.027
Sperm production efficiency (10^6/g testis/d) – F0	35.02	34.92	34.24	34.27
SD	0.70	1.04	1.00	1.00
Epididymal sperm concentration (10^6/g) – F0	861	884	892	919
SD	32	31	29	24
% Motile sperm – F0	82.5	78.3	85.7	81.7
SD	1.4	2.7	1.8	1.9
% Abnormal sperm – F0	1.21	5.1	1.74	1.49
SD	0.05	3.5	0.09	0.08
F1 adults – males
Testis weight (left) (g) – F1	1.924	1.891	1.892	1.924
SD	0.024	0.033	0.029	0.023
Sperm production efficiency (10^6/g testis/d) – F1	38.79	NE	NE	38.2
SD	1.08			1.02
Epididymal sperm concentration (10^6/g) – F1	1008.97	1039	1041	996
SD	25	34	20	28
% Motile sperm – F1	81.7	82.4	82	82.5
SD	1.9	1.6	1.5	1.9
% Abnormal sperm – F1	1.5	NE	NE	1.67
SD	0.08			0.10

NE: not evaluated.

(c) Litter data

GTL base oil	Dose (mg/kg/d)
F1 pups – females	0	50	250	1000
PND 0 weight (g)	6.8	6.5	6.7	6.8
SD	0.1	0.1	0.1	0.1
Adjusted ano-genital distance (mm)	0.96	0.81	0.71**	0.78*
SD	0.05	0.05	0.05	0.05
PND 21 weight (g)	57.4	56.2	56.9	55
SD	0.9	0.8	1.1	1.2
Adjusted age at vaginal patency (d)	31	31.5	31.5	31.3
SD	0.3	0.3	0.3	0.3
F1 pups – males
PND 0 weight (g)	7.2	6.9	7.1	7.1
SD	0.1	0.1	0.1	0.1
Adjusted ano-genital distance (mm)	1.97	1.84	1.71	1.82
SD	0.07	0.08	0.08	0.08
PND 21 weight (g)	61.1	58	59.6	58.1
SD	1	0.9	1	1.3
Adjusted age at preputial separation (d)	42.9	43.8	43.7	43
SD	0.4	0.4	0.4	0.4
F2 pups – females
PND 0 weight (g)	6.4	6.6	6.5	6.7
SD	0.1	0.1	0.2	0.1
Adjusted ano-genital distance (mm)	0.89	0.97	0.82	0.87
SD	0.06	0.06	0.08	0.06
PND 21 weight (g)	53.1	54.2	54.7	53.5
SD	0.7	0.9	1.7	1.2
F2 pups – males
PND 0 weight (g)	6.8	6.8	6.8	7.1
SD	0.1	0.1	0.2	0.1
Adjusted ano-genital distance (mm)	1.93	1.95	1.85	1.9
SD	0.07	0.07	0.09	0.07
PND 21 weight (g)	56.3	56.3	57.8	56
SD	0.7	0.8	1.8	1

* p < 0.05 (ANOVA with covariate [body weight]); **p < 0.01 (ANOVA with covariate body weight).

There were no adverse test item-related effects on F₀ and F₁ parental body weights, body weight changes, feed consumption, and food efficiency. Test item-related histopathological lesions were identified in the lungs of both males and females of the F₀ and F₁ generations (Table 9a). There was an increased incidence and severity of chronic interstitial/alveolus inflammation in the F₀ and F₁ males and females given 1000 mg/kg/d; this microscopic finding correlated with macroscopic observations in F₀ males and females as well as increased absolute and/or relative lung weights in F₀ and F₁ males and females. As these lung lesions were considered to be secondary to minor aspiration of the dose formulation and the chronic interstitial/alveolus inflammation finding was not unanticipated based on a previous 90-day repeat-dose study with this test item, the lungs from the low and mid-dose animals were not evaluated. A test item-related slight increase in the incidence of renal tubule hyaline droplets was seen in the F₁ males given 1000 mg/kg/d; however, only kidneys with gross lesions were examined in this study. The morphologic appearance of these droplets suggested hydrocarbon-induced α_2μ-globulin male rat nephropathy, an anticipated outcome of this study. No degenerative/necrotic renal tubule changes were observed in association with the hyaline droplets.

Ano-genital distance for female F₁, but not F₂, pups was statistically significantly decreased to a similar level in all test item-treated groups without adjustment for body weight, and also in groups given 250 and 1000 mg/kg/d when adjusted for body weight, compared with the control group (Table 9c). F₁ pup ano-genital distance is a non-standard endpoint, not required by the OECD testing guideline 416, therefore, no historical control ranges exist. However, these changes in ano-genital distance and adjusted ano-genital distance values spanned a range similar to the historical control range for ano-genital distance in F₂ pups and fell within the range noted for the F₂ females on this study. Other female parameters, such as vaginal patency and estrous cyclicity, were unaffected by test item administration. A decrease in ano-genital distance values in females is generally not considered toxicologically relevant. In addition, the biological meaning of this small change in ano-genital distance is not clear as hyper-feminization was not apparent based on other endpoints and also since there were no adverse effects on the F₁ reproductive parameters or the F₂ pup parameters. As a consequence, the small changes in ano-genital distance were not considered adverse. An equivocal, slight decrease in F₂ pup brain weights was noted. Macroscopic and microscopic findings in post-natal day 21 pups were not test item-related.

Based on the absence of significant adverse, test item-related findings on the integrity and performance of the male and female reproductive systems, the absence of adverse findings directly attributable to the test item in non-reproductive tissues, and the absence of adverse effects on in-life parameters (such as body weight, feed consumption, and clinical observations), the highest dosage level of 750 and 1000 mg/kg/d, for GTL gas oil and GTL base oil, respectively, were considered to be the no-observed-adverse-effect levels (NOAEL) for both reproductive and systemic toxicity for all generations (Faiola 2011a, 2011b; Boogaard & Roberts 2015).

Prenatal developmental toxicity

GTL gas oil.

The prenatal developmental toxicity of GTL gas oil was studied in the rat according to OECD testing guideline 414 (Prenatal Developmental Toxicity Study). The test material was dissolved in corn oil and administered by oral gavage (dosing volume 2.0 ml/kg body weight) at dose levels of 0 (control), 50, 200, and 750 mg/kg/d to groups of 24 mated Sprague–Dawley Crl:CD rats, between days 5 and 19 (inclusive) of gestation (Dunster 2014). During the study clinical signs, body weight changes, food, and water consumption were monitored and no significant effects were observed. At day 20 of gestation the animals were euthanized and subjected to gross necroscopy including examination of uterine contents. The number, position and type of implantations, numbers of corpora lutea, fetal weights, sex, and external and internal macroscopic appearance were recorded. Half of each litter underwent detailed skeletal development examination (following staining with alizarin red S) and the other half was subjected to detailed visceral examination. No treatment related effects were observed in any uterine parameter, fetal viability or in fetal growth and development. There were no treatment related abnormalities in external development or in type or incidence of skeletal or visceral findings (Table 10). It was concluded that oral administration of GTL gas oil to pregnant rats during gestation did not lead to any toxicologically significant effect on either the parental females or the offspring. As a consequence, the No-Observed Adverse Effect Level for both maternal toxicity and developmental toxicity was considered to be 750 mg/kg/d (Dunster 2014).

Table 10. Prenatal developmental toxicity evaluation of GTL gas oil.

GTL gas oil	Dose (mg/kg/d)
Maternal data	0	50	200	750
Group size	24	24	24	24
No. litters/group	22	23	22	24
Body weight gain, GD 5 – 20 (g)	124	123.9	127	116.5
SD	21.2	17.5	15.3	29.4
Corrected body weight gain, GD 5 – 20 (g)	44.7	42.4	41.8	35.1
SD	11.8	14	12.7	23.2
Gravid uterine weight (g)	79.2	81.5	85.2	81.4
SD	17.6	12.7	9.9	15.3
Implantation data
Mean corpora lutea	14.4	15.2	14.3	14.4
SD	1.7	1.4	1.5	2.1
No. implantations	12.9	13.6	13.5	13.4
SD	2.3	1.7	1.9	2.2
Resorptions per litter	0.6	0.7	0.4	0.8
SD	1.1	1.5	0.8	2
Live litter size	12.3	12.9	13.2	12.6
SD	3.1	2.4	1.8	2.9
Fetal data
Fetal body weight (g) – males	4.2	4.2	4.4	4.2
SD	0.3	0.3	0.2	0.4
Fetal body weight (g) – females	4.1	4	4.2	4.1
SD	0.3	0.3	0.2	0.4
Total external alterations – fetuses	2/271	2/297	1/278	6/302
Litters	2/22	2/23	1/21	1/24
Total visceral alterations	35/140	71/155	54/149	58/158
Litters	16/22	21/23	20/22	24/24
Total skeletal alterations	107/131	116/141	126/142	114/144
Litters	21/22	23/23	22/22	24/24

GTL did not impair pregnant dams or concepti exposed during GD 5 – 20. There was no impairment of maternal body weight gain or food consumption (not shown), viability or growth of implantations, or the structural development of fetuses. Although the incidences of total external, visceral, and skeletal alterations only are shown, no treatment-attributed individual findings were found either.

GTL base oil.

GTL base oil was also tested in the rat according to OECD testing guideline 414 (Prenatal Developmental Toxicity Study). The test material was dissolved in corn oil and administered by oral gavage (dosing volume 2.0 ml/kg body weight) at dose levels of 0 (control), 50, 200, and 1000 mg/kg/d to groups of 22 mated RccHan:WIST rats, between days 6 and 20 post-coitum (Senn 2014). Dams and fetuses were subjected to gross necroscopy. During the study, clinical signs, body weight changes, and food and water consumption were monitored and no significant effects were observed. At day 21 post-coitum, the dams were euthanized and the fetuses removed by Cesarean section. The number, position and type of implantations, numbers of corpora lutea, fetal weights, sex, and external and internal macroscopic appearance were recorded. Half of each litter underwent detailed skeletal development examination (following staining with Alizarin Red S) and the other half was subject to detailed visceral examination. No treatment related effects were observed in any uterine parameter, fetal viability, or in fetal growth and development. There were no treatment-related abnormalities in external development or in type or incidence of skeletal or visceral findings. However, the mean body weight of the live fetuses was slightly increased (5.1 ± 0.2 g) in the highest dose group (1000 mg/kg/d) compared with the controls (4.9 ± 0.2 g) and the low-dose (5.0 ± 0.2 g) and mid-dose (4.9 ± 0.3 g) groups. The minor increase in fetal body weight in the high-dose group was just outside the historical control range but well within the range of normal biological variability (Table 11). It was concluded that oral administration of GTL base oil to pregnant rats during gestation did not lead to any toxicologically significant effect on either the parental females or the offspring. As a consequence, the No-Observed Adverse Effect Level for both maternal toxicity and developmental toxicity was considered to be 1000 mg/kg/d (Senn 2014).

Table 11. Prenatal developmental toxicity evaluation of GTL base oil.

GTL base oil	Dose (mg/kg/d)
Maternal data	0	50	200	1000
Group size	22	22	22	22
No. litters/group	21	18	19	21
Body weight gain, GD 6 – 21 (g)	115	115	112	122
SD	22	20	24	25
Corrected body weight gain, GD 6 – 21 (g)	47	46	46	50
SD	3	7	7	5
Gravid uterine weight (g)	86.0	86.5	82.3	90.0
SD	11.0	18.1	17.8	16.7
Implantation data
Mean corpora lutea	13.1	13.3	12.5	13.2
SD	1.2	2.0	2.7	2.6
No. implantations	13.0	12.9	12.3	13.1
SD	1.2	2.3	2.7	2.6
Resorptions per litter	0.5	0.4	0.4	0.5
SD	0.7	0.8	0.6	1.0
Live litter size	12.5	12.5	11.8	12.6
SD	1.6	2.6	2.7	2.4
Fetal data
Fetal body weight (g) – males	5.1	5.1	5.0	5,2**
SD	0.2	0.2	0.3	0.3
Fetal body weight (g) – females	4.8	4.9	4.8	5,0**
SD	0.2	0.2	0.3	0.3
Total external alterations – fetuses	0/262	0/225	1/225	0/265
Litters	0/21	0/18	1/19	0/22
Total visceral alterations/variations	64/132	60/119	65/119	67/139
Litters	20/21	18/18	18/19	20/21
Total skeletal alterations	100/125	87/106	83/106	108/126
Litters	21/21	18/18	19/19	21/21

GTL did not impair pregnant dams or concepti exposed during GD 5 – 20. There was no impairment of maternal body weight gain or food consumption (not shown), viability or growth of implantations, or the structural development of fetuses. Although the incidences of total external, visceral, and skeletal alterations only are shown, no treatment-attributed individual findings were found either.

Mutagenicity

In vitro assessment of the GTL products for potential genetic toxicity has demonstrated no mutagenicity or clastogenicity. Additionally, GTL gas oil and GTL base oil did not cause chromosome aberrations in vivo. The absence of any genotoxicity or any other triggers indicative for a carcinogenic response, and GTL products are considered not to pose a carcinogenic hazard.

The basic GTL products, as listed in Table 1, the eight GTL solvents, as listed in Table 3, and the GTL wax SX30 were all tested for their mutagenic potential in the reverse mutation assay using Salmonella typhimurium (strains TA1535, TA1537, TA98, and TA100) and, with exception of SX30, also in Escherichia coli (strain WP2uvrA⁻), with and without metabolic activation by mammalian microsome fractions (rat liver S₉ homogenate), according the OECD testing guideline 471 (Bacterial Reverse Mutation Test). The GTL products did not cause visible reductions of cell growth in the tests systems at any dose level and were, therefore, tested up to the maximum recommended concentration of 5000 μg/plate. For all materials, all test results were negative and it was concluded that these materials were not genotoxic mutagens under the test conditions (Blowers 1993, May 1996, Bowles 2006a, 2006b, 2006c, 2006d, 2006e, 2006f, 2006g; Westerink 2014q, 2014r, 2014s, 2014t, 2014u, 2014v, 2014w, 2014x).

The basic GTL products, as listed in Table 1, were also tested in a micronucleus assay using human lymphocytes according to the draft OECD testing guideline 487 (In Vitro Mammalian Cell Micronucleus Test). The protocol was slightly modified to resemble more closely the one of OECD testing guideline 473 (In Vitro Mammalian Chromosome Aberration Test). The lighter GTL products (GTL naphtha, GTL kerosine, GTL and diesel) were tested at the maximum recommended dose level of 5000 μg/ml. The heavier materials (GTL base oil, GTL residual oil, GTL light waxy raffinate, and GTL wax) could not be tested at this concentration due to poor solubility which resulted in difficulties in formulating the test materials and were, therefore, tested at 2500 μg/ml. All materials tested negative indicating a lack of clastogenic potential (Durward 2006a, 2006b, 2006c, 2006d, 2006e, 2006f, 2006g).

In addition, the eight GTL solvents, as listed in Table 3, and GTL diesel were tested in a chromosome aberration test in human lymphocytes in vitro according to OECD testing guideline 473 The GTL solvents were tested at top concentrations that varied between 1350 and 2960 depending on the carbon chain length, GTL diesel was tested at concentration up to 1250 μg/ml (the maximum concentration that could be achieved with this material). As for the mutation assay, again for all materials, all test results were negative and it was concluded that these materials were not clastogenic under the test conditions (Lacey 2010, Sokolowski 2014, Bohnenberger 2014a, 2014b, 2014c, 2014d, 2014e, 2014f, Sokolowski 2015). In addition to these in vitro assays, GTL gas oil and GTL base oil were tested in a mammalian bone marrow test in vivo in male Wistar Han rats according to revised OECD testing guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test) (Morris 2011a, 2011b). The rats received oral gavage of 500, 1000, or 2000 mg/kg of either test material dissolved in arachis oil or cyclophosphamide (positive control), or arachis oil only (negative control). Positive control rats were killed 24 h later for assessment; treated rats receiving 2000 mg/kg were killed 48 h after the initiation of the experiment. The animals receiving the GTL gas oil or GTL base oil did not show any clinical signs at any of the dose levels. No significant decreases in the mitotic index mean values were seen in any of the test groups with either test material when compared with the vehicle control group. There was no evidence of any statistically significant increase in the incidence of cells with chromosomal aberrations excluding gaps in the animals treated with GTL gas oil or GTL base oil. There was also no statistically significant increase in the number of polyploidy cells at any dose level for either test material. These in vivo results confirm the in vitro data that GTL gas oil and GTL base oil are not clastogenic to rat bone marrow cells (Morris 2011a, 2011b).

Discussion

In general, GTL products are not expected to be hazardous to human health based on their chemistry since they consist almost entirely of alkanes. A concise overview of the testing performed on the various GTL products can be found in the Supplementary Material.

Acute effects

The acute oral toxicity studies, which tested a wide range of GTL products, covering the hydrocarbon chain lengths from C₄ to C₉₀, support this. Even at the highest limit dose of 5000 mg/kg body weight, no lethality was observed, or any gross pathology was observed upon necropsy in any of the studies. Also in acute dermal toxicity studies, no systemic toxicity was seen with any of the GTL products tested at the limit dose of 2000 mg/kg body weight. In some of the acute dermal toxicity studies, local effects were observed which were in line with the observations in primary skin irritation studies: slight skin effects, transient erythema, and some desquamation of the skin, which occurred primarily in female animals. These effects are generally also seen with conventional petroleum-derived equivalent products because they are all capable of defatting of the skin. Petroleum-derived middle distillates, such as kerosine, may exert dermal irritant properties (Kanikkannan, et al. 2001; Singh et al. 2003; Smulders 2006). The severity of the irritation and the effects (erythema and edema) are dependent on dose and duration of exposure and may be linked to saturated hydrocarbons with carbon chain lengths typical for kerosenes (Smulders & Boogaard 2006). The results obtained in the primary skin irritation studies with GTL products are fully in line with these observations in petroleum-derived middle distillates. Overall, several lighter GTL products, with predominantly linear and branched hydrocarbons with up to 12 carbons may induce some irritation of the rabbit skin and are consequently classified as Category 2 skin irritants under GHS. The heavier GTL products appear to be devoid of skin irritant properties. This observation was also investigated in vitro in a human three-dimensional epidermal model (Episkin-SM^®). The in vitro results from the tests with this epidermal model corroborate the in vivo studies, confirming the notion that GTL products that consist predominantly of alkanes with 12 or fewer carbon atoms may act as dermal irritants. Based on the data, GTL solvents GS160 and GS170 are classified as skin irritants (Category 2). Although there were no data generated for GTL naphtha, this material is expected to be a Category 2 skin irritant as well and, therefore, classified as such under GHS. GTL kerosine and SX30 both induced transient, very slight erythema, which was insufficient to trigger classification. Although GTL kerosine contains significant amounts of alkanes with chain lengths less than C₁₂, apparently the percentage of the lighter chains in the products is too low to exert more than just slight, transient irritation. This observation is in line with the in vitro test results obtained with GTL solvent GS170 which was borderline negative. In contrast, petroleum-derived kerosine may exert significant skin irritation with some kerosine types being moderate to severe irritants (Tagami & Ogino 1973; Broddle et al. 1996). In general, the lighter GTL products, such as GTL naphtha, GTL kerosene, and GTL gas oil, are less irritating to the skin than the analogous petroleum-derived products. GTL products with carbon chain lengths primarily longer than C₁₂ showed no skin irritation at all. In fact, the heavier GTL products, roughly with carbon chain lengths greater than C₂₅ are applied in skin cosmetics and considered emollients. This implies that, basically, the heavier GTL products do not differ essentially from their petroleum-derived analogs with respect to their effects on the skin.

Overall, the lighter GTL products had no irritant effect in the eye, but some of the heavier products caused slight, transient erythema of the eye and were considered to be slightly irritant, but, again, insufficient to trigger classification under GHS. Again, this is in line with conventional petroleum-derived analogous products.

Based on their chemistry, GTL products are also not expected to be skin sensitizers. Nevertheless, the results from the local lymph node assay and the Magnusson–Kligman maximization assay appeared to be conflicting for GTL light waxy raffinate. Both tests were well conducted according to OECD testing guidelines and under GLP, and neither of them should be considered fundamentally invalid. For the local lymph node, assay 2-butanone was used, as it dissolved the material best, and although this is not a commonly used solvent in this assay, there is no good reason to assume this would have caused a false positive result. The regulatory use of the local lymph node assay is not to classify all sensitizing substances but rather to identify those substances that pose a significant hazard to induce sensitization in exposed individuals. This is reflected in the application of cutoff values, in particular the required 3-fold stimulation index, which in the case of the light GTL waxy raffinate was only met when a 50 or 100% w/w solution were applied but not with a 25% w/w solution. In view of the negative Magnusson–Kligman maximization results for the GTL light waxy raffinate, it was concluded that the GTL light waxy raffinate is not expected to pose a skin sensitization hazard to humans. This conclusion was corroborated by the absence of any sensitizing property of the Sarawaxes SX30 and SX50, the full range GTL paraffin wax, GTL base oil, and the GTL solvents GS160, GS170, GS215, and GS310. In addition, petroleum-derived analog products, which have mainly been tested in Magnusson–Kligman Guinea pig maximization tests and in Buehler tests, do not exert sensitizing properties either. The in vivo findings for the eight GTL solvents, covering hydrocarbons from C₈ to C₂₄, were used to check the validity of an alternative, in vitro skin sensitization assay, the DPRA test (Gerberick et al. 2004, 2007, Jeong et al. 2013). Although the results of this alternative assay were in line with the obtained in vivo results, some of the in vitro results were difficult to interpret due to technical difficulties with the method, in particular with regard to the introduction of test substances that are virtually insoluble in aqueous systems. Unexpected outliers in the tested concentration range were found. This is possibly due the fact that at the end of the incubation period of 24 h at 37 °C, samples were at times cloudy and disturbing the readings. In addition, unexpected results may be due to pH changes because the introduction of a surfactant to this test system has not been validated (Natsch et al. 2007; Natsch & Gfeller 2008). Thus, although there was an attempt to modify the method to account for poor solubility, further research should focus on the introduction of alternative solvents and incubation period (Troutman et al. 2011).

Inhalation toxicity

Although GTL products were not toxic at the limit dose in either acute oral or acute dermal toxicity tests, some GTL products caused apparent inhalation toxicity at a concentration that would trigger classification under GHS. When GTL gas oil was tested initially, the test concentration was 5.61 mg/l, which was higher than the target concentration of 5.0 mg/l. At this concentration, one out of three male rats and one out of three female rats died. The surviving animals showed transient adverse effects (such as labored respiration, hunched posture, unkempt fur); however, they recovered completely within 8–9 d. When the study was repeated at a slightly lower concentration of 5.11 mg/l, none of the male rats and only one out of three female rats died and the surviving animals recovered fully with 7–8 d. Gross pathology did not show any systemic toxicity but the lungs were dark colored or had dark patches. Detailed histopathology showed pulmonary congestion and edema with clear signs of inflammation consistent with aspiration toxicity. Based on these results, which indicate an acute LC₅₀ value >5 mg/L (4 h), GTL gas oil appears to be less hazardous than some of the conventional, petroleum-derived analogs. Conventional petroleum-derived gas oil and low-viscosity base oils are classified as acute inhalation hazards, since some of these products were found to be acute inhalation toxicants with LC₅₀ values <5 mg/L (4 h). Petroleum products with a kinematic viscosity of less than 20.5 mm²/at 40 °C are considered to pose an aspiration hazard by GHS criteria. Since these products in general do not cause systemic effects in acute inhalation toxicity studies apart from local inflammation of the lungs, it has been argued that the observed inhalation toxicity is secondary to aspiration of the aerosols used in these studies. For instance, several conventional gas oil-streams have been reported to cause acute toxicity in inhalation studies with LC₅₀ values of 1.78 mg/L (4 h) for a straight-run middle distillate (CAS RN 64741–44-2) (API 1987), 4.65 and 4.80 mg/L (4 h) for light catalytically cracked distillates (CAS RN 64741–59-9) (API 1986a, 1986b) and 4.60 and 7.64 mg/L (4 h) for hydrodesulphurised middle distillates (CAS RN 64742–80-9) (API 1983a, 1983b). In cases, where histopathology was done on the lungs, the observed effects were consistent with inflammation as seen following aspiration of the material. A possible explanation for this difference might be that the aerosols from GTL gas oil are more stable (i.e. less likely to coalesce) than those of conventional petroleum-derived analogs or, alternatively, the absence of aromatic constituents. The latter explanation would be based on the fact that the gas oils that showed the most severe acute inhalation effects had an aromatic content between 25% and 40% whereas GTL gas oil is totally devoid of aromatic constituents. However, the first explanation is more likely since the effects observed are very similar to those seen in studies with both low-viscosity GTL base oils and GTL solvent and low-viscosity conventional, petroleum-derived base oils and solvents. In fact, the histopathological observations that the induced toxicity was due to mere aspiration is corroborated by the studies with GTL base oils where GTL base oil 4, with a kinematic viscosity of 18 mm²/at 40 °C (just below the standard cutoff viscosity of 20.5 mm²/at 40 °C for petroleum hydrocarbons), showed some transient toxic effects but caused no lethality whereas GTL base oil 3, with a kinematic viscosity of 10 mm²/at 40 °C, was found to have an LC₅₀ between 1.05 and 5.09 mg/l (4 h) without any systemic effects apart from pulmonary inflammation consistent with aspiration toxicity. Similar observations were made in the acute inhalation studies of GTL solvent GS310, with a viscosity of 5.9 mm²/at 40 °C, for which the LC₅₀ value was found to be close to 5 mg/l (4 h). Again, the only lesions observed were in the lung and fully consistent with aspiration. Based on the histopathological findings in the lungs – the only organ affected in all acute inhalation studies with GTL products – the most likely explanation is that the aerosols generated to conduct these inhalation experiments with these substances are unstable in the upper respiratory tract at the high concentrations required (5000 mg/m³) and coalesce to some extent forming a film of fluid in the trachea leading to aspiration and subsequently cause a chemical pneumonitis.

In conclusion, in all acute inhalation studies with GTL products where toxicity was observed, the histopathology showed that there were only effects in the lung and these were compatible with hydrocarbon aspiration induced inflammatory reactions (chemical pneumonitis). Hence, in the absence of other systemic effects, it appears that the mortality observed is due study design leading to aspiration of the products, secondary to coalescence of the aerosol droplets forming a thin film of the material in the upper respiratory tract of the exposed animals, rather than to systemic acute toxicity by inhalation of vapors. It should be noted that rats, unlike humans, are obligatory nasal breathers and have highly complex nasal turbinates compared with humans. Consequently, coalescence of aerosol droplets is much more likely to occur in rats upon inhalation of high concentrations of hydrocarbons than in humans. For these reasons, acute toxicity caused by inhalation of GTL products, including the lower viscosity base oils and solvents, is considered to be a highly unrealistic scenario in humans. However, GTL products with viscosities less than 20.5 mm²/s at 40 °C should be considered hazardous by aspiration.

Repeated-dose studies

Similar aspiration-induced effects as described above may be observed in oral repeated-dose studies where gavage is the administration method. In repeated-dose studies with GTL naphtha a higher incidence of alveolar infiltration of macrophages occurred at the highest dose level (750 mg/kg), but the results were not statistically significantly different from the controls. Infiltration of alveolar macrophages was also observed in the reproductive toxicity studies (by oral gavage) with GTL gas oil at the highest dose level (750 mg/kg/d) in both the F₀ and the F₁ generation, and in both the reproductive toxicity studies and the repeated-dose studies with oral gavage of GTL base oil at the highest two dose levels (200 and 1000 mg/kg/d). However, in these studies, the differences with the controls were statistically significant. The pathology and histopathology of the observations in the lungs was consistent with aspiration. It is thought that these effects result from small amounts of the material on the outside of the gavage needle. Although the outside surface of the needle is routinely wiped prior to dosing, the nature of the hydrocarbons tested is such that it easily creeps out the orifice of the metal needle and wets its outside surface. This material is potentially rubbed off on the surface of the oral cavity and esophagus and subsequently aspirated which leads to the observed effects.

To show that indeed the observed effects were not systemic effects but rather local effects, secondary to aspiration, a dietary repeated-dose study with GTL base oil was conducted. In this study, as expected, no lesions in the lungs were observed at even significantly higher dose levels (1267 mg/kg/d) confirming that the observed lung effects after oral gavage are not systemic effects but local effects secondary to aspiration. In this study, as in the oral gavage studies, no other treatment related effects with relevance to human health were found. In the dietary study with GTL base oil, microscopic findings were detected in the viscera, especially in Peyer’s patches, the spleen and mesenteric lymph nodes consisting of apoptosis/single cell necrosis. This was associated with an increased lymphocytolysis in the Peyer’s patches, spleen, and mesenteric lymph nodes and considered an exacerbation of the normal process of cell attrition which occurs in these tissues. Similar findings were not observed in the low- or mid-dose group males and in none of the females (McRae 2014). It is most likely that the high dose level (15 000 ppm, equivalent to a mean dose level of 1267 mg/kg body weight/d) of the test-item has led to an overload and, subsequently, to a type of corpus alienum reaction of the lymphoid system in the gut. However, since the observed processes in the male rats at the highest dose level may be considered to aggravate the normal process of cell attrition this dose level can, from a precautionary point of view, not be considered as a NOAEL, but rather as a lowest-observed adverse effect level (LOAEL). In the female rats and in the male rats in the low- and mid-dose groups (average dose levels of 63 and 308 mg/kg/d), no histopathological effects were observed. This implies that the real no-adverse effect level (NAEL) lies somewhere between the NOAEL of 308 mg/kg/d and the LOAEL of 1267 mg/kg/d. Based on the absence of similar histopathological effects in both the 90-d oral repeated-dose toxicity studies and the two-generation oral reproductive toxicity studies with GTL base oil at dose levels of 1000 mg/kg/d, the true NAEL must be between 1000 and 1267 mg/kg/d. Therefore, for human risk assessment purposes, the NOAEL for GTL base oil can reliably be set at 1000 mg/kg/d based on the combined results of the three available studies with repeated oral dosing (Dunster 2009; Faiola 2011a; McRae 2014). In the 90-d oral repeated dose study with GTL naphtha, renal toxicity was observed as expected based on previous studies with light hydrocarbons and the range-finding study with GTL naphtha. The renal lesions observed were fully consistent with light hydrocarbon-induced α_2μ-globulin induced nephropathy which is specific for male rats and has no relevance for human health (Alden 1986; Gibson & Bus 1988). For human risk assessment purposes, the NOAEL for GTL naphtha can, therefore, be set at the highest tested dose (750 mg/kg/d).

In the 28-d repeated-dose oral gavage toxicity study with GTL residual oil in the rat, no systemic effects were expected because aliphatic hydrocarbons with carbon chain lengths over C₄₀ are generally considered to be non-bioavailable (EFSA 2012). Nevertheless, the histopathology showed a small increase in extramedullary hematopoiesis in the spleen of the females rats treated with 300 or 1000 mg/kg/d of the test material. This histopathological effect was not observed in the low-dose group females or any of the males and was fully reversible (Dunster 2010). The reason for the observed effects in the spleen is unclear since hematopoiesis in the red pulp of the spleen may develop under several non-specific conditions in rodents and basically reflects an increased demand in erythrocytes. Since the effect is reversible it may be considered adaptive rather than adverse since as there was no concomitant effect on any of the hematological parameters (Greaves 2011). For human risk assessment purposes, the NOAEL can, therefore, be set at 1000 mg/kg/d.

Typically, petroleum-derived waxes and white oils trigger inflammatory reactions in the liver and histopathological changes in mesenteric lymph nodes of Fisher 344 rats but not of Sprague–Dawley rats or dogs and female rats are more sensitive than male rats (Miller et al. 1996; Smith et al. 1996; Scotter et al. 2003; Griffis et al. 2010). For that reason, the GTL waxes (Sarawaxes) were tested in female Fisher 344 and Sprague–Dawley rats in dietary 90-d repeated dose studies with the GTL waxes. Basically, the GTL waxes show a toxicological profile that is very consistent with what is observed in rats dosed with petroleum-derived waxes (paraffin waxes). This is not surprising since the GTL waxes are chemically virtually identical to the paraffin waxes in that they consist almost entirely of linear alkanes. The effects observed in the Fisher 344 rats seem to be strain-specific and not relevant to humans probably because the materials are less bioavailable in humans (Miller et al. 1996; Carlton et al. 2001; Boogaard et al. 2012).

Apart from the lung histopathology, which as explained is most probably secondary to aspiration of material deposited from the gavage needle in the mouth and esophagus, very few product-related effects were seen in reproductive toxicity studies with GTL gas oil and GTL base oil, both in two-generation reproductive toxicity studies and a prenatal developmental toxicity study. The few effects observed were generally not statistically significant or within the range of historical control data. Hence it was concluded that GTL products do not exert a negative effect on reproductive performance or prenatal development at the highest dose levels studied. In addition, in repeated dose studies with both GTL naphtha and GTL base oil, no histopathological changes were observed in either male or female reproductive organs. Again, this is in line with the observation for conventional petroleum-derived products that prenatal developmental toxicity as observed with some products, such as heavy fuel oils, is related to the concentration of 4- to 6-ring polycyclic aromatic constituents (Feuston et al. 1994; Hoberman et al. 1995a; Murray et al. 2013). Since GTL products are virtually devoid of aromatic constituents, prenatal developmental toxicity was not expected to occur which was confirmed by the studies conducted. Fertility is not an issue with conventional petroleum-derived products, even with products with high concentrations of polycyclic aromatic constituents (Hoberman et al. 1995b; Murray et al. 2013). Hence, it is not surprising that GTL products, which consist entirely of saturated hydrocarbons, have no effect on fertility either. As a consequence, for human risk assessment purposes, the NOAELs for reproductive toxicity can be set at the highest tested doses (750 and 1000 mg/kg/d for GTL gas oil and GTL base oil, respectively).

The GTL products have been extensively tested in a variety of in vitro mutagenicity and clastogenicity tests and some GTL products were tested for genotoxic effects in in vivo studies as well. All results were negative which was expected since GTL product consist of saturated hydrocarbons which do not trigger any alert for genotoxicity or carcinogenicity and, as a consequence, none of the GTL products are expected to be mutagenic or carcinogenic. In fact, also the conventional, petroleum-derived analogs, such as gas oil streams and lubricant base oils, are not carcinogenic if they have been treated to remove (polycyclic) aromatic constituents (Halder et al. 1984; Bingham 1988; Chasey & McKee 1993).

In conclusion, a wide range of GTL products, covering the entire portfolio of GTL products, have been tested for mammalian toxicity and were generally found to exert minimal health effects. The data collected also provide evidence for what is known about the mechanisms of mammalian toxicological health hazards of the analogous petroleum-derived substances. In the few cases where adverse effects were found with GTL products, these were usually less severe than the adverse effects observed with their conventional, petroleum-derived analogs.

Declaration of interest

The employment affiliation of the authors is as indicated on the review. The employing companies of the authors are involved in manufacturing and marketing the GTL products which are subject of this review, in addition to broader energy exploration and production activities. These materials have completed chemical compliance requirements as determined by various international regulatory agencies. The review was written by the authors whilst being employed by these companies without any external (non-employer) support. None of the authors have ever testified in legal proceedings related to the contents of the. The manuscript underwent internal review to ensure the absence of confidential business information. The strategy for preparing the paper, its contents, interpretations, and conclusions are the exclusive scientific reflection of the authors and may not necessarily be the views of their employers.

Supplemental material

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Acknowledgments

The authors gratefully acknowledge the extensive comments offered by four reviewers selected by the Editor and anonymous to the authors. The comments were very helpful in revising and improving the manuscript. In addition, the authors thank the scientific staff at the contract research facilities that performed the toxicology studies.