Toxicologic Assessment of Jet-Propulsion Fuel 8 (2003)

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11 Genotoxic Effects of Jet-Propulsion Fuel 8 This chapter summarizes the findings on genotoxicity of jet-propulsion fuel 8 (JP-8) presented in the National Research Council report Permissible Exposure Levels for Selected Military Fuel Vapors (NRC 1996) and reviews additional studies, some of which were completed after the 1996 report was published. The studies are summ arized in Table 11-1. The subcommittee used the body of evidence to assess the genotoxicity of JP-8 in humans. SUMMARY OF STUDIES DISCUSSED IN THE 1996 NATIONAL RESEARCH COUNCIL REPORT The National Research Council Subcommittee on Perm issible Exposure Levels for Military Fuels reviewed studies relevant to the evaluation of the genotoxicity of JP-5, JP-8, and diesel fuel marine. The review included data from in vitro and in vivo rodent genotoxicity testing of JP-4, JP-5, and JP-8 (NRC 1996). Among the studies discussed in the 1996 report, the battery of in vitro and in vivo assays used by Brusick and Matheson (1978a) in testing JP- 8 is the most relevant to the present assessment of JP-8, although the studies of JP-4 and JP-5 are also of interest. 130 

TABLE 11-1 Genotoxic Effects of JP-8 Fuel in Humans and Experimental Animals Species/ Exposu re Exposu re Fuel Type Cell Line Concentration Duration Effec ts Reference Hydro- Hu man (34 m ale Benzene, 0.10 9.77 yr No increases in SCE, M N, or ras p21 protein Pitarque et al. carbons, airport workers, ± 0.05 mg/m 3; (mean) levels were observed in expo sed workers; 1999 jet-fuel 11 unexposed toluene, 0.13 ± significant d ifferenc e in mean comet length derivatives controls) 0.01 mg/m 3; and in genetic-damage index observed xylenes, between exposed and unexposed workers 0.13±0.02 mg/m 3, measured at Barcelona airport JP-4, Hum an (58 All means At least 30 Exposu re well below threshold limit values; Lemasters et solvents aircraft- below 6 ppm, wk small but statistically significant increase in al. 1997, 1999 maintenance as measured frequency of SCE occurred after 30 wk of workers, 8 with indu strial- exposure in sheet-metal workers and pa inters; unexposed hygiene MN frequen cy in shee t-metal w orkers initially controls) methods showed statistically significant increase but had decreased by 30 wk JP-8 Salmonella strains, Microbial Microbial JP-8 not mutagenic in Ames-type reverse- Brusick and mou se assay, 0.001- assay, 48 hr; mutation assay in Salm onella strains in either Matheson lymphoma cells, 5.0 :l/plate; mou se presence or absence of metabolic activation 1978a hum an d iploid mou se lymphoma with rat liver S9; JP-8 toxic to most Salm onella WI-38 cells lymphoma assay, 4 hr; strains at above 1 :L/plate; no gene mutation assay, 0.01- unscheduled in mouse cells in L5178Y thymidine kinase (Continued) 131 

TABLE 11-1 Continued 132 Species/ Exposu re Exposu re Fuel Type Cell Line Concentration Duration Effec ts Reference 0.16 :l/m l; DNA mouse lymphoma cell assay; JP-8 produced a unscheduled synthesis significant, moderate increase in unscheduled DNA assay, 1.5 hr DNA synthesis in W I-38 cells synthesis assay, 0.1-5.0 :L/mL JP-8 H4IIE rat 1-20 :g/mL 4 hr JP-8 ind uced dose-dependent increase in Grant et al. hepatom a cells mean comet tail moments, indicative of DNA 2001 damage; comet tail lengths and DNA strand breaks accumulated in presence of DNA repair inh ibitors and JP-8; ne ither cytotoxicity nor significant apoptosis induced by JP-8 Various Salm onella strains 100-10,000 Not Jet fuel A, JP-4, JP-5, MD API81-07 not IARC 1989; middle :g/plate reported mutagenic in Salm onella reverse-mutation Brusick and distillates assays; M DF s showed no mutagenic activity Matheson in Salm onella; straight-run MDFs 1978b; nonmutagenic or m arginally m utagenic; lightly Pennzoil refined paraffinic oil and C10-C14 normal 1988; Nessel paraffins negative in Salm onella at up to 10,000 1999; :g/plate Deininger et al. 1991; McKee et al. 1994; McKee et al. 1989; 

Nessel et al. 1999 Various Mouse cells Not reported Not Jet A fuel induced gene mutation in mouse IARC 1989; middle (L5178Y reported cells in presence of metabolic activation Koschier distillates thymidine kinase (mouse or rat liver S9); straight-run kerosene 1999; mou se positive in mouse assay in presence of Brusick and lym phoma cell metabolic activation; JP -4 no t mu tagenic in Matheson assay) mouse assay; M D AP I 81-07 not m utagenic in 1978b; API mou se assay, did not induce SCE s in Chinese 1984; Skisak ham ster ovary cells 1991 Jet fuel A, CD-1 mice, Som e 1.0-5.0 Some 24-27 Inhalation exposure of jet fuel A induced Conaway et middle Sprague-Dawley g/kg, some hr, some not chromosomal aberrations in bone marrow of al. 1984; distillates rats not reported reported rats; exposure to turbo fuel A and C10-C14 IARC 1989; normal paraffins by gavage did not induce Koschier MNs in CD-1 m ouse bone m arrow test; 1999; McKee exposure of hydrodesulfurized kerosene by et al. 19 94; gavage indu ced chrom osom al aberrations in Nessel et al. bone marrow of mice; MD API 81-07 did not 1999; Skisak induce chromosomal aberrations in rat bone 1991 marrow but induced SCEs in B6C3F 1 mice Abbreviations: M N, micronucle us; SCE , sister chromatid exchange; MDF, m iddle distillate fraction. 133 

134 Toxicologic A ssessment of Jet-Propulsion F uel 8 Brusick and Matheson (1978a) observed that JP-8 was not mutagenic in the Ames-type reverse-mutation assay in Salmonella typhimurium strains TA1535, TA1537, TA 1538, TA98, and TA100 in the presence or absence of metabolic activation with rat liver S9. JP-8 was toxic to most of the Salmonella strains at concentrations above 1 :L/plate. JP-8 was not mutagenic when tested in a yeast forward-mutation assay with Saccharomyces cerevisiase strain D4. It did not induce gene mutation in mouse cells in the L5178Y thymidine kinase mouse lymphoma-cell assay in the presence or absence of m etabolic activation with mouse liver S9; it was moderately toxic at 0.16 :L/mL in this assay system. When tested for ability to induce unscheduled D NA synthesis (UDS) in WI-38 cells, a human diploid cell line, JP-8 produced a significant moderate increase in UD S, as measured by the incorporation of 3H-thymidine, in either the pres- ence or absence of mouse liver S9. The induction of UDS plateaued and was not dose-related; JP-8 toxicity was observed at 5 :L/mL. Brusick and Mathe- son interpreted the findings in W I-38 cells as suggesting that nonspecific DNA lesions were produced by JP-8. They tested JP-8 with the dominant-lethal test in rats and mice and reported that JP-8 was only moderately toxic at the doses tested and was negative in both m ice and rats. JP-4 was tested in the same battery of tests as those described for JP-8, with very similar results (positive solely in the WI-38-cell UDS assay) (Brusick and Matheson 1978b). JP-5 was not mutagenic in the Am es-type reverse- mutation assay in Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, and TA100 in the presence or absence of metabolic activation with rat or hamster liver S9 (N TP 1986). GENOTOXICITY IN HUMANS In a study of 34 male workers exposed to hydrocarbons and jet-fuel deriv- atives at low concentrations at the Barcelona airport and 11 unexposed con- trols, Pitarque et al. (1999) measured ras p21 plasma protein concentrations, sister chromatid exchanges (SCEs), micronuclei (MNs), and DNA strand breaks, as detected by the Comet assay, in peripheral blood lymphocytes. No increases in ras p21 prote in, SCEs, or MNs were observed in workers com- pared with controls. The frequency of binucleated cells with MNs was de- creased in workers com pared with controls. Statistically significant differences in mean Comet length and in the genetic damage index were observed between workers and controls. Confounding factors, such as age and sm oking status, may have contributed to the findings; the mean age of the workers was 47.91 ± 4.22 (SEM) years (yr) compared with 34.87 ± 1.11 yr in controls, and the 

Genotoxic E ffects of Jet-Propulsion F uel 8 135 percentage of workers that smoked was 56.4% compared with 37.5% of the controls. In a study of aircraft maintenance workers at a U.S. Air Force base ex- posed to solvents (Lemasters et al. 1997,1999) and JP-4 (Lemasters et al. 1997), 58 new hires w ere assessed for peripheral blood lymphocyte SCE and MN frequencies before starting work and again after 15 and 30 wk of work. Occu- pational exposures of the workers were well below the threshold limit values, with total solvent exposures all at less than 6 ppm, as measured by industrial- hygiene air methods. A time-dependent increase in SCEs was observed for the solvent- and fuel-exposed group compared with the unexposed group (n = 8). Most of the increases occurred in the sheet-metal (fu el-cell) workers (n = 6; p = 0.003), who had a 20% increase in SCEs, and the paint-shop workers (n = 6; p = 0.05). These workers had higher concentrations of solvents and fuel in their breath than workers in jet-fueling operations (n = 15) and the flight-line crew (n = 23). MNs in the jet-fueling operations workers went down over time. The authors concluded that the observations of increased SCEs in the exposed group might be due to chance, inasmuch as the increases w ere w ithin ranges reported in the general population (Lem asters et al. 1999). GENOTOXICITY STUDIES IN BACTERIA, YEAST, AND MAM MALIAN CELLS IN VITRO JP-8 No additional data were identified on the genotoxicity of JP-8 in bacteria or yeast, other than the studies of B rusick and M atheson (1978a) d iscussed in the 1996 National Research Council report. The ability of JP-8 to induce DNA dam age in cultured mamm alian cells has been investigated w ith the Com et (single-cell gel electrophoresis) assay. Grant et al. (2001) tested JP-8 in H4IIE rat hepatoma cells, w hich are capable of expressing many of the metabolic enzymes, including cytochrome P450- dependent oxidases, normally expressed in liver in vivo. JP-8, solu bilized in ethanol at 0.1% (v/v), was applied to the H4IIE cells at 0-20 :g/m L for 4 hr, after which DNA damage was assessed with the Comet assay. JP-8 induced a dose-dependent increase in mean Comet tail m oments in H4IIE cells; this indicates DNA dam age. The authors reported that comet tail lengths increased and DNA strand breaks accumulated in the presence of DN A-repair inhibitors and JP-8 and concluded that JP-8 induces DN A dam age, which can be m iti- gated by DNA repair. Neither cytotoxicity nor significant apoptosis was in- duced by JP-8. 

136 Toxicologic A ssessment of Jet-Propulsion F uel 8 Related Mixtures Other jet fuels and related middle distillate fractions (MDFs) have been tested in bacteria and in vitro mammalian cell assays. As summarized by IARC (1989), jet fuel A and JP-5 were not mutagenic in Salm onella reverse-mutation assays. Neither was MD A PI 81-07, a hydrodesulfurized kerosene sample (Pennzoil 1988, as reviewed by Skisak 1991). Nessel (1999) reviewed the data on several MDFs and found that they showed little or no mutagenic activity in Salm onella. Straight-run MDFs were either nonmutagenic (straight-run kerosenes: CON CAW E 1991, as cited by Nessel 1999) or marginally mutagenic (Deininger et al. 1991, as cited by Nessel 1999). McKee et al. (1994) evaluated five middle distillate materials, including turbo fuel A, in Salm onella strain TA98 in the presence or absence of hamster liver S9 and found that straight-run distillates were nonmutagenic; that is, they induced less than a doubling of revertant colonies. Lightly refined paraffinic oil (McKee et al. 1989) and C10-C14 normal paraffins (Nessel et al. 1999) were negative when tested in Salmonella at up to 10,000 :g/plate. Jet fuel A induced gene mutation in mouse cells in the L5178Y thymidine kinase mouse lymphoma-cell assay in the presence but not in the absence of metabolic activation (mouse or rat liv er S9), as reviewed by IARC (1989). Straight-run kerosene has also tested positive in the m ouse lym phoma assay in the presence of metabolic activation (as summ arized by Koschier 1999). MD API 81-07, a hydrodesulfurized kerosene, was not m utagenic in the m ouse lymphoma assay (API 1984, as reviewed by Skisak 1991), nor did it induce SCE in Chinese ham ster ovary cells (API 1988a, as reviewed by Skisak 1991). IN VIVO GENOTOXICITY STUDIES IN ANIMALS JP-8 No data were identified on the in vivo genotoxicity of JP-8 in animals other than the studies of Brusick and Matheson (1978b) discussed in the 1996 National Research Council report. Related Mixtures Other jet fuels and related MDFs have been tested for genotoxicity in anim als in vivo. Jet fuel A, administered by inhalation, induced chromosomal aberrations in the bone marrow of male and female Sprague-Daw ley rats (Conaway et al. 1984, as reviewed by IARC 1989, Koschier 1999). McKee et 

Genotoxic E ffects of Jet-Propulsion F uel 8 137 al. (1994) evaluated five middle distillate materials, including turbo fuel A, administered by gavage, in the CD-1 m ouse bone marrow micronucleus test. No increases in the frequency of MNs were observed for any of the test mate- rials in assessments 24, 48, or 72 hr after treatment. The authors did not see any evidence of bone marrow depression (McKee et al. 1994). C10-C14 nor- mal paraffins administered by gavage did not induce MNs in the CD-1 mouse bone marrow micronucleus test in assessments 24, 48, or 72 hr after treatment in ether male or female mice (Nessel et al. 1999). Koshier (1999) reported that hydrodesulfurized kerosene, administered to mice by gavage, induced chromo- somal aberrations in the bone marrow. Administration (route not specified) of MD A PI 81-07, a hydrodesulfurized kerosene, induced SCE in B6C3F 1 mice (API 1988b, as reviewed by Skisak 1991), but did not induce chromosomal aberrations in rat bone marrow (API 1984, as reviewed by Skisak 1991). CONCLUSIONS AND RECOMMENDATIONS The available data on genotoxicity in human populations exposed to jet fuels com e from two relatively sm all studies of people exposed to jet fuels and a number of other solvents—one am ong w orkers at the Barcelona airport (type of jet fuel not specified) and one among workers exposed to JP-4 at a U.S. Air Force base. Both studies found slight genotoxic effects associated with exposure, but interpretation of the findings in those studies is complicated by a variety of factors, including small number of subjects studied and the mu ltiple chemical exposures experienced by them in addition to exposure to jet fuel. In the case of the B arcelona airport study, the finding of increased DNA damage in workers is confounded by the higher mean age of workers than unexposed controls, and the higher percentage of workers than of con- trols who were smokers. In the case of the U.S. Air Force base study, the significance of observations of time-dependent increases in SCEs in some subgroups of workers is uncertain, given the sm all numbers of subjects in whom the increases were observed and the small magnitude of the increases (all were within the range of population controls). Available data on the genotoxicity of JP-8 in animals, cultured cells, and prokaryotes indicate that JP-8 does not induce dominant lethal mutations in Sprague-Dawley rats or CD-1 mice, or mutations in Salmonella typhimurium, Saccharomyces cerevisiae, or the mouse lymphom a assay system. In vitro JP-8 exposure has been shown to induce DN A damage in human and rat cell lines, namely, induction of UDS in hum an diploid cell line WI-38 and DNA damage in H4IIE rat hepatoma cells. No published data regarding the genetic toxicity of JP-8 in vivo w ere identified. 

138 Toxicologic A ssessment of Jet-Propulsion F uel 8 A larger database is available on the genotoxicity of other jet fuels and related MDFs. Other jet fuels—including jet fuel A, and JP-5, and several mid dle distillates—were not mutagenic in Salmonella typhimurium strains. Mixed results have been reported for the in vitro mouse lymphom a assay: some mate- rials tested positive (such as jet fuel A and straight-run kerosenes) and others negative (such as hydrodesulfurized kerosene). M ixed in vivo genotoxicity findings have been reported for other jet fuels and M DFs: inhalation exposure to jet fuel A induced chromosomal aberrations in rat bone marrow; gavage administration of hydrodesulfurized kerosene induced chromosomal aberra- tions in mice; gavage administration of turbo fuel A, C10-C14 normal paraf- fins, and other MD Fs did not induce chromosomal aberrations in mice; and MD API 81-07, a hydrodesulfurized kerosene, did not induce chromosomal aberrations in the bone marrow of rats but did induce SCEs in the bone mar- row of mice. The subcomm ittee concludes that the available data are insufficient to draw a conclusion regarding the genotoxicity of inhaled JP-8. JP-8 has been shown to induce DNA damage in cultured mammalian cells, and some related mixtures (such as jet fuel A and straight-run kerosene) but not others (such as JP-4 and MD API 81-07, a hydrodesulfurized kerosene) have been show n to induce mutations in cultured mouse lymphoma cells. Some related mixtures (jet fuel A in rats, hydrodesulfurized kerosene in mice, and another hydrodesulfurized kerosene, MD API 81-07, in mice) but not others (turbo fuel A, MDFs, and C10-C14 normal paraffins in mice and hydrodesulfurized kerosene MD API 81-07 in rats) have been shown to be clastogenic in vivo. Therefore, the subcomm ittee recomm ends that the Air Force conduct in vivo genotoxicity studies by the inhalation route in two animal species to determine whether JP-8 is mutagenic, clastogenic, or capable of inducing other types of DNA damage via inhalation. REFERENCES API (Am erican Petroleum Institute). 1984. Mutagenicity Evaluation Studies in the Rat Bone Marrow Cytogenetic Assay, in the Mouse Lymphoma Forward Mutation Assay: Hydrodesulfurized Kerosene, A PI Sam ple 81-07. API Med. Res. Publ. 32- 30240. Wa shington, D C: American Petroleum Institute, Medicine and Biological Science D ept. API (Am erican P etroleum Institute). 1988a. Sister Chromatid Exchange (SCE) Assay in Chinese Hamster Ovary (CHO) Cell with API 81-07: Hydrodesulfurized Kerosene. API Med. Res. Publ. 35-32482. Washin gton, DC : American Petro- leum Institute, Health and En vironm ental Sc ience Dept. 

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