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 summarized 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 Permissible 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.



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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 summarized 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 Permissible 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.

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TABLE 11-1 Genotoxic Effects of JP-8 Fuel in Humans and Experimental Animals Fuel Type Species/ Cell Line Exposure Concentration Exposure Duration Effects Reference Hydro-carbons, jet-fuel derivatives Human (34 male airport workers, 11 unexposed controls) Benzene, 0.10 ± 0.05 mg/m3; toluene, 0.13 ± 0.01 mg/m3; xylenes, 0.13±0.02 mg/m3, measured at Barcelona airport 9.77 yr (mean) No increases in SCE, MN, or ras p21 protein levels were observed in exposed workers; significant difference in mean comet length and in genetic-damage index observed between exposed and unexposed workers Pitarque et al. 1999 JP-4, solvents Human (58 aircraft-maintenance workers, 8 unexposed controls) All means below 6 ppm, as measured with industrial-hygiene methods At least 30 wk Exposure well below threshold limit values; small but statistically significant increase in frequency of SCE occurred after 30 wk of exposure in sheet-metal workers and painters; MN frequency in sheet-metal workers initially showed statistically significant increase but had decreased by 30 wk Lemasters et al. 1997, 1999 JP-8 Salmonella strains, mouse lymphoma cells, human diploid WI-38 cells Microbial assay, 0.001-5.0 μl/plate; mouse lymphoma assay, 0.01- Microbial assay, 48 hr; mouse lymphoma assay, 4 hr; unscheduled JP-8 not mutagenic in Ames-type reverse-mutation assay in Salmonella strains in either presence or absence of metabolic activation with rat liver S9; JP-8 toxic to most Salmonella strains at above 1 μL/plate; no gene mutation in mouse cells in L5178Y thymidine kinase Brusick and Matheson 1978a

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Fuel Type Species/ Cell Line Exposure Concentration Exposure Duration Effects Reference   0.16 μl/ml; unscheduled DNA synthesis assay, 0.1-5.0 μL/mL DNA synthesis assay, 1.5 hr mouse lymphoma cell assay; JP-8 produced a significant, moderate increase in unscheduled DNA synthesis in WI-38 cells   JP-8 H4IIE rat hepatoma cells 1-20 μg/mL 4 hr JP-8 induced dose-dependent increase in mean comet tail moments, indicative of DNA damage; comet tail lengths and DNA strand breaks accumulated in presence of DNA repair inhibitors and JP-8; neither cytotoxicity nor significant apoptosis induced by JP-8 Grant et al. 2001 Various middle distillates Salmonella strains 100-10,000 μg/plate Not reported Jet fuel A, JP-4, JP-5, MD API81-07 not mutagenic in Salmonella reverse-mutation assays; MDFs showed no mutagenic activity in Salmonella; straight-run MDFs nonmutagenic or marginally mutagenic; lightly refined paraffinic oil and C10-C14 normal paraffins negative in Salmonella at up to 10,000 μg/plate IARC 1989; Brusick and Matheson 1978b; Pennzoil 1988; Nessel 1999; Deininger et al. 1991; McKee et al. 1994; McKee et al. 1989;

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  Nessel et al. 1999 Various middle distillates Mouse cells (L5178Y thymidine kinase mouse lymphoma cell assay) Not reported Not reported Jet A fuel induced gene mutation in mouse cells in presence of metabolic activation (mouse or rat liver S9); straight-run kerosene positive in mouse assay in presence of metabolic activation; JP-4 not mutagenic in mouse assay; MD API 81-07 not mutagenic in mouse assay, did not induce SCEs in Chinese hamster ovary cells IARC 1989; Koschier 1999; Brusick and Matheson 1978b; API 1984; Skisak 1991 Jet fuel A, middle distillates CD-1 mice, Sprague-Dawley rats Some 1.0-5.0 g/kg, some not reported Some 24-27 hr, some not reported Inhalation exposure of jet fuel A induced chromosomal aberrations in bone marrow of rats; exposure to turbo fuel A and C10-C14 normal paraffins by gavage did not induce MNs in CD-1 mouse bone marrow test; exposure of hydrodesulfurized kerosene by gavage induced chromosomal aberrations in bone marrow of mice; MD API 81-07 did not induce chromosomal aberrations in rat bone marrow but induced SCEs in B6C3F1 mice Conaway et al. 1984; IARC 1989; Koschier 1999; McKee et al. 1994; Nessel et al. 1999; Skisak 1991 Abbreviations: MN, micronucleus; SCE, sister chromatid exchange; MDF, middle distillate fraction.

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Brusick and Matheson (1978a) observed that JP-8 was not mutagenic in the Ames-type reverse-mutation assay in Salmonella typhimurium strains TA1535, TA1537, TA1538, 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 μL/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 metabolic activation with mouse liver S9; it was moderately toxic at 0.16 μL/mL in this assay system. When tested for ability to induce unscheduled DNA synthesis (UDS) in WI-38 cells, a human diploid cell line, JP-8 produced a significant moderate increase in UDS, as measured by the incorporation of 3H-thymidine, in either the presence or absence of mouse liver S9. The induction of UDS plateaued and was not dose-related; JP-8 toxicity was observed at 5 μL/mL. Brusick and Matheson interpreted the findings in WI-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 mice 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 Ames-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 (NTP 1986). GENOTOXICITY IN HUMANS In a study of 34 male workers exposed to hydrocarbons and jet-fuel derivatives at low concentrations at the Barcelona airport and 11 unexposed controls, 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 protein, SCEs, or MNs were observed in workers compared with controls. The frequency of binucleated cells with MNs was decreased in workers compared 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 smoking 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

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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 exposed to solvents (Lemasters et al. 1997,1999) and JP-4 (Lemasters et al. 1997), 58 new hires were assessed for peripheral blood lymphocyte SCE and MN frequencies before starting work and again after 15 and 30 wk of work. Occupational 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 (fuel-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 were within ranges reported in the general population (Lemasters et al. 1999). GENOTOXICITY STUDIES IN BACTERIA, YEAST, AND MAMMALIAN 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 Brusick and Matheson (1978a) discussed in the 1996 National Research Council report. The ability of JP-8 to induce DNA damage in cultured mammalian cells has been investigated with the Comet (single-cell gel electrophoresis) assay. Grant et al. (2001) tested JP-8 in H4IIE rat hepatoma cells, which are capable of expressing many of the metabolic enzymes, including cytochrome P450-dependent oxidases, normally expressed in liver in vivo. JP-8, solubilized in ethanol at 0.1% (v/v), was applied to the H4IIE cells at 0-20 μg/mL for 4 hr, after which DNA damage was assessed with the Comet assay. JP-8 induced a dose-dependent increase in mean Comet tail moments in H4IIE cells; this indicates DNA damage. The authors reported that comet tail lengths increased and DNA strand breaks accumulated in the presence of DNA-repair inhibitors and JP-8 and concluded that JP-8 induces DNA damage, which can be miti-gated by DNA repair. Neither cytotoxicity nor significant apoptosis was induced by JP-8.

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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 Salmonella reverse-mutation assays. Neither was MD API 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 Salmonella. Straight-run MDFs were either nonmutagenic (straight-run kerosenes: CONCAWE 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 Salmonella 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 μg/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 liver S9), as reviewed by IARC (1989). Straight-run kerosene has also tested positive in the mouse lymphoma assay in the presence of metabolic activation (as summarized by Koschier 1999). MD API 81-07, a hydrodesulfurized kerosene, was not mutagenic in the mouse lymphoma assay (API 1984, as reviewed by Skisak 1991), nor did it induce SCE in Chinese hamster 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 animals in vivo. Jet fuel A, administered by inhalation, induced chromosomal aberrations in the bone marrow of male and female Sprague-Dawley rats (Conaway et al. 1984, as reviewed by IARC 1989, Koschier 1999). McKee et

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al. (1994) evaluated five middle distillate materials, including turbo fuel A, administered by gavage, in the CD-1 mouse bone marrow micronucleus test. No increases in the frequency of MNs were observed for any of the test materials 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 normal 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 chromosomal aberrations in the bone marrow. Administration (route not specified) of MD API 81-07, a hydrodesulfurized kerosene, induced SCE in B6C3F1 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 come from two relatively small studies of people exposed to jet fuels and a number of other solvents—one among workers 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 multiple chemical exposures experienced by them in addition to exposure to jet fuel. In the case of the Barcelona 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 controls 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 small 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 lymphoma assay system. In vitro JP-8 exposure has been shown to induce DNA damage in human and rat cell lines, namely, induction of UDS in human 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 were identified.

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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 middle distillates—were not mutagenic in Salmonella typhimurium strains. Mixed results have been reported for the in vitro mouse lymphoma assay: some materials tested positive (such as jet fuel A and straight-run kerosenes) and others negative (such as hydrodesulfurized kerosene). Mixed in vivo genotoxicity findings have been reported for other jet fuels and MDFs: inhalation exposure to jet fuel A induced chromosomal aberrations in rat bone marrow; gavage administration of hydrodesulfurized kerosene induced chromosomal aberrations in mice; gavage administration of turbo fuel A, C10-C14 normal paraffins, and other MDFs 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 marrow of mice. The subcommittee 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 shown 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 subcommittee recommends 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 (American Petroleum Institute). 1984. Mutagenicity Evaluation Studies in the Rat Bone Marrow Cytogenetic Assay, in the Mouse Lymphoma Forward Mutation Assay: Hydrodesulfurized Kerosene, API Sample 81-07. API Med. Res. Publ. 32-30240. Washington, DC: American Petroleum Institute, Medicine and Biological Science Dept. API (American Petroleum 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. Washington, DC: American Petroleum Institute, Health and Environmental Science Dept.

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API (American Petroleum Institute). 1988b. In Vivo Sister Chromatid Exchange (SCE) Assay with API 81-07: Hydrodesulferized Kerosene . API Med. Res. Publ. 36-30043. Washington, DC: American Petroleum Institute, Health and Environmental Science Dept. Brusick, D.J., and D.W. Matheson. 1978a. Mutagen and Oncogen Study on JP-8. AMRL-TR-78-20. Prepared by Litton Bionetics, Inc., Kensington, MD, for the Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, OH. Brusick, D.J., and D.W. Matheson. 1978b. Mutagen and Oncogen Study on JP-4. AMRL-TR-78-24. Prepared by Litton Bionetics, Inc., Kensington, MD, for the Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, OH. Conaway, C.C., C.A. Schreiner, and S.T. Cragg. 1984. Mutagenicity evaluation of petroleum hydrocarbons. Pp 89-197 in Advances in Modern Environmental Toxicology, Vol. 6. Applied Toxicology of Petroleum Hydrocarbons, H.N. MacFarland, C.E. Holdsworth, J.A. MacGregor, R.W. Call, and M.L. Lane, eds, Princeton, NJ: Princeton Scientific. CONCAWE (The Oil Companies’ European Organization for Environment, Health, Safety). 1991. Middle Distillates – A Review of the Results of a CONCAWE Programme of Short-Term Biological Studies. Report 91/51. CONCAWE, Brussels, Belgium. Deininger, G., H. Jungen, and R.P. Wenzel-Hartung. 1991. Middle Distillates: Analytical Investigations of Mutagenicity Studies. Research Reports No. 412-1. DGMK, Hamburg, Germany (as cited in Nessel et al 1999). Grant, G.M., S.M. Jackman, C.J. Kolanko, and D.A. Stenger. 2001. JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells. Mutat. Res. 490(1):67-75. IARC (International Agency for Research on Cancer). 1989. Jet fuel. Pp. 203-264 in Occupational Exposures in Petroleum Refining, Crude Oil and Major Petroleum Fuels. IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Vol. 45. Lyon: International Agency for Research on Cancer, World Health Organization. Koschier, F.J. 1999. Toxicity of middle distillates from dermal exposure. Drug Chem. Toxicol. 22(1):155-164. Lemasters, G.K., G.K. Livingston, J.E. Lockey, D.M. Olsen, R. Shukla, G. New, S.G. Selevan, and J.H. Yiin. 1997. Genotoxic changes after low-level solvent and fuel exposure on aircraft maintenance personnel. Mutagenesis 12(4):237-243. Lemasters, G.K., J.E. Lockey, D.M. Olsen, S.G. Selevan, M.W. Tabor, G.K. Livingston, and G.R. New. 1999. Comparison of internal dose measures of solvents in breath, blood, and urine and genotoxic changes in aircraft maintenance personnel . Drug Chem. Toxicol. 22(1):181-200. McKee, R.H., M.A. Amoruso, J.J. Freeman, and R.T. Przygoda. 1994. Evaluation of the genetic toxicity of middle distillate fuels. Environ. Mol. Mutagen. 23(3):234-238. McKee, R.H., R.T. Plutnick, and R.T. Przygoda. 1989. The carcinogenic initiating and promoting properties of a lightly refined paraffinic oil. Fundam. Appl. Toxicol. 12:748-756.

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Nessel, C.S. 1999. A comprehensive evaluation of the carcinogenic potential of middle distillate fuels. Drug Chem. Toxicol. 22(1):165-180. Nessel, C.S., J.J. Freeman, R.C. Forgash, and R.H. McKee. 1999. The role of dermal irritation in the skin tumor promoting activity of petroleum middle distillates. Toxicol. Sci. 49(1):48-55. NRC (National Research Council). 1996. Permissible Exposure Levels for Selected Military Fuel Vapors. Washington, DC: National Academy Press. NTP (National Toxicology Program). 1986. Toxicology and Carcinogenesis Studies of Marine Diesel Fuel and JP-5 Navy Fuel (CAS No. 8008-20-6) in B6C3F1 Mice (Dermal Studies). NTP 310. NIH 86-2566. Research Triangle Park, NC: National Toxicology Program/National Institutes of Health. Pennzoil. 1988. Mutagenicity Test on API 81-07 in the Modified Salmonella Micro-some Mutation Assay for Petroleum Samples. Prepared for Pennzoil by Hazelton Laboratories America, Kesington, MD. Pitarque, M., A. Creus, R. Marcos, J.A. Hughes, and D. Anderson. 1999. Examination of various biomarkers measuring genotoxic endpoints from Barcelona airport personnel. Mutat. Res. 440(2):195-204. Skisak, C. 1991. The role of chronic acanthosis and subacute inflammation in tumor promotion in CD-1 mice by petroleum middle distillates. Toxicol. Appl. Pharmacol. 109(3):399-411.