OEL's Scoping Study

Home , Metal deactivator

ANNEX 1b

to the Final Report of the SCOEL Support Project

AA/31887/DF/EMPL - ARES 30/11/2010 – No 84933

Aviation Fuels

Scoping Study on behalf of DG EMPL

Ispra, September 2012

September 2012

Dimosthenis Papameletiou Dimosthenis Axiotis

European Commission Joint Research Centre, Institute for Health and Consumer Protection, Chemical Assessment and Testing Unit (I.1) Competence Group: Risk Analysis,

Ispra, Italy

Table of contents

Acronyms ...... v About the scoel support project...... ix Executive summary...... xi 1. Background ...... 1 2. Existing OELs and major risk assessment studies on aviation fuels...... 3 3. Substance identification and caRacterization...... 6 3.1 Fuel types and uses...... 6 3.2 Fuel composition...... 7 3.3 Statistical data about the consumption of aviation fuels and the employment in the air- transport sector ...... 11 4. Physico-chemical properties ...... 13 4.1 Commercial aviation fuels (Jet A / Jet A-1 fuels)...... 13 4.2 Military aviation fuels (JP-8) ...... 14 5. Specification of exposure sources, chemicals involved and levels of exposures...... 15 5.1 Sources of occupational Exposure to aviation fuels...... 15 5.1.1 Fuel cell maintenance ...... 15 5.1.2 Fuelling ...... 16 5.2 Key factors influencing the exposure and health effects...... 16 5.3 Categories of personnel who work directly with fuel ...... 17 5.4 Chemicals of major concern...... 18 5.4.1 Aerosols...... 19 5.4.2 Combustion products...... 19 5.5 Exposure Levels ...... 21 5.5.1 Inhalation Exposure ...... 21 5.5.2 Dermal exposure ...... 22 5.5.3 Biomonitoring studies ...... 23 5.5.3.1 Exhaled breath measurements...... 23 5.5.3.2 Blood ...... 25 5.5.3.3 Urine...... 25 5.5.3.4 Dermal studies...... 27 6. Health effects...... 29 6.1 Effects on the respiratory system ...... 30

6.1.1 1996 assessment (COT 1996)...... 30 6.1.2 2003 assessment (COT 2003)...... 30 6.1.3 Human studies...... 31 6.1.4 Animal studies ...... 32 6.1.5 Recent studies to be considered by SCOEL ...... 33 6.2 Effects on the nervous system...... 36 6.2.1 1996 assessment (COT 1996)...... 36 6.2.2 2003 assessment (COT 2003)...... 36 6.2.3 Human data...... 37 6.2.4 Animal data ...... 39 6.2.5 Recent studies to be considered by SCOEL ...... 40 6.3 Effects on immune system ...... 43 6.3.1 1996 Assessment (COT 1996) ...... 43 6.3.2 2003 Assessment (COT 2003) ...... 43 6.3.3 Human data...... 44 6.3.4 In vitro data...... 45 6.3.5 Animal data ...... 45 6.3.6 Recent studies to be considered by SCOEL ...... 47 6.4 Effects on liver ...... 53 6.4.1 1996 assessment (COT 1996)...... 53 6.4.2 2003 assessment (COT 2003)...... 53 6.4.3 Human data...... 54 6.4.4 In vitro data...... 55 6.4.5 Animal data ...... 55 6.4.6 Recent studies to be considered by SCOEL ...... 56 6.5 Effects on kidney...... 57 6.5.1 1996 assessment (COT 1996)...... 57 6.5.2 2003 assessment (COT 2003)...... 57 6.5.3 Human data...... 57 6.5.4 Animal data ...... 58 6.5.5 Recent studies to be considered by SCOEL ...... 59 6.6 Effects on reproduction and development...... 60 6.6.1 1996 assessment (COT 1996)...... 60 6.6.2 2001 assessment (COT 2001)...... 60

6.6.3 2003 assessment (COT 2003)...... 60 6.6.4 Human data...... 61 6.6.5 Animal data ...... 61 6.6.6 Recent studies to be considered by SCOEL ...... 62 6.7 Effects on cardiovascular system ...... 63 6.7.1 1996 Assessment (COT 1996) ...... 63 6.7.2 2003 Assessment (COT 2003) ...... 63 6.7.3 Human data...... 63 6.7.4 Animal data ...... 64 6.7.5 Recent studies to be considered by SCOEL ...... 64 6.8 Genotoxic effects...... 65 6.8.1 1996 Assessement (COT 1996) ...... 65 6.8.2 2003 Assessment (COT 2003) ...... 65 6.8.3 Human data...... 65 6.8.5 Animal data ...... 66 6.8.6 Recent studies to be considered by SCOEL ...... 66 6.9 Carcinogenic effects...... 69 6.9.1 1996 Assessment (COT 1996) ...... 69 6.9.2 2003 Assessment (COT 2003) ...... 69 6.9.3 Human data...... 71 6.9.4 Animal data ...... 71 6.9.5 Studies to be considered by SCOEL...... 71 6.10 Dermal effects and toxicity ...... 72 7. Conclusions ...... 76 References ...... 77

iii

Acronyms

ACGIH – American Conference of Governmental Industrial Hygienists AFB – Air Force Base ALH - Amplitude of Lateral Head displacement ALT – Alanine Aminotransferase ANCOVA – Analysis of Co-Variance AST – Aspartate Aminotransferase ASTM - American Society for Testing and Materials ATSDR – Agency for Toxic Substances and Disease Registry AvGas – Aviation Gasoline BLVs – Biological Limit Values BTEX - Benzene, toluene, ethylbenzene, m-lp xylene, o-xylene CA – Chromosomal Aberration CD – Criteria Document CNS – Central Nervous System COT - Committee of Toxicology CR – Conditioned Response CTL - Cytotoxic T-lymphocyte (response) DINNSA – Dinonylnaphthylsulfonic acid DFM – Diesel Fuel Marine DNA - Deoxyribonucleic Acid DOD – Department of Defence EC – European Commission ELISA – Enzyme-linked Immunosorbent Assay EPA – Environmental Protection Agency FSII – Fuel System Icing Inhibitor GST – Glutathione-S-trasferase HC – HydroCarbon HDS - Hydrodesulfurization

IARC – International Agency for Research on Cancer IATA - International Air Transport Association IC50 - Half Maximal Inhibitory Concentration ICAO – International Civil Aviation Organization IL-8 – InterLeukin iNOS – Inducible Nitric Oxide Synthase IRIS – Integrated Risk Information System JRC – Joint Reasearch Centre KLH – Keyhole-Limpet Hemocyanin LAK - Lymphokine-Activated Killer (response) MEAA - (2-methoxyethoxy)acetic acid MN - MicroNucleus MRLs – Minimum Risks Levels mRNA – Messenger Ribonucleic Acid NATO - North Atlantic Treaty Organization NHEK – Normal Human Epidermal Keratinocyte NIOSH – National Institute for Occupational Safety and Health NK – Natural Killer (activity) NKA - Naphthyl–Keratin Adduct NOAEL – No Observed Adverse Effect Level NQOI – Quinone Oxidoreductase NRC – National Research Council NTP – National Toxicology Program OEL – Occupational Exposure Limit OELV - Occupational Exposure Limit Value OR – Odds Ratio OSHA – Occupational Safety and Health Administration PAHs – Polycyclic Aromatic Hydrocarbons PEL – Permissible Exposure Limit

PFC – Plaque-Forming Cell PM – Particulate Matter

RD50 - Exposure concentration producing a 50% respiratory rate decrease RMMs – Risk Management Measures REL – Recommended Exposure Limit SBS – Structural Business Statistics SCE – Sister-Chromatid Exchanges SCGE - Single-Cell Gel Electrophoresis SCOEL – Scientific Committee on Occupational Exposure Limits SRBC – Sheep Red Blood Cell SS – Scoping Study STEL – Short-Term Exposure Limit SUM – Summary document SUMMA - Stainless steel electropolished passivated vessel (refers to canisters) TLV - Threshold Llimit Value TM – Tail Moment TNF – Tumour Necrosis Factor TWA – Time Weighted Average VOCs – Volatile Organic Compounds

vii

viii

ABOUT THE SCOEL SUPPORT PROJECT

The Commission has established and has been operating over the last two decades a Scientific Committee on Occupational Exposure Limits (SCOEL) in the framework of setting occupational exposure limit values.

The mandate of SCOEL is to examine available information on toxicological and other relevant properties of chemical agents, evaluate the relationship between the health effects of the agents and the level of occupational exposure, and where possible recommend values for occupational exposure limits which it believes will protect workers from chemical risks. Members of SCOEL are selected following an invitation from the European Commission to the Member States that requests the nomination of suitable candidates. All SCOEL members act as independent scientific experts, not as representatives of their national Governments. SCOEL membership includes, inter-alia, experts in chemistry, toxicology, epidemiology, occupational medicine and industrial hygiene.

At national level, Member States active in the area of OELs – Occupational Exposure Limits - development, have been setting up, in addition to their Scientific Committee, specific support infrastructures to tackle the production of criteria documents, which are basic inputs to the work of the Scientific Committees. At EU level, the operation of SCOEL has not been supported by such an infrastructure.

The increasing amount of workload imposes to the Commission the need to outsource the preparation of preliminary scientific documents. These documents which were the basis for the scientific evaluation carried out by SCOEL have been prepared through studies by external experts.

The scientific support to be provided by JRC will replace these studies and will provide a long term and more consistent scientific support to SCOEL.

To this end, DG EMPL considered necessary to take a concrete initiative to set up a cooperation framework with JRC and requests the assistance of it for the establishment of a support activity for SCOEL in the JRC.

The overall objective of the SCOEL support project is to develop at the JRC Institute of Health and Consumers Protection (IHCP), an infrastructure that will provide technical and scientific services to DG EMPL and to the SCOEL in the area of Occupational Exposure Limit Values (OELVs) for individual hazardous chemicals in the workplace in accordance

with article 3 of Directive 98/24/EC on chemical agents and Directive 2004/37/EC on carcinogens and mutagens.

The support activity is focused on the preparation of the following types of documents:

• Criteria Documents (CDs): summarize in a single document the entire relevant scientific information base for a given hazardous chemical that is available for the OEL setting by SCOEL. • Summary Documents (SUMs): screen out the key information that is finally used for the derivation of a proposed OEL for a given hazardous chemical. • Scoping Studies (SSs): are carried out in complex areas, where exploratory analysis is necessary for establishing the feasibility and specifications of future work on criteria documents.

EXECUTIVE SUMMARY

Recently, questions have been raised in the EU Parliament regarding occupational exposure and adverse health effects arising from aviation fuels. As a response, DG EMPL requested the JRC to investigate this issue in the frame of the present scoping study with the main objective to assist SCOEL on setting OELs (Occupational Exposure Limits) in this area in the EU.

The present report reviews the following topics:

• Characterization of aviation fuels • Uses, exposure sources and levels • Existing OELs and major assessment studies • Health effects • Toxicology

Currently the most widely used fuel types are Jet A and Jet A-1 for the commercial aviation and JP-8 for the military aviation, which replaced earlier specifications (JP-4 and JP-5). JP-8 is the single largest source (9.58 billion litres in 2000) of chemical exposure to military personnel in the United States. It is estimated that 2.0 million commercial and military airline workers in the USA are exposed to jet fuels. In Europe the number of persons employed only in the commercial air transport field is about 380,000.

Regulatory activity for the setting of OELs in the area of aviation fuels are known only from the USA. The earliest known OEL is attributed to the Department of Defence (DOD) and was recommended before 1996 as an interim permissible exposure level (PEL), a time-weighted exposure concentration in workplace air averaged over an 8hr shift, of 350 mg/m3 and a 15 min short-term exposure limit (STEL) of 1800 mg/m3 for vapours from JP-5, JP-8, and DFM (diesel fuel marine) (COT 1996).

Following the wider introduction of JP-8 by the end of the 90’s a new request was made by the US Air Force to US National Research Council to independently review the scientific basis of the PEL of 350 mg/m3. The review was carried out in 2003 again by the Committee on Toxicology (COT) and the Subcommittee on Permissible Exposure Levels for Military Fuels. On the basis of the available toxicologic data, the review concluded that the interim PEL of 350 mg/m3 for JP-8 might be too high to be protective of human health.

Exposure of military and civil aviation workers to aviation fuels may cause adverse health effects that may be attributed to a number of chemicals contained in the fuels. Constituents of major concern are:

• Naphthalene. • Benzene, toluene, ethylbenzene, m-lp xylene, o-xylene (BTEX). • N-hexane. • Aerosols and combustion products.

The issue of aerosols/combustion products appears to be covered to some extend by investigations regarding the measurement of exposure levels. However, most toxicological studies were carried out by exposing animals to vapours from aviation fuels. In this light, these studies may not be representative for the effects of combustion products and therefore it was decided to deal with the combustion products from aviation fuels in a separate scoping study.

Based on the analysis of the available information on exposure levels, of the OELs set to date, mainly in the USA, and the literature that has emerged since the most recent US risk assessment, the present report proposes the following options and/or alternative scenarios for further discussion by SCOEL:

• Adopt a similar approach as the US risk assessments by COT (1996), (2003) and update the OEL’s level according to the findings of the recent literature. • Establish health based biological limit values (BLVs) in the light of recent breakthroughs in biomonitoring (outlined in Chapter 5.5.3.) • Assign a skin notation, in the light of recent breakthroughs in dermal exposure assessment and evaluation of related health effects. • To distinguish OELs related to vapours and aerosols. • To consider particular action for individual chemicals, of major concern, such as naphthalenes.

xii

1. BACKGROUND

Aviation is powered by petroleum fuels, a choice based on petroleum's recognized advantages. The early aircraft engines were similar to those used in automobiles and burned the same fuel. However, the need for increased power led to the development of specialized engines and aviation gasoline (avgas) tailored to their requirements. In the 1940's, the turbine engine emerged as the answer to the quest for still power and in a replay of avgas development, kerosene – the fuel used in the first aircraft turbine engines – was eventually replaced by specialized aviation turbine fuels (jet fuels). In 1951, a wide cut taken of the crude oil distillation, labelled JP-4 (standardized under MIL-F-5624A) issued for widespread use. The main problem of JP-4 was revealed after war experience when aircrafts using JP-4 had more combat losses than aircrafts using JP-5. The main difference was the higher volatility of JP-4 compared to JP-5 (used by US Navy).

Nowadays two types of aviation fuels are commonly used in military and civil aviation: Jet Propellant (JP) – 8 and Jet – A respectively. Jet Fuels are similar in composition to kerosene and contain over 200 aliphatic and aromatic compounds including thousands of isomeric forms and several non-hydrocarbon performance additives.

The main difference between commercial (Jet–A) and military fuels (JP-8) is the essential performance additives (usually 3 types) included into the latter.

The North Atlantic Treaty Organization forces and U.S. military services use an estimated 5 billion gallons of JP-8 each year and 2 million people worldwide are exposed to 60 billion gallons of JP-8 annually. That trend follows a continuous increase for commercial and military purpose

• Naphthalene, a polycyclic aromatic hydrocarbon, is found in in JP-8 mixtures in low levels (McDougal et al., 2000; Smith et al., 2010), and is classified as Group 2B: “possibly carcinogenic to humans” (IARC, 2002). • Benzene, toluene, ethylbenzene, m-lp xylene, o-xylene (BTEX). Although benzene is found at concentrations below 0.02% in JP-8, it is a known human carcinogen and is arguably together with naphthalene among the most hazardous components of jet fuel 8 Egeghy et al., 2003). All of the BTEX chemicals can produce neurological impairment, and exposure to benzene can additionally cause hematological effects including aplastic anemia and acute myelogenous leukemia. The critical nature of the neurotoxicity (i.e., the noncancer effect expected to occur at the lowest exposure levels) is reflected by the use of neurological impairment as the basis for 9 of 13 MRLs for BTEX chemicals, including 6 of 8 inhalation MRLs (ATSDR 1995, 1997, 1999, 2000). The carcinogenic (leukemogenic) potential of benzene is well established as indicated by its consensus classification as a human carcinogen by the National Toxicology Program (NTP 2001), U.S. Environmental Protection Agency (EPA) (IRIS 2001), and International Agency for Research on Cancer (IARC 1987). Ethylbenzene is possibly carcinogenic to humans based on a recent assessment by IARC (2000). Toluene and xylenes have been categorized as not classifiable as to human carcinogenicity by both EPA (IRIS 2001) and IARC (1999a, 1999b), reflecting the lack of evidence for the carcinogenicity of these two chemicals (ATSDR, 2004). • N-hexane, a neurotoxicant, which was present in JP-4, does not appear to be present in JP-8, or it is at extremely low concentrations (Pilot Study, 1998). • Aerosols /combustion products; the vapour was found to represent the lower weight components of JP-8 while the aerosol was composed of higher molecular weight components. Therefore, it has been suggested that vapour and aerosol should be treated as two discrete forms of exposure to aviation fuels.

Risk assessments have been carried out to date only in the USA, COT (1996), (2003). These actions reviewed the current scientific knowledge about health effects caused by aviation fuels and established an interim permissible exposure limit – PEL of 350 mg/m3. In this light, the objective of the present scoping study is to investigate and report on the complexity of the scientific assessment of possible adverse health effects outlined in the recent literature and to suggest practical solutions on how to set OELs in this area in the EU.

2. EXISTING OELS AND MAJOR RISK ASSESSMENT STUDIES ON AVIATION FUELS

Regulatory activity for the setting of OELs in the area of aviation fuels are known only from the USA.

The earliest known OEL is attributed to the Department of Defence (DOD) and was recommended before 1996 as an interim permissible exposure level (PEL), a time-weighted exposure concentration in workplace air averaged over an 8-hr shift, of 350 mg/m3 and a 15 min short-term exposure limit (STEL) of 1800 mg/m3 for vapours from JP-5, JP-8, and DFM (diesel fuel marine) (COT 1996).

This interim PEL by was evaluated systematically by the Committee on Toxicology (COT) and the Subcommittee on Permissible Exposure Levels for Military Fuels of the National Research Council (NRC) in 1996. The evaluation was carried out by reviewing data on the toxicity of the vapours from JP-5, JP-8, and DFM in experimental animals and humans. COT concluded that the PEL of 350 mg/m3 for the fuel vapours was found adequate to protect naval personnel exposed to them occupationally. Data needed to evaluate the adequacy of the 15-min STEL of 1,800 mg/m3 for the three fuels are sparse. The subcommittee considered the acute CNS effects (e.g., dizziness, headache, nausea, and fatigue) in the Swedish jet-motor factory workers to be the most critical health effects for determining the adequacy of the STELs. Based on the limited information on exposure concentrations and the attribution of CNS symptoms to peak exposures of approximately 1,000 mg/m3 or higher, the subcommittee recommends that the Navy's current STEL be lowered from 1,800 mg/m3 to 1,000 mg/m3 to avoid acute CNS toxicity. The STEL of 1,000 mg/m3should also be considered as an interim recommendation until further research is completed. To this end, COT highlighted several uncertainties and recommended the PEL to be considered as interim until further research would provide the missing data in the following areas:

• On exposures during operational procedures, including exposures to respirable aerosols of unburned fuels • On possible effects of high level acute and low-level chronic exposure to fuel vapours on the central nervous system • On the effect of fuel vapours on hepatotoxicity in experimental animals to help identify a no-observed –adverse –effect level for JP-8 with greated confidence.

Following the wider introduction of JP-8 by the end of the 90’s a new request was made by the US Air Force to NRC to independently review the scientific basis of the PEL of 350 mg/m3. The review was carried out again by the Committee on Toxicology (COT) and the Subcommittee on Permissible Exposure Levels for Military Fuels and the results were published in 2003 (COT, 2003). The health-effects data on JP-8 and related fuels were reviewed for the following end points: respiratory tract toxicity, neurotoxicity, immunotoxicity, liver toxicity, kidney toxicity, reproductive and developmental toxicity, cardiovascular toxicity, genotoxicity, and carcinogenicity. JP-8 was found to be potentially toxic to the immune system, respiratory tract, and nervous system at exposure concentrations near the interim PEL of 350 mg/m3. On the basis of the available toxicologic data, the subcommittee concluded that the interim PEL of 350 mg/m3 for JP-8 might be too high to be protective of human health. However, it was beyond the charge to the subcommittee to propose a specific PEL for JP-8; such decisions necessarily involve more than scientific considerations. In addition, further studies on JP-8 are necessary to provide the requisite data to establish a PEL with greater confidence. Because JP-8 vapors and aerosols have different toxic potencies, the Air Force should consider developing separate PELs for vapors and aerosols.

The subcommittee further concluded that in addition to inhalation exposures, the potential exists for a substantial contribution to the overall JP-8 exposure by the dermal route, including mucous membranes and the eyes, either by contact with vapours and aerosols or by direct skin contact with JP-8.

Further assessment work related to aviation fuels has been carried out by a number of institutions:

• A Toxicological Profile for Jet Fuels (JP-5 and JP-8) was established by ATSDR (Agency for Toxic Substances and Disease Registry), 1998. • The National Institute for Occupational Safety and Health (NIOSH) set an 8-hr recommended exposure limit (REL) time-weighted average (TWA) of 350 mg/m3 for petroleum distillates. NIOSH has established a REL TWA of 100 mg/m3 for kerosene (NIOSH 1997). • The Occupational Safety and Health Administration (OSHA) set a PEL TWA of 2,000 mg/m3 (NIOSH 1997; OSHA 29 CFR (1910. 1000) 1997).

• The American Conference of Governmental Industrial Hygienists (ACGIH) proposed a Threshold Limit Value for kerosene and jet fuels (as a total hydrocarbon vapour) of 200 mg/m3 (ACGIH 2002). ACGIH classified kerosene and jet fuels as “confirmed animal carcinogens with unknown relevance to human skin” (ACGIH 2002). • ExxonMobil Biomedical Sciences, Inc., has set occupational exposure levels for kerosene and other middle distillate fuels of 500 mg/m3 for vapors and 5 mg/m3 for aerosols (ExxonMobil BS, 2001). • The International Agency for Research on Cancer concluded that jet fuel is “not classifiable” as to its carcinogenicity in humans (IARC 1989). • A systematic literature review was published recently (Witten et al., 2010)

3. SUBSTANCE IDENTIFICATION AND CARACTERIZATION

3.1 Fuel types and uses

Aviation fuel is a specialized type of petroleum-based fuel used to power aircraft. It is generally of a higher quality than fuels used in less critical applications, such as heating or road transport, and often contains additives to reduce the risk of icing or explosion due to high temperatures, among other properties.

Aviation fuels can be divided into two main categories, depending on the types of engines used in the aircrafts:

• Aviation Gasoline, abbreviated AvGas, is used in engines with spark plugs, i.e. piston and Wankel rotary engines, • Jet Fuels are used in aircrafts with jet turbine engines. Jet fuels are subdivided in categories specific to commercial and to the military aviation.

Alcohol, alcohol mixtures and other alternative fuels may be used experimentally, but alcohol is not permitted in any certified aviation fuel specification.

Avgas, which is sold in much lower volumes compared to jet fuel, is out of the scope of the present study, which is exclusively focused on jet fuels.

Jet fuels can further be categorised into

• Commercial grades: JetA, JetA-1 'kerosene Type' and JetB 'wide cut=a blend of e.g.: gasoline and kerosene' (other grades: Jet TS-1_Former Soviet Union, Jet TH_Romania, Jet Fuel No 3_China). • Military grades: JP-4 'wide cut' similar to JetB, JP-5, JP-7, JP-8 'kerosene type' similar to JetA-1, JP-9 Missile fuel-higher energy per unit volume).

Currently the most widely used fuel types are JeT A and Jet A-1 for the commercial aviation and JP-8 for the military aviation, which replaced earlier specifications (JP-4 and JP-5).

Jet A and Jet A-1 fuels have to comply with international standardized set specifications:

• Jet A-1 Fuel must meet the specification for DEF STAN 91-91 (Jet A-1), ASTM specification D1655 (Jet A-1) and IATA Guidance Material (Kerosene Type), NATO Code F-35.

• Jet A Fuel must reach ASTM specification D1655 (Jet A).

Jet A fuel specification has been used in the United States since the 1950s and is only available in the United States (and at Gander Airport in Newfoundland by Shell Aviation), whereas Jet A-1 is the standard specification fuel used in the rest of the world.

Jet B is a fuel in the naphtha-kerosene region that is used for its enhanced cold-weather performance. However, Jet B's lighter composition makes it more dangerous to handle. For this reason it is rarely used, except in very cold climates. A blend of approximately 30% kerosene and 70% gasoline, it is known as wide-cut fuel. It has a very low freezing point of - 60 degrees Celsius and a low flash point as well. It is primarily used in US and some military aircraft.

Jet A-1 is classified by EC as flammable, harmful, irritant and dangerous for the environment. It is slightly irritating to respiratory system and breathing of high vapour concentrations may cause central nervous system depression resulting in dizziness, light headedness, headache and nausea. In addition it is irritating to skin and harmful as it may cause lung damage if swallowed (Shell datasheet, 2010).

JP-8 jet fuel is essentially the same as commercial jet fuel (Jet A and A-1, ASTM D1655 and DEFSTAN 91-91, respectively) except for the inclusion of several additives in JP-8 that inhibit icing, reduce corrosion, and dissipate static electricity. As a multipurpose fuel, JP-8 was developed by the U.S. military for use as a single “universal” fuel in ground vehicles, tanks, generators, tent heaters, cooking stoves, and other equipment, as well as in aircraft of the entire U.S. military and the forces of the North Atlantic Treaty Organization (NATO). JP- 8 is the primary fuel used by the U.S. Department of Defense (DOD), although the Navy procures significant quantities of high-flash-point JP-5 (MIL-DTL-5624U) and naval distillate (marine diesel) fuel F-76 (MIL-DTL-16884L). The DOD also uses significant amounts of commercial jet fuel. A good source for data and information on military fuels are the annual reports of the Petroleum Quality Information System located at the Defense Energy Support Center, which procures all fuels for the DOD. The US Air Force started transitioning to JP-8 from JP-4 (Jet Propulsion Fuel 4) in the 1980s. JP-4 was a “wide-boiling” fuel with a much lower boiling point than current “kerosene” jet fuels.

3.2 Fuel composition

Jet fuels are complex mixtures of hydrocarbons produced by distillation of crude oil. They contain hundreds of hydrocarbons as well as many additives. The actual composition of any given fuel varies depending upon source of the crude oil, refinery processes, and product specifications. The hydrocarbons in jet and diesel fuels are less volatile than those in gasoline.

Jet fuels typically contain a number of additives, including antioxidants to prevent gumming, antistatic agents to dissipate static electricity and prevent sparking, and fuel system icing inhibitor agents:

The DEF STAN 91-91 (UK) and ASTM D1655 (international) specifications allow for certain additives to be added to jet fuel, including.

• Antioxidants to prevent gumming, usually based on alkylated phenols, e.g., AO-30, AO-31, or AO-37; • Antistatic agents, to dissipate static electricity and prevent sparking; Stadis 450, with dinonylnaphthylsulfonic acid (DINNSA) as the active ingredient, is an example • Corrosion inhibitors, e.g., DCI-4A used for civilian and military fuels, and DCI- 6A used for military fuels; • Fuel System Icing Inhibitor (FSII) agents, e.g., Di-EGME; FSII is often mixed at the point-of-sale so that users with heated fuel lines do not have to pay the extra expense. • Biocides are to remediate microbial (i.e., bacterial and fungal) growth present in aircraft fuel systems. Currently, two biocides are approved for use by most aircraft and turbine engine original equipment manufacturers (OEMs); Kathon FP1.5 Microbiocide and Biobor JF. • Metal deactivator can be added to remediate the deleterious effects of trace metals on the thermal stability of the fuel. The one allowable additive is N,N’-disalicylidene 1,2- propanediamine.

Generally the composition of jet, gasoline, and diesel fuels is not controlled explicitly by their specifications, except for a few specific components (Witten et al., 2011). These specifications have implicitly limited jet fuel hydrocarbons to carbon numbers between about 8 and 16, but still jet fuels contain hundreds of hydrocarbons. Common characteristics of jet fuels are depicted in Table 3.1.

Table 3.1. Common characteristics of jet fuels (Simon et al., 2008). Name Specification Description Flash point. C Freeze point C ASTM D1655, Standard Jet A-1 UK DefStan 91- commercial jet >38 <-47 91 fuel US domestic jet Jet A ASTM D1655 >38 <-40 fuel US military jet MIL-DTL- JP-8 fuel (Jet A-1 + 3 >38 <-47 83133 additives) MIL-DTL- US Navy high JP-5 >60 <-46 83133 flash jet fuel TS-1 GOST 10227-86 Russian jet fuel >28 <-50

To identify and compare existing jet fuels it would be necessary to take into account the most important manufacturers and their data sheets for respective fuels. For that reason 'World Fuel Sampling Program took place in 2006, releasing a document that contains the data from a fuel sampling and testing program conducted jointly by Boeing, Goodrich, General Electric, Chevron Texaco, and the United States Air Force (CRC, 2006). In general the composition of JP-8 is 33-61% hydrocarbons, 12-22% aromatics (benzene, substituted benzenes, napthalene and substituted naphthalene), 10-45% alicyclic hydrocarbons (cycloalkanes), 0.5-5% olefins and 0-0.3% sulphur-containing heterocyclics. (Gregg et al., 2007).

Table 3.2. Mean hydrocarbon composition from STM D2425 in world survey of jet fuels

Composition %

Paraffins (n- + iso-) 58.78 Monocycloparaffins 10.89 Dicycloparaffins 9.25 Tricycloparaffins 1.08 Alkyl benzenes 13.36 Indans/tetralins 4.9 Naphtalene 0.13 Substituted naphthalenes 1.55

The kerosene/jet fuel category consists of complex petroleum refinery streams derived from crude oil that have similar composition and carbon ranges (C9 to C16)..

Similarities in the composition of the streams are expected to result in similar physicochemical, environmental fate and toxicological properties (EPA US, 2011).

Table 3.3. Constituents of Jet-A specified by gas Chromatography /Mass Spectrometry (Witten et al., 2011). Name CAS No. n-Heptane 142-82-5 Methylcyclohexane 108-87-2 2-Methylheptane 592-27-8 Toluene 108-88-3 cis-1,3-Dimethylcyclohexane 638-04-0 n-Octane 111-65-9 1 1,2,4-Trimethylcyclohexane 2234-75-5 4 - Methyloctane 2216-34-4 1,2-Dimethylbenzene 95-47-6 n-Nonane 111-84-2 4-Methylnonane 17301-94-9 1-Ethyl-3-methylbenzene 620-14-4 2,6-Dimethyloctane 2051-30-1 1-Methyl-3-(2-methylpropyl) cyclopentane 29053-04-1 1-Ethyl-4-methylbenzene 622-96-8 1-Methyl-2-propylcyclohexane 4291-79-6 1,2,4-Trimethylbenzene 95-63-6 n-decane 124-18-5 1-Methyl-2-propylbenzene 1074-17-5 4-Methyl decane 2847-72-5 1,3,5-Trimethylbenzene 108-67-8 2,3-Dimethyldecane 17312-44-6 1-Ethyl-2,2,6-trimethylcyclohexane 71186-27-1 1-Methyl-3-propylbenzene 1074-43-7 5-Methyldecane 13151-35-4 2-Methyldecane 6975-98-0 3-Methyldecane 13151-34-3 1-Methyl-(4-methylethyl)benzene 99-87-6 n-Undecane 1120-21-4 1-Ethyl-2,3-dimethylbenzene 933-98-2 n-Dodecane 112-40-3 2,6-Dimethylundecane 17301-23-4

3.3 Statistical data about the consumption of aviation fuels and the employment in the air-transport sector

Global oil production is roughly 81.5 million barrels per day, which is equivalent to an annual output of 3905.9 million tonnes (Nygren, 2009). Jet fuel production, originating from crude oil, was 5,269 barrels per day in 2008 (Figure 3.1). Scenarios show (Figure 3.2) that aviation fuel demand will continue to increase. However, the increase is believed to not be very high as jet fuel demand and aviation traffic growth are not strictly correlated, since the efficiency of aircraft and air traffic management are improving.

Figure 3.1. Annual word jet fuel consumption (source: http://www.indexmundi.com).

Figure 3.2. Aviation fuel demand (Nygren, 2009).

JP-8 is the single largest source (9.58 billion litres in 2000) of chemical exposure to military personel in the United States (Henz, 1998, Egeghy et al. 2003, Richie et al 2003).

In the occupational section, about 2.0 million commercial and military airline workers in the USA are exposed to the fuels (COT 2003). In Europe more than 400,000 people were employed in the air transport sector in 2002, according to Eurostat’s Structural Business Statistics (SBS) (Figure 3.3).

Figure 3.3. Key indicators, air transport, EU-27, 2009 (Eurostat, 2009).

4. PHYSICO-CHEMICAL PROPERTIES

4.1 Commercial aviation fuels (Jet A / Jet A-1 fuels)

Jet A specification fuel has been used in the United States since the 1950s and is only available in the United States (and at Gander Airport in Newfoundland by Shell Aviation), whereas Jet A-1 is the standard specification fuel used in the rest of the world. Both Jet A and Jet A-1 have a flash point higher than 38 °C, with an auto-ignition temperature of 210 °C. This shows a significant advantage that the fuel is safer to handle than traditional avgas.

The primary differences between Jet A and Jet A-1 are the higher freezing point of Jet A (−40 °C vs −47 °C for Jet A-1), and the mandatory requirement for the addition of an anti-static additive to Jet A-1.

Table 4.1. Typical physical properties for Jet A / Jet A-1 fuel: Properties Jet A-1 Jet A Flash point 42 °C 51.1 °C Auto ignition temperature 210 °C Freezing point −47 °C −40 °C Open air burning temperatures 260-315 °C Density at 15 °C (59 °F) .804 kg/L .820 kg/L Specific energy 43.15 MJ/kg 43.02 MJ/kg Energy density 34.7 MJ/L 35.3 MJ/L Source: DEFSTAN 91-91, D1655 ASTM, IATA Guidance Material

4.2 Military aviation fuels (JP-8)

In the next tables the typical physical and chemical properties of JP-8 are depicted.

Table 4.2. Typical physical and chemical properties for JP-8 fuel (COT 2003). Molecular » 180 weight: Jet fuel JP-8, kerosene, aviation kerosene, fuel oil no. 1, jet kerosene, Synonyms: turbo fuel A, straight-run kerosene, distillate fuel oil–light, MIL-T- 83133B, AVTUR, NATO F-34. CAS registry 8008-20-6 (kerosene)/70892-10-3 (fuel oil 1) number: Freezing point, -47ºC maximum: Boiling point: 175-300ºC 10% recovered, 205ºC maximum: End point, 300ºC maximum: Flash point, 38ºC minimum: 0.52 mm Hg (10ºC), Vapour pressure: 1.8 mm Hg (28ºC) Specific gravity,

kg/L, 15ºC, minimum: 0.775 maximum: 0.840 Heating value,

Btu/lb, minimum: 18,400 Viscosity, maximum at - 8 20ºC: Physical state: Liquid Color: Clear and bright Solubility in 5 mg/L (kerosene) water: Vapor density (air 4.5-5 = 1): Liquid density 0.788-0.845 kg/L (water = 1): Odor: Kerosene-like

5. SPECIFICATION OF EXPOSURE SOURCES, CHEMICALS INVOLVED AND LEVELS OF EXPOSURES

5.1 Sources of occupational Exposure to aviation fuels

Exposure to chemicals originating from aviation fuels in both military and civilian occupational settings can occur through contact to several types of media:

• Raw fuel • Vapour phase • Aerosol phase • Fuel combustion exhaust • Some combinations of the above

According to Kim et al. 2007, and COT (2003), the most important sources for occupational exposures can be listed as follows: • Spills during transportation and storage of fuel • Fuelling • General maintenance and operation of aircrafts and vehicles • Cold engine starts • Performance testing • Cleaning and decreasing of parts with fuel

5.1.1 Fuel cell maintenance Fuel cell maintenance personnel have the highest exposure to JP-8 because in order to perform repair work, they must enter the aircraft fuel tanks where fuel vapours accumulate, and residual fuel covers the bottom of the tank. Before maintenance can be performed, the polyurethane foam, which is saturated with fuel that fills the fuel cell, must be removed (Carlton and Smith, 2000). This foam is in fuel cells to improve the safety and stability of the aircraft. Fuel cell maintenance personnel work in groups of three and consist of an entrant (enters the fuel tank, removes polyurethane foam from the tank, and performs maintenance work inside the tank); an attendant (assists entrant from outside the fuel tank and hands foam to the runner); and a runner (moves foam to a temporary location and provides tools to the attendant) (Serdar et al. 2004).

5.1.2 Fuelling Aviation fuel is often dispensed from a tanker or bowser, which is driven up to parked aircraft and helicopters. Some airports have pumps similar to filling stations up to which aircraft must taxi. Some airports also have permanent piping to parking areas for large aircraft.

Regardless of the method, aviation fuel is transferred to an aircraft via one of two methods: over-wing or under-wing. Over-wing fuelling is used on smaller planes, helicopters, and all piston-engine aircraft. Over-wing fuelling is similar to car fuelling — one or more fuel ports are opened and fuel is pumped in with a conventional pump. Under-wing fuelling, also called single-point, is used on larger aircraft and for jet fuel exclusively. For single-point fuelling, a high-pressure hose is attached and fuel is pumped in at 40 psi and a maximum of 45 psi for most commercial aircraft. Pressures for military aircraft, especially fighters, range up to 60 psi maximum. Air being displaced in the tanks is usually vented overboard through a single vent on the aircraft. Since there is only one attachment point, fuel distribution between tanks is either automated or it is controlled from a control panel at the fuelling point or in the cockpit. A dead man's switch is also used to control fuel flow.

Because of the danger of confusing the fuel types, a number of precautions are taken to distinguish between avgas and jet fuel beyond clearly marking all containers, vehicles, and piping. Avgas is treated with either a red, green, or blue dye, and is dispensed from nozzles with a diameter of 40 mm (49 mm in the USA). The aperture on fuel tanks of piston-engined aircraft cannot be greater than 60 mm in diameter. Jet fuel is clear to straw- coloured, and is dispensed from a special nozzle called a "J spout" that has a rectangular opening larger than 60 millimetres in diameter, so as not to fit into avgas ports. However, some jet and turbine aircraft, such as some models of the Astar helicopter, have a fuelling port too small for the J spout, and thus require a smaller nozzle to be installed to be refuelled efficiently.

5.2 Key factors influencing the exposure and health effects

• Fuel type and lot • Work tasks • Environmental conditions, such as temperature and humidity • Noise, may interact with exposure to influence the functioning of the nervous system (Morata et al. 1993, Odkvist et al. 1986)

• Risk Management Measures (RMMs), such as workplace procedure protocols, use of protective gear, such as goggles, gloves and coveralls • Lifestyle factors, such as smoking and alcohol use and genetic variability in terms of enzyme polymorphisms

5.3 Categories of personnel who work directly with fuel

• Fuel cell maintenance personnel • Crew chiefs perform inspections on the aircraft before and after flight operations and may be exposed to aerosols during low ambient temperature engine starts. • Mechanics are exposed to aviation fuels when working with engine or other fuel- soaked parts that need repair. • Fuel specialists are responsible for receiving aviation fuels, refuelling aircraft on the flight line, and checking the fuel for contamination and proper additive concentrations. Exposure occurs when handling the fuel and during fuel spills. • All personnel on base, regardless of their occupation, had measurable levels of jet fuel exposure (Pleil et al., 2000; Tu et al., 2004). Individuals who do not directly work with aviation fuels, such as hospital staff, military police, office workers are exposed to aviation fuels, because it is present in the ambient air or from contact with directly exposed individuals.

Several studies (Egeghy et al., 2003; Smith et al. 2010) carrying out exposure assessment measurements in military air force bases, have been categorising the active duty personnel into the following three exposure groups:

• High exposure group: aircraft fuel systems maintenance workers with routine direct contact with JP-8 fuel. • Moderate exposure group: workers with regular contact with JP-8 via fuel handling (fuel storage, distribution, laboratory testing) or refuelling maintenance (performed maintenance on fuel distribution tanks). • Low exposure group: personnel working in office jobs (health clinic) with no regular contact with JP-8.

5.4 Chemicals of major concern

Aviation fuels are a complex mixture of numerous hydrocarbons and additives.

Concentrations of different constituents vary across the different fuel types and within the same fuel type from lot to lot. For example, JP-8 contains less benzene, has a higher flashpoint, and is less volatile compared to its predecessor formulations, namely JP-4 and JP-5 (COT, 2003).

Aviation fuels consist of blends of over a thousand chemicals, primarily hydrocarbons (paraffins, olefins, naphthenes, and aromatics), as well as additives, such as antioxidants and metal deactivators, and impurities. Principal components include n-heptane and isooctane. Like other fuels, blends of aviation fuel used in spark-ignited piston-engined aircraft are often described by their octane rating.

Naphthalene, a polycyclic aromatic hydrocarbon, is found in JP-8 mixtures in low levels (McDougal et al., 2000; Smith et al., 2010), and is classified as Group 2B: “possibly carcinogenic to humans” (IARC, 2002).

Benzene, toluene, ethylbenzene, m-lp xylene, o-xylene (BTEX). Although benzene is found at concentrations below 0.02% in JP-8, it is a known human carcinogen and is arguably together with naphthalene among the most hazardous components of JP-8 (Egeghy et al., 2003). All of the BTEX chemicals can produce neurological impairment, and exposure to benzene can additionally cause hematological effects including aplastic anaemia and acute myelogenous leukaemia. The critical nature of the neurotoxicity (i.e., the non cancer effect expected to occur at the lowest exposure levels) is reflected by the use of neurological impairment as the basis for 9 of 13 Minimum Risk Levels (MRLs) for BTEX chemicals, including 6 of 8 inhalation MRLs (ATSDR 1995, 1997, 1999b, 2000). The carcinogenic (leukaemogenic) potential of benzene is well established as indicated by its consensus classification as a human carcinogen by the National Toxicology Program (NTP 2001), U.S. Environmental Protection Agency (EPA) (IRIS, 2001), and International Agency for Research on Cancer (IARC, 1987). Ethylbenzene is possibly carcinogenic to humans based on a recent assessment by IARC (2000). Toluene and xylenes have been categorized as not classifiable as to human carcinogenicity by both EPA (IRIS 2001) and IARC (1999a, 1999b), reflecting the lack of evidence for the carcinogenicity of these two chemicals (ATSDR, 2004).

N-hexane, a neurotoxicant, which was present in JP-4, does not appear to be present in JP-8, or it is at extremely low concentrations (Pilot Study, 1998).

5.4.1 Aerosols Methods for the characterisation of vapours and aerosols resulting from JP-8 were investigated by Gregg et al. (2007). The vapour was found to represent the lower weight components of JP-8, while the aerosol was composed of higher molecular weight components. Therefore, the study concluded that vapour and aerosol should be treated as two discrete forms of exposure to aviation fuels.

Issues associated with the generation of a jet fuel atmosphere including the vapour and aerosol phase are discussed by Tremblay et al. 2008 (Witten et al., 2011). The study reveals that typically, at no point during the fuel exposure is the vapour or aerosol composition absolutely predictable in terms of representing the known neat fuel composition. In a given example, measured composition of vapour and aerosol phases generated for a JP-8 inhalation exposure is compared to the composition of neat JP-8. The volatile compounds (methylcyclohexane, toluene, 2-methylheptane, and octane) account for nearly 10% of the vapour phase, although they only represent about 3% of the neat fuel. A similar but reverse discordance between the neat fuel composition and aerosol droplet composition is observed for low vapour pressure compounds in the aerosol.

In another example, is showed the ratio aerosol to gas/vapour phase, for a series of detected compounds ordered by molecular weight for a targeted 1000-mg/m3 exposure to S-8 synthetic jet fuel made through the Fischer-Tropsch process. Over 90% of the octane is in the gas phase while 70% to 80% of the heavier hydrocarbons are in the aerosol phase.

5.4.2 Combustion products Combustion products from aviation fuels, relevant to the present study, mainly consist of the following chemical components (Ritchie, 2003):

• Inorganic gases: CO, CO2, NOx, SO2

• Volatile organic compounds (VOCs)

• Raw fuel

• Oxygenated organics

• Polycyclic aromatic hydrocarbons (PAHs)

• Alcohols

• Ozone

• Particulate matter

Ultrafine particles appear to be a key issue among the above compounds in combustion products from aviation fuels. The following recent studies and reports discuss the relationship between aviation activities and ultrafine particulate matter:

• Hsu et al., 2012;

• Westerdahl, 2008;

• Fanning et al., 2007;

• Danish Ecocouncil, 2012;

• Ellerman et al., 2011;

5.5 Exposure Levels

Assessing JP-8 exposure is difficult because it is a mixture of aliphatic hydrocarbons (81%) and aromatic compounds (19%).

Several methods have been developed to estimate human exposure to JP-8 and they utilize different sampling media, such as ambient air, skin samples, exhaled breath, blood, and urine. Marker compounds like naphthalene and benzene, JP-8 fingerprint compounds (aliphatic: nonane, decane, undecane, and dodecane; aromatic: benzene, toluene, ethylbenzene, m- xylene, o-xylene, and p-xylene), or their metabolites have been measured in various media.

5.5.1 Inhalation Exposure Breathing zone concentrations: Concentrations of pollutants originating from aviation fuels have been measured, by a number of studies using portable samplers in the breathing zone of personnel working in several locations within commercial airports and military air force bases (AFBs):

• Puhala E, et al. (1997): Measurement of breathing zone ambient concentrations over whole working shifts at three AFBs. Mean ambient levels for Total Hydro-Carbons (THC) were 1.33 ppmv and 0.01 ppmv for benzene. • Yeung P. et al. (1997): Measurement of vapour concentrations inside B747 aircraft fuel tanks were found to reach 2823 mg/m3 THC and 1.3 ppm benzene. • Pleil J. D. et al. (2000): Measurements were carried out in a number of US AFBs, at the indoor of AFB shops, at the outdoor during aircraft cold-starts, around the aircrafts during fuel tank maintenance, and inside the fuel tanks during fuel tank maintenance. Mean values for benzene and toluene are the following: o indoor of AFB shops: 1.05 ppbv benzene, 2.51 ppbv toluene o outdoor during aircraft cold-starts: 13.04 ppbv benzene, 8.87 ppbv toluene o around the aircrafts during fuel tank maintenance: 17.64 ppbv benzene, 53.15 ppbv toluene o inside the fuel tanks during fuel tank maintenance: 2.987 ppbv benzene, 16.026 ppbv toluene. • Carlton, G.N. and L. B. Smith (2000): Jet fuel and benzene vapor exposures were measured during aircraft fuel tank entry and repair at twelve U.S. Air Force bases. Breathing zone samples were collected on the fuel workers who performed the repair.

In addition, instantaneous samples were taken at various points during the procedures with SUMMA canisters and subsequent analysis by mass spectrometry. The highest eight-hour time-weighted average (TWA) fuel exposure found was 1304 mg/m3; the highest 15-minute short-term exposure was 10,295 mg/m3. The results indicate workers who repair fuel tanks containing explosion suppression foam have a significantly higher exposure to jet fuel as compared to workers who repair tanks without foam (p<0.001). It is assumed these elevations result from the tendency for fuel, absorbed by the foam, to volatilize during the foam removal process. Fuel tanks that allow flow-through ventilation during repair resulted in lower exposures compared to those tanks that have only one access port and, as a result, cannot be ventilated efficiently. The instantaneous sampling results confirm that benzene exposures occur during fuel tank repair; levels up to 49.1 mg/m3 were found inside the tanks during the repairs. As with jet fuel, these elevated benzene concentrations were more likely to occur in foamed tanks. The high temperatures associated with fuel tank repair, along with the requirement to wear vapour-permeable cotton coveralls for fire reasons, could result in an increase in the benzene body burden of tank entrants. • Egeghy P.P. et al. (2003): Benzene and naphthalene were measured in air and breath of 326 personnel in the US Air Force, who had been assigned a priori into low, moderate, and high exposure categories for JP-8. Median air concentrations for persons in the low, moderate, and high exposure categories were 3.1, 7.4, and 252 µg benzene/m3 air, 4, 1.9, 10.3, and 485 µg naphthalene/m3 air. In the moderate and high exposure categories, 5% and 15% of the benzene air concentrations, respectively, were above the 2002 threshold limit value (TLV) of 1.6 mg/m3. • Smith K. W. et al. (2010): Personal air samples (breathing zone) were collected from each worker over 3 consecutive days (72 worker-days) and analyzed for total hydrocarbons (THC), benzene, toluene, ethylbenzene, xylenes, and naphthalene. Air samples were collected from inside the fuel tank and analyzed for the same analytes- THC exposure levels were generally lower than those reported previously.

5.5.2 Dermal exposure Because JP-8 is less volatile than its predecessor JP-4, contact with liquid fuel on skin and clothing may result in increased dermal exposures. Until recently, the only method for quantification of dermal exposure to aviation fuels consisted in the measurement on

unmetabolized fuel components on the skin surface using the 'Tape stripping' method according to Chao and Nylander French (2004), Chao et al. (2005) and Mattorano et al. (2004).

Initial results were reported by McDougal et al. (2000). The purpose of this investigation was to measure the penetration and absorption of JP-8 and its major constituents with rat skin, so that the potential for effects with human exposures can be assessed. Concentrations absorbed into the skin at 3.5 hr ranged from 0.055 μg per gram skin (tetradecane) to 0.266 μg per gram skin (undecane). These results suggest: (1) that JP-8 penetration will not cause systemic toxicity because of low fluxes of all the components; and (2) the absorption of aliphatic components into the skin may be a cause of skin irritation.

The most cited dermal exposure work has been carried out on US Air force fuel cell maintenance workers (Chao et al., 2005). The study investigated the magnitude of dermal exposure to jet propulsion fuel 8 (JP-8), using naphthalene as a surrogate, on the US Air Force fuel-cell maintenance workers. Dermal exposure of 124 workers routinely working with JP-8 was measured using a non-invasive tape-strip technique coupled with gas chromatography- mass spectrometry analysis. The contribution of job-related factors to dermal exposure was determined using multiple linear regression analyses. Average whole body dermal exposure to naphthalene (as a marker for JP-8) was 7.61 +/- 2.27 ln(ng m(-2)). Significant difference (P < 0.0001) between the high-exposure group [8.34 +/- 2.23 ln(ng m(-2))] and medium- and low- exposure groups [6.18 +/- 1.35 ln(ng m(-2)) and 5.84 +/- 1.34 ln(ng m(-2)), respectively] was observed reflecting the actual exposure scenarios. Skin irritation, use of booties, working inside the fuel tank and the duration of JP-8 exposure were significant factors explaining the whole body dermal exposure. This study demonstrated the efficiency and suitability of the tape-strip technique for the assessment of dermal exposure to JP-8 and that naphthalene can serve as a useful marker of exposure and uptake of JP-8 and its components. It also showed that the skin provides a significant route for JP-8 exposure and that actions to reduce exposure are required.

5.5.3 Biomonitoring studies

5.5.3.1 Exhaled breath measurements

Pleil et al. (2000) analysed pre-exposure and post-exposure breath samples for a number of chemicals, including benzene, toluene, ethylbenzene, etc. These data were combined and

compared to ambient/breathing zone measurements, demonstrating measurable exposure levels and the efficiency of risk management measures. JP-8 exposure in fuel systems workers as measured in their breath is equivalent for tank entry and attendant personnel, yet the ambient (potential) exposures are 40 times greater inside the fuel tanks. Therefore, it was concluded that the full-face forced-air respirators worn by tank entry personnel (only while they are inside the tank) are extremely effective in eliminating inhalation exposure and that the JP-8 in their breath is primarily from their activity in the vicinity of the aircraft outside the fuel tanks (while they are not wearing respiratory protection). This is supported by the similar JP-8 breath levels found in exhaust workers during indoor pre-flight activity.

Egeghy et al. (2003) reported median concentrations for persons in the low, moderate, and high exposure categories in the levels 4.6, 9.0, and 11.4 µg benzene/m3 breath, and 0.73, 0.93, and 1.83 µg naphthalene/m3 breath, respectively. In the moderate and high exposure categories multiple regression analyses of air and breath levels revealed prominent background sources of benzene exposure, including cigarette smoke. However, naphthalene exposure was not unduly influenced by sources other than JP-8. Among heavily exposed workers, dermal contact with JP-8 contributed to air and breath concentrations along with several physical and environmental factors.

While concentrations of naphthalene were higher than concentrations of benzene in air, median naphthalene levels were lower than those of benzene in “post-exposure” breath. This points to the lower vapour pressure of naphthalene compared to benzene (0.082 v 95.3 mm Hg at 25°C) and the much higher estimated blood-air partition coefficient (based on log octanol-water partition coefficients of 3.39 for naphthalene and 2.13 for benzene).

Concentrations of both benzene and naphthalene were significantly higher in “post-exposure” breath than in “pre-exposure” breath in the moderate and high exposure categories. This provides further evidence that JP-8 was a significant source of exposure to both compounds among Air Force personnel whose jobs required at least incidental contact with jet fuel. In the moderate exposure category, the difference between “pre-exposure” and “post-exposure” breath levels was more significant for naphthalene than for benzene, but in the high exposure category the difference was highly significant for both compounds (p<0.0001).

Comparisons of “post-exposure” breath concentrations across exposure categories showed significant differences not only between the high and low exposure categories but also between the high and moderate exposure categories for both naphthalene and benzene. The level of significance for the difference between benzene concentrations in the moderate and

high exposure categories was weaker than that for naphthalene (p<0.01 v p<0.0001), presumably because benzene is more abundant in cigarette smoke. The difference in “post- exposure” breath levels between the low and moderate exposure categories was not significant for either compound (p>0.05) despite a highly significant difference in external exposures (p<0.0001).

Despite the low concentration of benzene in fuel, benzene exposure was significant among subjects having regular contact with JP-8. Indeed, 5% and 15% of benzene air measurements were above the 2002 TLV among workers in the moderate and high exposure categories, respectively.

Airborne naphthalene was highly correlated with a priori categories of JP-8 exposure and, unlike benzene, was not unduly influenced by background sources and cigarette smoking. Furthermore, among highly exposed personnel, several factors known to increase exposure to and uptake of JP-8 also increased exposure to and uptake of naphthalene. This suggests that naphthalene may be a good surrogate for JP-8 in studies of health effects associated with jet fuel. Whereas benzene was predominantly absorbed by inhalation, naphthalene was absorbed by a combination of inhalation and dermal contact. Thus, biomonitoring of naphthalene or its products in breath or urine should reflect both respiratory and dermal uptake of JP-8 and might be preferred to air monitoring for exposure assessment.

5.5.3.2 Blood

Analysis of blood samples from aviation workers for trace level amounts (ppt) of VOC fuel components including benzene, ethyl benzene, m-/p-/o-xylenes and toluene was reported by Proctor et al. (2011). The presence of glutathione-S-trasferase (GST) enzyme polymorphisms, specifically deletion of GSTM1, was examined as a potential marker of susceptibility of exposure. As another potential marker of exposure, levels of peripheral blood DNA methylation patterns were also determined.

5.5.3.3 Urine

Benzene, naphthalene, and hydroxynaphthalene metabolites were measured in urine before and after exposure (Serdar et al., 2003). Urinary concentrations of benzene and naphthalene may be good surrogates of internal exposure because they reflect inhalation and skin exposure. Preliminary results indicated that, in the high-exposure group, there was strong correlation between workplace air and exhaled-air concentrations of each of these chemicals.

A high correlation was also observed for urinary naphthalene and its metabolite. Urinary concentrations of these chemicals were higher in smokers, but the effect of smoking became less significant as the exposure to JP-8 increased. That air and pre-exposure urine samples did not show a positive correlation suggests that pre-exposure urinary benzene and naphthalene concentrations were the result of sources other than occupational exposures.

Urinary metabolites of naphthalene have been suggested for use as short-term biomarkers of exposure to jet fuel (jet propulsion fuel 8 (JP-8)). In a recent study by Smith et al. (2012), urinary biomarkers of JP-8 were evaluated among US Air Force personnel. Personnel (n=24) were divided a priori into high, moderate, and low exposure groups. Pre- and post-shift urine samples were collected from each worker over three workdays and analysed for metabolites of naphthalene (1- and 2-naphthol). Questionnaires and breathing-zone naphthalene samples were collected from each worker during the same workdays. Linear mixed-effects models were used to evaluate the exposure data. Post-shift levels of 1- and 2-naphthol varied significantly by a priori exposure group (levels in high group>moderate group>low group), and breathing-zone naphthalene was a significant predictor of post-shift levels of 1- and 2- naphthol, indicating that for every unit increase in breathing-zone naphthalene, there was an increase in naphthol levels. These results indicate that post-shift levels of urinary 1- and 2- naphthol reflect JP8 exposure during the work-shift and may be useful surrogates of JP-8 exposure. Among the high exposed workers, significant job-related predictors of post-shift levels of 1- and 2-naphthol included entering the fuel tank, repairing leaks, direct skin contact with JP8, and not wearing gloves during the work-shift. The job-related predictors of 1- and 2-naphthol emphasize the importance of reducing inhalation and dermal exposure through the use of personal protective equipment while working in an environment with JP-8.

B’Hymer et al. (2005, 2011) report about the utility of the urinary metabolite (2- methoxyethoxy)acetic acid (MEAA) as a biomarker of exposure. 2-(2-methoxyethoxy)ethanol [diethylene glycol monomethyl ether] is an anti-icing agent used in the formulation of JP-8, and it is added at a known uniform 0.1% (v/v) concentration to each batch lot. JP-8 is a kerosene-based fuel containing different compounds that vary in the content of every batch/lot of fuel; thus, the study suggests that MEAA has the potential to be a more specific and a consistent quantitative biomarker for JP-8 exposure.

5.5.3.4 Dermal studies

Chao et al., (2006) investigated the contributions of dermal and inhalation exposure to JP-8 to the total body dose of U.S. Air Force fuel-cell maintenance workers using naphthalene as a surrogate for JP-8 exposure and conclude that dermal exposure to JP-8 significantly contributes to the systemic dose and affects the levels of urinary naphthalene metabolites. In addition, Kim et al. (2006) investigated dermal exposure in vitro in order to understand the absorption and penetration of aromatic and aliphatic components of JP-8 in humans. Rat and pig models of the skin over predict the internal dose of JP-8 components in humans.

Naphthyl-Keratin Adducts as biomarkers for Jet-Fuel Exposure: Studies to investigate the relative contribution of dermal uptake of JP-8 on total body dose and the toxicokinetics of dermal exposure to JP-8 have been carried out recently (Kang-Sickel et al., 2008; Kang-Sickel et al., 2010, Kang-Sickel et al., 2011). These studies have also shown that highly specific polyclonal antibodies to naphthalene metabolites can be used for a sensitive enzyme-linked immunosorbent assay (ELISA) for quantification of naphthyl-keratin adducts as biomarkers of dermal exposure to jet fuel. In dermal tape-strip samples collected from 105 individuals exposed to JP-8, naphthyl-conjugated keratin peptides were detected at levels from 0.004 to 6.104 pmol adduct/μg keratin, but were undetectable in unexposed volunteers. The promising feature of this approach is that only naphthalene through dermal route of exposure can induce formation of NKAs in the skin (i.e., these adducts are route specific indicators of exposure; inhalation exposure will not contribute to the formation of these adducts). Therefore, quantitation of naphthyl-keratin protein adducts in the skin of jet fuel exposed individuals allows to investigate the importance of dermal exposure, penetration, metabolism, and adduction of naphthalene and to predict more accurately the contribution of chronic dermal exposure to total body burden for use in exposure assessment models. The total NKA levels were affected by both the work scenarios and extrinsic and intrinsic personal factors, and were associated with the urine naphthalene levels, indicating their potential as quantitative biomarkers of dermal exposure. In addition, this novel dermal exposure biomarker represents the past exposure that occurred 20 – 28 days prior to sampling, and can be utilized in exposure assessment for other common PAHs and environmental pollutants. Further studies are needed in regard to the relationship between skin NKAs, biological effect markers, and JP-8 health effects to further explore the potential application of these adducts as biomarkers of naphthalene exposure and related health effects.

Chao et al. (2005) demonstrated the efficiency and suitability of the tape-strip technique for the assessment of dermal exposure to JP-8 and that naphthalene can serve as a useful marker of exposure and uptake of JP-8 and its components. It was also showed that the skin provides a significant route for JP-8 exposure and that actions to reduce exposure are required. Finally, Chou et al. (2006) revealed that human keratinocyte responds to a single dose of JP-8 insult and several cellular processes previously not associated with jet fuel exposure.

6. HEALTH EFFECTS

Depending on the route of exposure - inhalation, oral and dermal- health effects can be categorized as follows (COT 1996, COT 2003):

1. Effects on the respiratory system

2. Effects on the nervous system

3. Effects on immune system

4. Effects on liver

5. Effects on kidney

6. Effects on reproduction and development

7. Effects on cardiovascular system

8. Genotoxic effects

9. Carcinogenic effects

Reviewing the recent literature revealed several new studies with focus on dermal effects and toxicity. Therefore a separate chapter was added in the present scoping study under the title 'Dermal effects and toxicity' for further consideration by SCOEL.

6.1 Effects on the respiratory system

6.1.1 1996 assessment (COT 1996) This assessment did not include a review of the effects of JP-8 on the respiratory tract.

6.1.2 2003 assessment (COT 2003)

• No respiratory-tract effects were found in F344 rats and C57BL/6 mice exposed to JP- 8 vapour at 500 or 1,000 mg/m3 for 90 days. • Several animal studies conducted in F344 rats and C57BL/6 mice suggest that mixtures of JP-8 vapors and aerosols can result in pulmonary inflammation and alterations in pulmonary functions. • Toxic effects have been reported in C57BL/6 mice exposed at concentrations as low as 50 mg/m3 for 1 hr per day for 7 days. The results from those studies suggest that JP-8 aerosol is more toxic to the respiratory tract than JP-8 vapour.

COT (2003) reviewed the methods used to generate the exposure atmospheres in the studies using mixtures of vapors and aerosols and suspects that the JP-8 concentrations in the atmosphere may have been underreported. However, even if the actual concentration was 20 times as high (i.e., if exposure was at a concentration of 1,000 mg/m3), the observation of positive effects from a short exposure duration (1 hr/day for 7 days) at that concentration leads the subcommittee to conclude that the interim PEL of 350 mg/m3 might be too high to be protective of human health (assuming the application of commonly used uncertainty factors).

Because there are concerns about the characterization of the exposure atmospheres in the studies using mixtures of vapours and aerosols, COT (2003) recommended an examination of the methods of characterizing the exposure atmosphere. Future studies involving exposures to aerosols should be designed in collaboration with scientists knowledgeable in aerosol generation, aerosol physics, and quantification of vapours and aerosols to ensure accurate characterization of exposure atmospheres. COT (2003) recommended further that respiratory- system toxicity be evaluated in experimental animals exposed to JP-8 vapors and mixtures of vapors and aerosols by the inhalation route. Because the composition of JP-8 varies from batch to batch, scientists with expertise in petroleum toxicology should be consulted to design the best approach for testing the respiratory-system toxicity of JP-8 (e.g., testing JP-8 samples

at the extremes of their composition ranges or testing JP-8 samples so that the concentrations of component classes can be correlated with toxic end points).

6.1.3 Human studies Tunnicliff et al. (1999) investigated 222 full-time airport employees divided into three exposure groups; high-exposure group, 56 participants exposed for most of day; the moderate- exposure group included 83 participants exposed for 1 hr/day and the “ no-exposure group” 86 participants. The results showed that adjusted odds ratios (OR) for cough with phlegm (3.5) and for runny nose (2.9) were significantly associated with frequent exposure; adjusted odds ratios for symptoms of watering eyes, stuffy nose, wheezing, and shortness of breath were not significant. COT (2003) criticised the Tunnicliff et al. (1999) study mainly because of lack of quantitative exposure assessment, elimination of evaluation of the unexposed group, limited end-point evaluation, lack of correction for subject bias, and the relatively small number of participants. The hypothesis that symptoms of respiratory irritation were due more to jet exhaust than to fuel should be taken with caution; more-recent studies have determined that JP-8 vapor can cause upper respiratory tract irritation in mice (U.S. Department of the Air Force, 2001).

Gipson et al. (2001a) carried out a comparison of medical records of Air Force personnel occupationally exposed to JP-8 with records of unexposed (control) personnel. The exposed group consisted of 5,706 people (242 women and 5,464 men), and the control group consisted of 5,706 people (2,853 women and 2,853 men) randomly selected from a cohort of 20,244 Air Force unexposed personnel. The results showed that numbers of medical visits related to respiratory problems were not markedly different between the exposed and unexposed groups. Specific diseases, including respiratory illnesses, were examined, but no marked differences were found between the groups. COT (2003) criticised the study by Gipson et al. (2001a) is limited by many factors, including limited information on potential confounders, completeness of health-event recording, differences among personnel in availability of health care, consequences of taking sick leave for health care visits, differences in health-care- seeking behavior, and differences in amount of self-care or sensitivity to symptoms of illness.

Gibson et al. (2001b) conducted a health survey of 328 Air Force personnel (276 men and 52 women) with a self-assessment questionnaire. Measurements taken in breathing zones of subjects resulted in median concentration of naphthalene, 1.9 μg/m3 (low-exposure group), 10.4 μg/m3 (moderate-exposure group), 447 μg/m3(high-exposure group); median

concentration of benzene, 3.1 μg/m3(low-exposure group), 7.45 μg/m3 (moderate-exposure roup), 242 μg/m3 (high-exposure group). High- and moderate-exposure groups had persistent exposure to JP-8 (defined as at least 1 hr twice per week for at least 9 months); the low- exposure group had no significant exposure to jet fuel or solvents. Only in one case the symptom of “difficulty breathing,” was related to the effects of jet fuel on the respiratory tract. Preliminary results showed no statistical differences (adjusted for age, gender, and smoking history) between the high- and moderate-exposure groups compared with the low- exposure group in the reported symptom of “difficulty breathing.” This study was considered by COT (2003) as limited by the fact that the symptoms were self-reported, allowing for bias.

Todd and Buick, (2000) reported that prolonged exposure to kerosene vapors may result in development of asthma and other respiratory tract symptoms. Three families (six adults, three children) were exposed to kerosene vapors for 4-8 months as a result of a spill near their homes. Exposures occurred for an estimated 100 hr/wk. Concentrations in one home were measured at 5.6-79.7 mg/m3. Three of the children and one adult developed asthma that persisted for more than 2 yr. The remaining adults developed other respiratory tract symptoms, such as sore throat, cough, watery eyes, stuffed noses, and chest tightness. However, this study was considered by COT (2003) as limited because only a small group of people (n = 9) were exposed.

6.1.4 Animal studies Newton et al. (1991) investigated the effects of JP-4 exposure on F344 rats at concentrations of 1,000 mg/m3 during 90 days continuously. The exposure resulted in no effects on lung volumes, dynamic resistance and compliance, quasistatic compliance, partial and full forced vital capacities, carbon monoxide diffusion capacity, and closing volume; no effects on deposition or clearance of inhaled 51Cr-labeled microspheres; no evidence of pulmonary disease in control and exposed rats.

Mattie et al. (1991) investigated the effects of JP-8 exposure on F344 rats, C57BL/6 mice at concentrations of 500, 1,000 mg/m3 (vapor) during 90 days. The results well characterized with regard to concentration and chemical composition No respiratory tract effects were attributed to JP-8.

Hays et al. (1995); Pfaff et al. (1995) investigated the effects of JP-4 exposure on F344 rats at concentrations of 495-520, 813-1,094 mg/m3(aerosol-vapor mixture)during 1 hr/day, 5 days/wk for 7, 28 and 56 days. Pulmonary resistance increased in 7- and 28-day exposure

groups; lung-permeability data indicated lung injuries peaking at 28 d of exposure; all groups had interstitial oedema resulting from endothelial damage; all groups had activated thickening of alveolar septa and alveolar macrophages.

Robledo and Witten (1998); Robledo et al. (2000); Wang et al. (2001) investigated the effects of JP-8 exposure on C57BL/6, B6.A.D. (Ahrd/Natsknockout) mice at concentrations of Up to 118 mg/m3aerosol; vapor concentration during 1 hr/day for 7 days. Exposures resulted in increases in total protein and LDH among groups at high concentrations; minimal morphologic changes after inhalation; damage to bronchiolar epithelium resulting in perivascular oedema and damaged Clara cells at highest concentrations.

US Department of the Air Force (2001) investigated the effects of JP-4, JP-8, and JP-8 +100exposure on Swiss-Webster mice at concentrations of JP-4: 685-11,430 mg/m3, JP-8: 681-3,613 mg/m3, JP-8 + 100:777-2,356 mg/m3(vapor and aerosol) during 30 min. Exposure to jet fuels caused breathing patterns characteristic of upper airway sensory irritation at all concentrations but no apparent deep lung irritation at any concentration; RD50 determined to be 4,842 mg/m3 for JP-4, 2,876 mg/m3for JP-8, 1,629 mg/m3 for JP-8 +100.

Witzmann et al. (1999) investigated the effects of JP-8 exposure on Swiss-Webster mice at concentrations of 1,000, 2,500 mg/m3 (vapor-aerosol mixture) during 1 hr/day for 7 days. Of 796 proteins analyzed, 42 were altered by exposure to JP-8 at 2,500 mg/m3. 8 were increased, 34 were decreased in abundance; 1 of 42 proteins altered at 2,500 mg/m3 was also altered at 1,000 mg/m3.

6.1.5 Recent studies to be considered by SCOEL

2003: Yang et al., Adverse respiratory and irritant health effects in airport workers in Taiwan, Journal of Toxicology and Environmental Health - Part A, 66 (9), pp. 799-806. ABSTRACT: Airport workers are potentially exposed to aviation fuel or jet stream exhaust. The purpose of this study was to assess if there was an excess of adverse health outcomes among airport workers. Self-reported adverse chronic respiratory symptoms and acute irritative symptoms were assessed in a cross-sectional study among 106 airport workers (exposure group) and 305 terminal or office workers (control group) at the Kaohsiung International Airport (KIA), Taiwan. The prevalence rates for acute irritative symptoms were not significantly different between groups. A possible explanation may be that the concentration of volatile organic compounds (VOCs) that airport workers are exposed to is not sufficient to induce acute irritative symptoms, although this is not known for certain since data on the concentration of VOCs are lacking in this study. Chronic respiratory symptoms (cough and dyspnea), however, were significantly more common among the exposed group.

2003: Drake et al., JP-8 jet fuel exposure alters protein expression in the lung, Toxicology, 191 (2-3), pp. 199-210. ABSTRACT: The purpose of this study was to investigate the proteomic mechanisms of Jet Propulsion-8 (JP-8) toxicity in the lung, specifically relating to lung epithelial cell apoptosis and edema. Male Swiss-Webster mice were exposed to 1 h/day aerosolized JP-8 jet fuel at concentrations of 250, 1000, and 2500 mg/m3 for 7 days.

Lung cytosol and whole lung samples were solubilized, separated via large scale, high-resolution two- dimensional electrophoresis, and processed for analysis. Significant quantitative differences in lung protein expression were found as a result of JP-8 exposure. At 250 mg/m3 JP-8 concentration, 31 proteins exhibited increased expression, while 10 showed decreased expression. At 1000 mg/m3 exposure levels, 21 lung proteins exhibited increased expression and 99 demonstrated decreased expression. At 2500 mg/m3, 30 exhibited increased expression, while 135 showed decreased expression. Several of the proteins were identified by peptide mass fingerprinting, and were found to relate to cell structure, cell proliferation, protein repair, and apoptosis. These data demonstrate the significant stress JP-8 jet fuel puts on lung epithelium. Furthermore, there was a decrease in α1-anti-trypsin expression suggesting that JP-8 jet fuel exposure may have implications for the development of pulmonary disorders. © 2003 Elsevier Ireland Ltd. All rights reserved.

2006: Herrin et al., A reevaluation of the threshold exposure level of inhaled JP-8 in mice, Journal of Toxicological Sciences, 31 (3), pp. 219-228. ABSTRACT: C57BL/6 mice were nose-only exposed to JP-8 jet fuel at average concentrations of 45, 267, and 406 mg JP-8/m3 for 1 hr/d for 7 days to further test the hypothesis that exposure to JP-8 concentrations below the current permissible exposure level (PEL) of 350 mg/m3 will induce lung injury, and to validate a new "in- line, real-time" total hydrocarbon analysis system capable of measuring both JP-8 vapor and aerosol concentrations. Pulmonary function and respiratory permeability tests were performed 24 to 30 hr after the final exposures. No significant effects were observed at 45 or 267 mg/m3. The only significant effect observed at 406 mg/m3 was a decrease in inspiratory dynamic lung compliance. Morphological examination and morphometric analysis of distal lung tissue demonstrated that alveolar type II epithelial cells showed limited cellular damage with the notable exception of a significant increase in the volume density of lamellar bodies (vacuoles), which is indicative of increased surfactant production, at 45 and 406 mg/m 3. The terminal bronchial epithelium showed initial signs of cellular damage, but the morphometric analysis did not quantify these changes as significant. The morphometric analysis techniques appear to provide an increased sensitivity for detecting the deleterious effects of JP-8 as compared to the physiological evidence offered by pulmonary function or respiratory permeability tests. These observations suggest that the current 350 mg/m3 PEL for both JP-8 jet fuel and for other more volatile petroleum distillates should be reevaluated and a lower, more accurate PEL should be established with regard human occupational exposure limits.

2006: Espinoza et al., Expression of JP-8-induced inflammatory genes in AEII cells is mediated by NF-kB and PARP-1, American Journal of Respiratory Cell and Molecular Biology, 35 (4), pp. 479-487. ABSTRACT: Lung epithelial cells are critical in the regulation of airway inflammation in response to environmental pollutants. Altered activation of NF-κB is associated with expression of several proinflammatory factors in respiratory epithelial cells in response to an insult. Here we show that a low threshold dose (8 μg/ml) of the jet fuel JP-8 induces in a rat alveolar epithelial cell line (RLE-6TN) a prolonged activation of NF-κB as well as the increased expression of the proinflammatory cytokines TNF-α and IL-8, which are regulated by NF- κB. The up-regulation of IL-6 mRNA in cells exposed to JP-8 appears to be a reaction of RLE-6TN cells to reduce the enhancement of proinflammatory mediators in response to the fuel. Moreover, lung tissues from rats exposed to occupational levels of JP-8 by nasal aerosol also showed dysregulated expression of TNF-α, IL-8, and IL-6, confirming the in vitro data. The poly(ADP-ribosyl)ation of PARP-1, a coactivator of NF-κB, was coincident with the prolonged activation of NF-κB during JP-8 treatment. These results evidenced that a persistent exposure of the airway epithelium to aromatic hydrocarbons may have deleterious effects on pulmonary function.

2007: Espinoza et al., Prolonged poly(ADP-ribose) polymerase-1 avtivity refilate JP-8- induced sustained cytokine expression in alveolar macrophages, Free Radical Biology and Medicine, 42 (9), pp. 1430-1440. ABSTRACT: Environmental pollutants inducing oxidative stress stimulate chronic inflammatory responses in the lung leading to pulmonary tissue dysfunction. In response to oxidative stress, alveolar macrophages produce both reactive oxygen species and reactive nitrogen species, which induce the expression of a wide variety of immune-response genes. We found that a prolonged exposure of alveolar macrophages to a nonlethal dose (8 μg/ml) of JP-8, the kerosene-based hydrocarbon jet fuel, induced the persistent expression of IL-1, iNOS, and COX-2, as well as cell adhesion molecules (ICAM-1 and VCAM-1). Because poly(ADP-ribose) polymerase (PARP-1), a coactivator of NF-κB, regulates inflammatory responses and associated disorders in the airways, we determined whether JP-8 induces the poly(ADP-ribosyl)ation automodification of PARP-1 in alveolar macrophages. We observed that PARP-1 is activated in a time-dependent manner, which was temporally coincident with the prolonged activation of NF-κB and with the augmented expression of the proinflammatory

factors described above. The 4 μg/ml dilution of JP-8 also increased the activity of PARP-1 as well as the expression of iNOS and COX-2, indicating that lower doses of JP-8 also affect the regulation of proinflammatory factors in pulmonary macrophages. Together, these results demonstrate that an extensive induction of PARP-1 might coordinate the persistent expression of proinflammatory mediators in alveolar macrophages activated by aromatic hydrocarbons that can result in lung injury from occupational exposure. © 2007 Elsevier Inc. All rights reserved.

2008: Wong et al., In vivo comparison of epithelial responses for S-8 versus JP-8 jet fuels below permissible exposure limit, Toxicology, 254 (1-2), pp. 106-111. ABSTRACT: This study was designed to characterize and compare the pulmonary effects in distal lung from a low-level exposure to jet propellant-8 fuel (JP-8) and a new synthetic-8 fuel (S-8). It is hypothesized that both fuels have different airway epithelial deposition and responses. Consequently, male C57BL/6 mice were nose- only exposed to S-8 and JP-8 at average concentrations of 53 mg/m3 for 1 h/day for 7 days. A pulmonary function test performed 24 h after the final exposure indicated that there was a significant increase in expiratory lung resistance in the S-8 mice, whereas JP-8 mice had significant increases in both inspiratory and expiratory lung resistance compared to control values. Neither significant S-8 nor JP-8 respiratory permeability changes were observed compared to controls, suggesting no loss of epithelial barrier integrity. Morphological examination and morphometric analysis of airway tissue demonstrated that both fuels showed different patterns of targeted epithelial cells: bronchioles in S-8 and alveoli/terminal bronchioles in JP-8. Collectively, our data suggest that both fuels may have partially different deposition patterns, which may possibly contribute to specific different adverse effects in lung ventilatory function.

2010: Robb et al., In vitro time- and dose-effect response of JP-8 and S-8 jet fuel on alveolar type II epithelial cells of rats, Toxicology and Industrial Health, 26 (6), pp. 367- 374. ABSTRACT: This study was designed to characterize and compare the effects of jet propellant-8 (JP-8) fuel and synthetic-8 (S-8) on cell viability and nitric oxide synthesis in cultured alveolar type II epithelial cells of rats. Exposure times varied from 0.25, 0.5, 1, and 6 hours at the following concentrations of jet fuel: 0.0, 0.1, 0.4, and 2.0 μg/mL. Data indicate that JP-8 presents a gradual decline in cell viability and steady elevation in nitric oxide release as exposure concentrations increase. At a 2.0 μg/mL concentration of JP-8, nearly all of the cells are not viable. Moreover, S-8 exposure to rat type II lung cells demonstrated an abrupt fall in percentage cell viability and increases in nitric oxide measurement, particularly after the 2.0 μg/mL was reached at 1 and 6 hours. At 0.0, 0.2, and 0.4 μg/mL concentrations of S-8, percentage viability was sustained at steady concentrations. The results suggest different epithelial toxicity and mechanistic effects of S-8 and JP-8, providing further insight concerning the impairment imposed at specific levels of lung function and pathology induced by the different fuels.

6.2 Effects on the nervous system

6.2.1 1996 assessment (COT 1996) COT, (1996) found that data on potential nervous system effects of jet fuels are sparse. In several Swedish studies conducted by Knave and his colleagues, acute CNS symptoms were reported in workers who were employed in jet factories where they were potentially exposed to jet fuels designated Jet A-1 and JP-1 (Knave et al. 1976, 1978, 1979). Industrial-hygiene measurements of up to 3,200 mg/m3 were reported for a variety of job activities. Although the one-time air measurements reflected various activities, COT (1996) found the exposures were not well characterized over time or by individual.

Knave et al., (1978) investigated 30 Swedish workers potentially exposed to jet fuels at a motor factory for an average of 17 years (yr), workers reported acute symptoms of exposure to vapors and performance degradation associated with long-term exposure. The study reported an approximate time-weighted average (TWA) of 300 mg/m3. The findings of performance degradation said to be attributable to long-term exposure were considered by COT (1996) as unreliable for a number of reasons, including weak and inconsistent evidence of impairment, inadequate methods of evaluation, inadequate consideration of confounding, a small cohort of workers, and a lack of quantitative information on exposure over time.

6.2.2 2003 assessment (COT 2003) The assessment considered preliminary results of a recent epidemiologic study on Air Force personnel occupationally exposed to JP-8 indicated that JP-8 exposure for 1 hr per day, 2 times per wk for 9 months may produce neurotoxic effects. Key findings and conclusions include

• In a self-assessment questionnaire, JP-8-exposed Air Force personnel reported more headaches, dizziness, trouble concentrating, balance problems, walking difficulties, forgetfulness, and trouble with gripping objects than an unexposed (control) group. In that study, JP-8-exposed Air Force personnel also showed lower performance than a control group on several neurobehavioral tests and disturbances of balance and altered eye-blink conditioning response.

• The lack of exposure information makes it difficult to determine the extent of the health risk.

Animal studies have investigated the effects of several jet fuels on a number of neurobehavioral end points. Key findings and conclusions include

• Several studies showed neurobehavioral effects in F344 and Sprague-Dawley rats exposed to JP-8 and JP-5 vapours at concentrations of about 1,000 mg/m3 for 6 hr per day, 5 days per week for 6 wk or to JP-8 aerosols at concentrations of 1,059 mg/m3 for 1 hr per day, 5 days per week for 4 wk.

• No dose-response relationships were demonstrated in the studies.

• The relevance of the observed neurobehavioral effects to humans is not known, and these positive findings need to be validated against other well-established neurotoxicity end points.

• The findings provide an indication that the interim PEL of 350 mg/m3 might be too high to be protective of human health.

COT (2003) recommended additional research to measure ambient and breathing-zone concentrations of JP-8 and its constituents (such as naphthalene and toluene) and to determine body burden through assays of biologic samples for JP-8 constituents and metabolites. The findings should be correlated with acute and chronic symptoms and signs experienced by JP- 8-exposed people. Preliminary positive findings reported in two neurologic tests (eye-blink and postural-sway tests) conducted as part of the Air Force human study should be validated with standard neurologic tests.

COT (2003) also recommended studies to be carried out in experimental animals to examine the potential neurotoxic effects of JP-8. Specifically, the subcommittee recommends that neurologic (histologic, physiologic, and behavioral) measures be included in inhalation- toxicity tests with JP-8 vapours and mixtures of vapours and aerosols.

6.2.3 Human data Anger and Storzbach (2001) investigated JP-8 exposure effects to humans on the nervous system. Measurements were taken in breathing zones of subjects; median concentration of naphthalene, 1.9 μg/m3(low-exposure group), 447 μg/m3 (high-exposure group); median concentration of benzene, 3.1 μg/m3(low-exposure group), 242 μg/m3 (high-exposure group). High-exposure group had persistent exposure to JP-8 (defined as 1 hr twice per wk for at least 9 mo); low-exposure group had no significant exposure to jet fuel or solvents. The subjects

were given seven neurobehavioral tests in Behavioural Assessment and Research System; before exposure, high-exposure group had significantly lower performance on digit-span forward and backward test, symbol digit-latency test, and tapping test than low-exposure group; results of tests did not correlate with breath or passive naphthalene or benzene exposure; effects may be result of carryover from previous exposure; when pre- and post- exposure test results were compared, passive naphthalene exposure was significantly associated with performance on Oregon Dual Task Procedure, Match to Sample, and Tapping Trial 2.

Bekkedal et al., (2001) investigated JP-8 exposure effects to humans on the nervous system. Measurements were taken in breathing zones of subjects. The median concentration of naphthalene was 1.9 μg/m3 for the “low-exposure group”, 447 μg/m3 for the “high-exposure group”; the median concentration of benzene, 3.1 μg/m3for the “low-exposure group” and 242 μg/m3 for the “high-exposure group”. The “high-exposure group” had persistent exposure to JP-8; the ”low-exposure group” had no significant exposure to jet fuel or solvents. The subjects were given eye-blink conditioning response test; the morning session showed that the “high-exposure group” had statistically significant differences in percentage CR (Conditioned Response), CR peak latency, and CR onset latency; high-exposure group also had fewer CRs than low-exposure group; no statistically significant exposure-based differences were observed in the afternoon session.

Bhattacharya et al., (2001) investigated JP-8 exposure effects to humans on the nervous system. Measurements were taken in breathing zones of subjects. The median concentration of naphthalene was 1.9 μg/m3 for the “low-exposure group”, 447 μg/m3 for the “high- exposure group”; the median concentration of benzene, 3.1 μg/m3for the “low-exposure group” and 242 μg/m3 for the “high-exposure group”. The “high-exposure group” had persistent exposure to JP-8; the ”low-exposure group” had no significant exposure to jet fuel or solvents. Subjects were given postural sway tests; post-log sway length, based on ANCOVA (Analysis of Co-Variance) after controlling for cofactors, was significantly associated with passive naphthalene exposure for “eyes closed no foam” and “eyes closed bending” tests.

Gibson et al. (2001a) investigated JP-8 exposure effects to humans on the nervous system. The exposed group (5,706) had potential occupational exposure to JP-8; control group (5,706) did not work in occupations in which exposure to JP-8 would occur; all subjects were active duty members of U.S. Air Force. The review of the medical records of the investigated

subjects showed no differences between exposed and control groups in neurologic and mental illnesses.

Gibson et al. (2001b) investigated JP-8 exposure effects to humans on the nervous system. Measurements were taken in breathing zones of subjects. The median concentration of naphthalene was 1.9 μg/m3 for the “low-exposure group”, 447 μg/m3 for the “high-exposure group”; the median concentration of benzene, 3.1 μg/m3for the “low-exposure group” and 242 μg/m3 for the “high-exposure group”. The “high-exposure group” had persistent exposure to JP-8; the ”low-exposure group” had no significant exposure to jet fuel or solvents. In self- assessment questionnaire, subjects in high-and moderate-exposure groups reported more headaches, dizziness, trouble concentrating, balance problems, walking difficulties, forgetfulness, and trouble in gripping objects

6.2.4 Animal data Baldwin et al. (2000) investigated the effects of JP-8 (aerosol) exposure to F344 rats at concentrations of 1,059 mg/m3 for first 25 days, 2,491 mg/m3 for final 3 days during 1 hr/day, 5 days/wk for 28 days. Neurologic measures were assessed with functional observation battery; exposed rats had significant differences in spontaneous activity and CNS excitability from controls; exposed rats exhibited greater velocity of swimming in Morris swimming task.

Ritchie et al. (2000), (2001a,b) investigated the effects of JP-8 (vapour) exposure to Sprague- Dawley rats at concentrations in the range of 11,000 - 5,000 mg/m3 during 6 hr/day, 5 days/wk for 6 wk, followed by no exposure for 64 days. The “high-dose group” was significantly impaired relative to the “low-dose group” in difficult task involving pressing one or more levers after auditory cue and in task involving complicated repeated acquisition; no differences observed between two groups in simple auto-shaping and fixed-ratio or spatial- reversal tasks; the “low-dose group” exhibited superior performance relative to control group in test requiring three or four lever presses in three-lever array.

Rossi et al. (2001) investigated the effects of JP-8 and JP-5 (vapour) exposure to Sprague- 3 Dawley rats at concentrations in the range of 1,000 (JP-8) or 1,200 (JP-5) mg/m during 6 hr/day, 5 days/wk for 6 wk, followed by no exposure for 65 days. Significant differences were observed in JP-8-exposed group in appetitive reinforcer approach sensitization compared with JP-5-exposed group and control group; JP-5-exposed group showed increased forelimb grip strengths compared with JP-8-exposed group and the control group; neurotransmitter concentrations were also measured; JP-8 exposure was associated with decreased

concentrations of 3,4-dihydroxyphenylacetic in cerebellum and brainstem; JP-5 exposure was associated with increased concentrations of dopamine and 3,4-dihydroxyphenyl-acetic acid in hippocampus and cortex, respectively, and with decreased concentrations of homovanillic acid in hippocampus; blood samples contained increased and decreased concentrations of 5- hydroxyindoleacetic acid in JP-5 and JP-8 exposed groups, respectively.

Nordholm et al. (1999) investigated the effects of JP-4 (vapour) exposure to Sprague-Dawley 3 rats at concentrations in the range of 2,000 mg/m during 6 hr/day for 14 days, followed by no exposure for 14 days or 60 days. Significant increase in appetitive reinforcer approach sensitization was observed for in short-recovery-period group, but not long-recovery-period group; long-recovery-period group exhibited significant differences in prepulse inhibition trial and treadmill response compared with controls and decrease in total locomotor activity compared with short-recovery-period group; no other differences in neurologic measures were observed; blood serotonin concentrations were increased in short-recovery-period group; blood 5-hydroxyindoleacetic acid was significantly increased in short- and long-recovery- period groups; serotonin and 5-hydroxyindoleacetic acid concentrations were increased in short- and long-recovery-period groups in cerebellum, brainstem, and hippocampal regions; those chemicals were also increased in striated region in short-recovery-period group and in cortical regions in long-recovery-period group.

Koschier (1999) investigated the effects of Hydro-desulfurized kerosene exposure to rats at concentrations in the range of 165, 330, 495 mg/kg (dermal) during 5 days/wk for 13 wk. Animals were evaluated immediately after exposure period ended and after 4-wk recovery period; no significant differences were observed in functional observed battery and motor activity, startle response, and histologic evaluations.

6.2.5 Recent studies to be considered by SCOEL

2004: Tu et al., Human exposure to the jet fuel, JP-8, Aviation Space and Environmental Medicine, 75 (1), pp. 49-59. ABSTRACT: Introduction: This study investigates anecdotal reports that have suggested adverse health effects associated with acute or chronic exposure to jet fuel. Methods: JP-8 exposure during the course of the study day was estimated using breath analysis. Health effects associated with exposure were measured using a neurocognitive testing battery and liver and kidney function tests. Results: Breath analysis provided an estimate of an individual's recent JP-8 exposure that had occurred via inhalation and dermal routes. All individuals studied on base exhaled aromatic and aliphatic hydrocarbons that are found in JP-8. The subject who showed evidence of the most exposure to JP-8 had a breath concentration of 11.5 mg · m-3 for total JP-8. This breath concentration suggested that exposure to JP-8 at an Air Guard Base is much less than exposure observed at other Air Force Bases. This reduction in exposure to JP-8 is attributed to the safety practices and standard operating procedures carried out by base personnel. The base personnel who exhibited the highest exposures to JP-8 were fuel cell workers, fuel specialists and smokers, who smoked downwind from the flightline. Discussion: Although

study-day exposures appear to be much less than current guidelines, chronic exposure at these low levels appeared to affect neurocognitive functioning. JP-8-exposed individuals performed significantly poorer than a sample of non-exposed age- and education-matched individuals on 20 of 47 measures of information processing and other cognitive functions.

2005: Bell et al., JP-8 jet fuel exposure and divided attention test performance in 1991 Gulf War veterans, Aviation Space and Environmental Medicine, 76 (12), pp. 1136-1144. ABSTRACT: Introduction: Previous research indicates that a large cohort of veterans from the 1991 Gulf War report polysymptomatic conditions. These syndromes often involve neurocognitive complaints, fatigue, and musculoskeletal symptoms, thus overlapping with civilian illnesses from low levels of environmental chemicals, chronic fatigue syndrome, and fibromyalgia. Methods: To test for time-dependent changes over repeated intermittent exposures, we evaluated objective performance on a computerized visual divided attention test in chronically unhealthy Gulf War veterans (n = 22 ill with low-level chemical intolerance (CI); n = 24 ill without CI), healthy Gulf War veterans (n = 23), and healthy Gulf War era veterans (n = 20). Testing was done before and after each of three weekly, double blind, low-level JP-8 jet fuel or clean air sham exposure laboratory sessions, including acoustic startle stimuli. Results: Unhealthy veterans receiving jet fuel had faster mean peripheral reaction times over sessions compared with unhealthy veterans receiving sham clean air exposures. Unhealthy Gulf veterans with CI exhibited faster post- vs. pre-session mean central reaction times compared with unhealthy Gulf veterans without CI. Findings were controlled for psychological distress variables. Discussion: These data on unhealthy Gulf veterans show an acceleration of divided attention task performance over the course of repeated low-level JP-8 exposures. The present faster reaction times are consistent with rat neurobehavioral studies on environmental toxicant cross-sensitization and nonlinear dose-response patterns with stimulant drugs, as well as some previous civilian studies using other exposure agents. Together with previous research findings, the data suggest involvement of central nervous system dopaminergic pathways in affected Gulf veterans.

2007: Baldwin et al., Repeated aerosol-vapor JP-8 jet fuel exposure affects neurobehavior and neurotransmitter levels in a rat model, Journal of Toxicology and Environmental Health - Part A: Current Issues, 70 (14), pp. 1203-1213. ABSTRACT: Four groups of Fischer Brown Norway hybrid rats were exposed for 5, 10, 15, or 20 d to aerosolized-vapor jet propulsion fuel 8 (JP-8) compared to freely moving (5 and 10-d exposures) or sham- confined controls (15 and 20-d exposures). Behavioral testing utilized the U.S. Environmental Protection Agency Functional Observational Battery. Exploratory ethological factor analysis identified three salient factors (central nervous system [CNS] excitability, autonomic 1, and autonomic 2) for use in profiling JP-8 exposure in future studies. The factors were used as dependent variables in general linear modeling. Exposed animals were found to engage in more rearing and hyperaroused behavior compared to controls, replicating prior JP-8 exposure findings. Exposed animals also showed increasing but rapidly decelerating stool output (autonomic 1), and a significant increasing linear trend for urine output (autonomic 2). No significant trends were noted for either of the control groups for the autonomic factors. Rats from each of the groups for each of the time frames were randomly selected for tissue assay from seven brain regions for neurotransmitter levels. Hippocampal DOPAC was significantly elevated after 4-wk JP-8 exposure compared to both control groups, suggesting increased dopamine release and metabolism. Findings indicate that behavioral changes do not appear to manifest until wk 3 and 4 of exposure, suggesting the need for longitudinal studies to determine if these behaviors occur due to cumulative exposure, or due to behavioral sensitization related to repeated exposure to aerosolized-vapor JP-8.

2011: Proctor et al., The Occupational JP8 Exposure Neuroepidemiology Study (OJENES): Repeated workday exposure and central nervous system functioning among US Air Force personnel, NeuroToxicology, 32 (6), pp. 799-808. ABSTRACT: One of the most prevalent workplace chemical exposures historically and currently confronting the global military and civilian workforce is jet propellant (JP) fuel (e.g., JP4, JP5, JP8, jet A1), a complex mixture of numerous hydrocarbon compounds and additives. To date, numerous protective and preventive strategies (e.g., federal exposure limits, workplace procedure protocols, protective gear such as goggles, respirator use, gloves, and coveralls) have been put in place to minimize acutely toxic exposure levels. However, questions remain regarding the effect of repeated exposures at lower (than regulated) levels of JP fuel. The Occupational JP8 Exposure Neuroepidemiology Study (OJENES) was designed to examine the relationships between occupational JP8 exposure over multiple, repeated workdays and specific aspects of central nervous system (CNS) functioning among Air Force (AF) personnel. In this report, we present the OJENES methodology, descriptive findings related to participant characteristics, JP8 exposure levels observed over a work week among

higher and lower exposure groups, and neuropsychological task performances at the first study assessment. Results indicated minimal differences between participants in the high and lower exposure groups in terms of descriptive characteristics, other than daily JP8 exposure levels (p< 0.001). In addition, neuropsychological task performances for most task measures were not found to be significantly different from reported reference ranges. These findings demonstrated that confounding and misclassification of exposure and outcome status are not major concerns for the study. Therefore, future OJENES analyses targeting the more focused research questions regarding associations between JP8 exposure and CNS functioning are likely to provide valid conclusions, as they will be less influenced by these research biases.

6.3 Effects on immune system

6.3.1 1996 Assessment (COT 1996) This assessment did not specifically consider the immunotoxic effects of JP-8 or related fuels

6.3.2 2003 Assessment (COT 2003)

• No histopathologic effects related to the immune system were found in F344 rats and C57BL/6 mice exposed continuously to JP-8 vapors at concentrations up to 1,000 mg/m3 for 90 days. No additional studies that tested the toxicity of JP-8 vapors in experimental animals were located.

• Studies by Harris et al. (1997a, 1997b, 1997c and 2000) were found to raise concern about the potential of JP-8 to cause immunotoxicity. These studies reported that inhalation exposure of C57BL/6 mice to JP-8 aerosols at a concentration of 100 mg/m3 for 1 hr/day for 7 days led to decreased cellularity of the thymus, exposure at 500 mg/m3 for 1 hr/day for 7 days led to decreased spleen weight and cellularity, and exposure at 1,000 mg/m3 for 1 hr/day for 7 days led to decreased ability of spleen cells to mediate several immune responses.

• Dermal exposure of mice to JP-8 in multiple small doses (50 μL/day for 4-5 days) or in larger single doses (300 μL) resulted in local and systemic effects on the immune system (e.g., suppressed contact- and delayed-hypersensitivity responses).

COT (2003) reviewed the methods used to generate the exposure atmospheres in the studies by Harris et al. and noticed that the total JP-8 concentration in the atmosphere may have been underreported. However, even if the actual concentration was 10 times as high as the lowest concentration at which effects were observed (100 mg/m3) (i.e., if exposure was at a concentration of 1,000 mg/m3), the observation of positive effects from a short exposure duration (1 hr/day for 7 days) at that concentration leads the subcommittee to conclude that the interim permissible exposure level of 350 mg/m3 might be too high to be protective of human health (assuming the application of commonly used uncertainty factors).

COT (2003) recommended:

• that experimental animal studies examining the immunotoxicity of JP-8 via the inhalation route be conducted with careful control of vapor and aerosol concentrations in the atmosphere and with consideration of appropriate controls,

• human blood samples from JP-8-exposed persons be assayed for indicators of immunotoxicity to determine whether effects in experimental animals are observed in humans,

• that military personnel avoid direct, prolonged skin contact with JP-8.

6.3.3 Human data Rhodes et al. (2001) investigated immunosuppressive effects of JP-8 inhalation exposure in humans. Measurements were taken in breathing zones of subjects; median concentration of naphthalene, 1.9 μg/m3 (low-exposure group), 447 μg/m3 (high-exposure group); median concentration of benzene, 3.1 μg/m3 (low-exposure group), 242 μg/m3 (high-exposure group). High-exposure group, persistent exposure to JP-8 (defined as at least 1 hr twice per wk for at least 9 months); low-exposure group, no significant exposure to jet fuel or solvents. High- exposure group had higher white-cell counts than low-exposure group; there were increased numbers of neutrophils and monocytes but no differences in total lymphocytes, T cells, NK cells, B cells; white cell, neutrophil, and monocyte counts in high-exposure group did not exceed range of normal values.

Gibson et al. (2001b) investigated immunosuppressive effects of JP-8 inhalation exposure in humans. The exposed group (5,706 people) had potential occupational exposure to JP-8. Control group (5,706 people) did not work in occupations in which exposure to JP-8 would occur. Health-event analysis did not find differences in immunologic measures (such as infections) between exposed and control groups.

Gibson et al. (2001a) investigated immunosuppressive effects of JP-8 inhalation exposure in humans. Measurements taken in breathing zones of subjects; median concentration of naphthalene, 1.9 μg/m3 (low-exposure group), 10.4 μg/m3(moderate-exposure group), 447 μg/m3(high-exposure group); median concentration of benzene, 3.1 μg/m3(low-exposure group), 7.45 μg/m3(moderate-exposure group), 242 μg/m3 (high-exposure group). High- and moderate-exposure groups, persistent exposure to JP-8; low exposure group, no significant.

Analysis of self-assessment questionnaire did not report differences among groups in immunologic-related illnesses.

6.3.4 In vitro data Rosenthal et al. (2001) investigated immunosuppressive effects of JP-8 dermal exposure in vitro using NHEK (Normal Human Epidermal Keratinocyte) with single doses of 80-200 μg/mL diluted in absolute ethanol. The results demonstrated that JP-8 induced necrosis and cell death in human keratinocytes in vitro.

Allen et al. (2000) investigated immunosuppressive effects of JP-8 dermal exposure in vitro using NHEK (Normal Human Epidermal Keratinocyte) with single doses of 0.1% single doses for 24 hr. The results demonstrated that JP-8 increased production of proinflammatory cytokines TNFα ( Tumor Necrosis Factor) and IL-8 (InterLeukin).

6.3.5 Animal data Mattie et al. (1991) investigated immunosuppressive effects of JP-8 inhalation exposure in F344 rat and C57Bl/6 mouse at concentrations of 500 and 1,000 mg/m3 during 90 days continuously, followed by recovery until approximately 24 mo of age. No treatment-related changes in spleen weight or hematology were observed.

Harris et al. (1997a) investigated immunosuppressive effects of JP-8 inhalation exposure in C57BL/6 mouses at concentrations of 100, 250, 500, 1,000, 2,500 mg/m3 (aerosol) during 1 hr/day for 7 days (nose-only). Exposure at 100 mg/m3 led to decreased cellularity of thymus; exposure at 500 mg/m3 led to decreased spleen weight, cellularity; splenic T cells, B cells, macrophages were also affected by exposure at 100 and 500 mg/m3; splenic T cells, B cells, macrophages were also decreased in JP-8-exposed mice; bone marrow cellularity increased after exposure at 100, 250 mg/m3 but decreased after exposure at higher concentrations; exposure at 250 mg/m3 led to reduced spleen cell proliferation responses in vitro after stimulation with Con A or Con A + IL-2.

Harris et al. (1997b) investigated immunosuppressive effects of JP-8 inhalation exposure in C57BL/6 mouses at concentrations of 1001,000, 2,500 mg/m3(aerosol) during 1 hr/day for 7 days (nose-only). In mice exposed at both doses, spleen cellularity and spleen cell proliferation persisted for more than 21 days; spleen cells in mice exposed at 1,000 mg/m3were suppressed in ability to mediate NK activity, LAK responses, CTL responses.

Harris et al. (1997c) investigated immunosuppressive effects of JP-8 inhalation exposure in C57BL/6 mouses at concentrations of 250-2,500 mg/m3 JP-8 aerosol + 1 μM or 1nM substance P aerosol during 1 hr/day for 7 days (nose-only). Substance P administration prevented loss of spleen and thymus cellularity after exposure to JP-8; it also partially restored proliferative response of spleen cells to Con A + IL-2.

Harris et al. (2000) investigated immunosuppressive effects of JP-8 inhalation exposure in C57BL/6 mouses at concentrations of 1,000 mg/m3(aerosol) during 1 hr/day for 7 days (nose- only). Mice showed significantly decreased NK (Natural Killer cell) cell function, significantly suppressed generation of LAK (Lymphokine-Activated Killer cell) cell activity, suppressed generation of CTL (Cytotoxic T Lymphocytes) cells from precursor T cells, inhibited helper T cell activity.

Ullrich (1999) investigated immunosuppressive effects of JP-8 dermal exposure in C3H/HeN Mouses at doses of 50, 250-300 μL during 5 days (50 μL) and with single doses (250-300 μL). Induction of contact hypersensitivity was impaired in dose-dependent manner regardless of whether contact allergen was applied directly to treated skin or at distant unrelated site; generation of classic delayed hypersensitivity reaction to Borellia burgdorferi (bacterial antigen) injected into subcutaneous space was suppressed by dermal application of JP-8 at distant site; ability of splenic T lymphocytes from JP-8-treated mice to proliferate in response to plate-bound monoclonal anti-CD3 (monoclonal antibody) was significantly suppressed; IL- 10 (InterLeukin) was found in the serum of JP-8-exposed mice.

Ullrich and Lyons (2000) investigated immunosuppressive effects of JP-8 dermal exposure in C3H/HeN Mouses with single doses of 50-300 μL undiluted or diluted in acetone. Splenic T- cells were cultured in vitro with antibody T-cell receptor; T cells from JP-8-exposed mice had reduced proliferative response; T-cell-dependent antibody responses to KLH (Keyhole- Limpet Hemocyanin) antigen injected in Freund’s adjuvant were not altered by exposure to JP-8.

Kabbur et al. (2001) investigated immunosuppressive effects of JP-8 dermal exposure in F344 rats with single doses of 0.25 mL. IL-1α (InterLeukin), iNOS ( Inducible Nitric Oxide Synthase) expression were induced in isolated skin samples.

Keil et al. (2001) investigated immunosuppressive effects of JP-8 oral exposure in B6C3F1 mouses with doses of 1,000, 2,000 mg/kg per day administered to pregnant mice on days 6-

15 of gestation. Significant suppression of PFC (plaque-forming cell) response in offspring when tested at age of 8 weeks. Dudley et al. 2001) investigated immunosuppressive effects of JP-8 oral exposure in B6C3F1 and DBA/2 mouse with doses of 1,000, 2,000 mg/kg per day administered at 1 dose/day for 7 or 14 days. Significant immunologic alterations in thymic weight and antibody PFC (plaque-forming cell) response to SRBC ( sheep red blood cell).

6.3.6 Recent studies to be considered by SCOEL

2003: Keil et al., Immunological function in mice exposed to JP-8 jet fuel in utero, Toxicological Sciences, 76 (2), pp. 347-356. ABSTRACT: Immunological parameters, host resistance, and thyroid hormones were evaluated in F1 mice exposed in utero to jet propulsion fuel-8 (JP-8). C57BL/6 pregnant dams (mated with C3H/HeJ males) were gavaged daily on gestation days 6-15 with JP-8 in a vehicle of olive oil at 0, 1000, or 2000 mg/kg. At weaning (3 weeks of age), no significant differences were observed in body, liver, spleen, or thymus weight, splenic and thymic cellularity, splenic CD4/CD8 lymphocyte subpopulations, or T-cell proliferation. Yet, lymphocytic proliferative responses to B-cell mitogens were suppressed in the 2000 mg/kg treatment group. In addition, thymic CD4-/CD8+ cells were significantly increased. By adulthood (8 weeks of age), lymphocyte proliferative responses and the alteration in thymic CD4-/CD8+ cells had returned to normal. However, splenic weight and thymic cellularity were altered, and the IgM plaque forming cell response was suppressed by 46% and 81% in the 1000 and 2000 mg/kg treatment groups, respectively. Furthermore, a 38% decrease was detected in the total T4 serum hormone level at 2000 mg/kg. In F1 adults, no significant alterations were observed in natural killer cell activity, T-cell lymphocyte proliferation, bone marrow cellularity and proliferative responses, complete blood counts, peritoneal and splenic cellularity, liver, kidney, or thymus weight, macrophage phagocytosis or nitric oxide production, splenic CD4/CD8 lymphocyte subpopulations, or total T3 serum hormone levels. Host resistance models in treated F1 adults demonstrated that immunological responses were normal after challenge with Listeria monocytogenes, but heightened susceptibility to B16F10 tumor challenge was seen at both treatment levels. This study demonstrates that prenatal exposure to JP-8 can target the developing murine fetus and result in impaired immune function and altered T4 levels in adulthood.

2003: Rhodes et al., The effects of jet fuel on immune cells of fuel system maintenance workers, Journal of Occupational and Environmental Medicine, 45 (1), pp. 79-86. ABSTRACT: Jet fuel is a common occupational exposure among commercial and military maintenance workers. JP-8 jet fuel, a military formulation, has shown immunotoxic effects in mice, but little data exist for humans. The aim of this cross-sectional study was to determine whether immune cell counts in the peripheral blood were altered among tank entry workers at three Air Force bases. After adjusting for covariates, fuel system maintenance personnel (n = 45) were found to have significantly higher counts of white blood cells (P = 0.01), neutrophils (P = 0.05), and monocytes (P = 0.02) when compared with a low-exposure group (n = 78), but no differences were noted in the numbers of total lymphocytes, T-cells, T-helper cells, T-suppressor cells, natural killer cells, and B-cells. Investigations are needed to evaluate the functional ability of these cells to produce lymphokines and cytokines and modulate the immune system.

2004: Wong et al., Inflammatory responses in mice sequentially exposed to JP jet fuel and influenza virus, ABSTRACT: To examine the hypothesis that Jet Propulsion Fuel (JP-8) inhalation potentiates influenza virus- induced inflammatory responses, we randomly divided female C57BL/6 mice (4-weeks old, weighing approximately 24.6g) into the following groups: air control, JP-8 alone (1023mg/m3 of JP-8 for 1h/day for 7 days), A/Hong Kong/8/68 influenza virus (HKV) alone (a 10μl aliquot of 2000 viral titer in the nasal passages), and a combination of JP-8 with HKV (JP-8 + HKV). The HKV alone group exhibited significantly increased total cell number/granulocyte differential in bronchoalveolar lavage fluid (BALF) compared to controls whereas the JP-8 alone group did not. The JP-8 + HKV group further exacerbated the HKV alone-induced response. However, increases in pulmonary microvascular permeability and pathological alterations in JP-8 + HKV just matched the sum of JP-8 alone- and HKV alone-induced response. Increases in BALF substance P in the JP-8

alone group and BALF leukotriene B4 or total lung compliance in the HKV alone group, respectively were similar to the changes in the JP-8 + HKV group. These findings suggest that changes in the JP-8 + HKV group may be attributed to either JP-8 inhalation or HKV treatment and indicate the different physiological responses to either JP-8 or HKV exposure. Taken together, most of the data did not provide supporting evidence that JP-8 inhalation synergizes influenza virus-induced inflammatory responses.

2004: Rogers et al., The cytotoxicity of volatile JP-8 jet fuel components in keratinocytes, Toxicology, 197 (2), pp. 139-147. ABSTRACT: In vitro models are being used to evaluate the toxic and irritating effects of JP-8, a kerosene-based jet fuel. JP-8 components are volatile, which makes in vitro studies difficult to evaluate dose-response relationships due to changes in chemical dosimetry caused by evaporation from the exposure medium. An in vitro approach testing volatile chemical toxicity that we have recently developed was used to evaluate the toxicity of the JP-8 components m-xylene, 1-methylnaphthalene (1-MN), and n-nonane in keratinocytes. Partition coefficients were measured and used to estimate the chemical concentration in the keratinocytes. The EC50 for m-xylene and 1-MN decreased significantly (P≤0.05) at 1, 2, and 4h. For n-nonane, no significant decreases in the EC50 values were observed over time; marginal cytotoxicity of n-nonane in keratinocytes was observed at 1h. Within 4h, about 75-90% of each volatile chemical was observed to be lost from the exposure medium when tissues were exposed in unsealed 24-well plates. This decrease resulted in significantly higher medium chemical concentrations needed to obtain EC50 values when compared to tissues exposed in sealed vials. This study demonstrates that chemical evaporation during in vitro exposures can significantly affect toxicological endpoint measurements. Ultimately, relating target cell chemical concentration to cellular responses in vitro could be used in determining an equivalent external dose using a biologically-based mathematical model.

2004: Gallucci et al., JP-8 je fuel exposure induces inflammatory cytokines in rat skin, International Immunopharmacology, 4 (9), pp. 1159-1169. ABSTRACT: The Department of Defense (DoD) has identified that one of the main complaints of personnel exposed to JP-8 jet fuel is irritant dermatitis. The purpose of this investigation is to describe the JP-8-induced inflammatory cytokine response in skin. JP-8 jet fuel or acetone control (300 μl) was applied to the denuded skin of rats once a day for 7 days. Skin samples from the exposed area were collected 2 and 24 h after the final exposure. Histological examination of skin biopsies showed neutrophilic inflammatory infiltrate. Reverse transcription-polymerase chain reaction (RT-PCR) was performed utilizing skin total RNA to examine the expression of various inflammatory cytokines. The CXC chemokine GROα was significantly upregulated at both time points, whereas GROβ was only increased 2 h post final exposure. The CC chemokines MCP-1, Mip-1α, and eotaxin were induced at both time points, whereas Mip-1β was induced only 24 h post exposure. Interleukins-1β and -6 (IL-1β and IL-6) mRNAs were significantly induced at both time points, while TNFα was not significantly different from control. Enzyme-linked immunosorbent assay (ELISA) of skin protein confirmed that MCP-1, TNFα, and IL-1β were modulated as indicated by PCR analysis. However, skin IL-6 protein content was not increased 2 h post exposure, whereas it was significantly upregulated by jet fuel after 24 h. Data from the present study indicate that repeated (7 days) JP-8 exposure induces numerous proinflammatory cytokines in skin. The increased expression of these cytokines and chemokines may lead to increased inflammatory infiltrate in exposed skin, resulting in JP-8-induced irritant dermatitis.

2005: Witzmann et al., Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture, Toxicology Letters, 160 (1), pp. 8-21. ABSTRACT: Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p < 0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

2007: Harris et al., JP-8 jet fuel exposure rapidly induces high levels of IL-10 and PGE2 secretion and is correlated with loss of immune function, Toxicology and Industrial Health, 23 (4), pp. 223-230. ABSTRACT: The US Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Previous work has demonstrated that JP-8 exposure is immunosuppressive. In the present study, the potential mechanisms for the effects of JP-8 exposure on the immune system were investigated. Exposure of mice to JP-8 for 1 h/day resulted in immediate secretion of two immunosuppressive agents; namely, interleukin-10 (IL-10) and prostaglandin E2 (PGE2). JP-8 exposure rapidly induced a persistently high level of serum IL-10 and PGE2 at an exposure concentration of 1000 mg/m3. IL-10 levels peaked at 2h post-JP-8 exposure and then stabilized at significantly elevated serum levels, while PGE2 levels peaked after 2-3 days of exposure and then stabilized. Elevated IL-10 and PGE2 levels may at least partially explain the effects of JP-8 exposure on immune function. Elevated IL-10 and PGE2 levels, however, cannot explain all of the effects due to JP-8 exposure (e.g., decreased organ weights and decreased viable immune cells), as treatment with a PGE2 inhibitor did not completely reverse the immunosuppressive effects of jet fuel exposure. Thus, low concentration JP-8 jet fuel exposures have significant effects on the immune system, which can be partially explained by the secretion of immunosuppressive modulators, which are cumulative over time.

2007: Harris et al., Effects of in utero JP-8 jet fuel exposure on the immune systems of pregnant and newborn mice, Toxicology and Industrial Health, 23 (9), pp. 545-552. ABSTRACT: The US Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Previous work has reported that JP-8 exposure is immunosuppressive. In the present study, the effects of in-utero JP-8 jet fuel exposure in mice were examined to ascertain any potential effects of jet fuel exposure on female personnel and their offspring. Exposure by the aerosol route (at 1000 mg/mm3 for 1 h/day; similar to exposures incurred by flight line personnel) commencing during the first (d7 to birth) or last (d15 to birth) trimester of pregnancy was analyzed. It was observed that even 6-8 weeks after the last jet fuel exposure that the immune system of the dams (mother of newborn mice) was affected (in accordance with previous reports on normal mice). That is, thymus organ weights and viable cell numbers were decreased, and immune function was depressed. A decrease in viable male offspring was found, notably more pronounced when exposure started during the first trimester of pregnancy. Regardless of when jet fuel exposure started, all newborn mice (at 6-8 weeks after birth) reported significant immunosuppression. That is, newborn pups displayed decreased immune organ weights, decreased viable immune cell numbers and suppressed immune function. When the data were analyzed in relation to the respective mothers of the pups the data were more pronounced. Although all jet fuel-exposed pups were immunosuppressed as compared with control pups, male offspring were more affected by jet fuel exposure than female pups. Furthermore, the immune function of the newborn mice was directly correlated to the immune function of their respective mothers. That is, mothers showing the lowest immune function after JP-8 exposure gave birth to pups displaying the greatest effects of jet fuel exposure on immune function. Mothers who showed the highest levels of immune function after in-utero JP-8 exposure gave birth to pups displaying levels of immune function similar to controls animals that had the lowest levels of immune function. These data indicated that a genetic component might be involved in determining immune responses after jet fuel exposure. Overall, the data showed that in-utero JP-8 jet fuel exposure had long-term detrimental effects on newborn mice, particularly on the viability and immune competence of male offspring.

2007: Ramos et al., Dermal exposure to jet fuel suppresse delayed-type hypersensitivity: A critical role for aromatic hydrocarbons, Toxicological Sciences, 100 (2), pp. 415-422. ABSTRACT: Dermal exposure to military (JP-8) and/or commercial (Jet-A) jet fuel suppresses cell-mediated immune reactions. Immune regulatory cytokines and biological modifiers, including platelet activating factor (PAF), prostaglandin E2, and interleukin-10, have been implicated in the pathway of events leading to immune suppression. It is estimated that approximately 260 different hydrocarbons are found in jet fuel, and the exact identity of the active immunotoxic agent(s) is unknown. The recent availability of synthetic jet fuel (S-8), which is refined from natural gas, and is devoid of aromatic hydrocarbons, made it feasible to design experiments to address this problem. Here we tested the hypothesis that the aromatic hydrocarbons present in jet fuel are responsible for immune suppression. We report that applying S-8 to the skin of mice does not upregulate the expression of epidermal cyclooxygenase-2 (COX-2) nor does it induce immune suppression. Adding back a cocktail of seven of the most prevalent aromatic hydrocarbons found in jet fuel (benzene, toluene, ethylbenzene, xylene, 1,2,4-trimethlybenzene, cyclohexylbenzene, and dimethylnaphthalene) to S-8 upregulated epidermal COX-2 expression and suppressed a delayed-type hypersensitivity (DTH) reaction. Injecting PAF receptor antagonists, or a selective cycloozygenase-2 inhibitor into mice treated with S-8 supplemented with the aromatic

cocktail, blocked suppression of DTH, similar to data previously reported using JP-8. These findings identify the aromatic hydrocarbons found in jet fuel as the agents responsible for suppressing DTH, in part by the upregulation of COX-2, and the production of immune regulatory factors and cytokines.

2008: Larabee et al., Serum profiling of rat dermal exposure to JP-8 fuel reveals an acute-phase response, Toxicology Mechanisms and Methods, 18 (1), pp. 41-51. ABSTRACT: Dermal exposure to JP-8 petroleum jet fuel leads to toxicological responses in humans and rodents. Serum profiling is a molecular analysis of changes in the levels of serum proteins and other molecules in response to changes in physiology. This present study utilizes serum profiling approaches to examine biomolecular changes in the sera of rats exposed to dermal applications of JP-8 (jet propulsion fuel-8). Using gel electrophoresis and electrospray ionization (ESI) mass spectrometry (MS), levels of serum proteins as well as low-mass constituents were found to change after dermal exposures to JP-8. The serum protein levels altered included the acute-phase response proteins haptoglobin, ceruloplasmin, α1-inhibitor III, and apolipoprotein A- IV. Haptoglobin levels increased after a 1-day JP-8 dermal exposure and continued to increase through 7 days of exposure. Ceruloplasmin levels increased after 5 days of exposure. Serum α1-inhibitor III was reduced after a 1- day exposure and the depletion continued after 7 days of exposure. Apolipoprotein A-IV increased after a 1-day exposure and then returned to basal levels after 3- and 5-day exposures of JP-8. Levels of the acute-phase protein α2-macroglobulin were found to not vary over these time course studies. Using ESI-MS analysis directly on the sera from rats exposed to dermal JP-8, low-mass sera constituents were found to correlate with control (acetone) or JP-8 exposure.

2008: Mann et al., Immunotoxicity evaluation of jet A jet fuel in female rats after 28-day dermal exposure, Journal of Toxicology and Environmental Health - Part A: Current Issues, 71 (8), pp. 495-504. ABSTRACT: The potential for jet fuel to modulate immune functions has been reported in mice following dermal, inhalation, and oral routes of exposure; however, a functional evaluation of the immune system in rats following jet fuel exposure has not been conducted. In this study potential effects of commercial jet fuel (Jet A) on the rat immune system were assessed using a battery of functional assays developed to screen potential immunotoxic compounds. Jet A was applied to the unoccluded skin of 6- to 7-wk-old female Crl:CD (SD)IGS BR rats at doses of 165, 330, or 495 mg/kg/d for 28 d. Mineral oil was used as a vehicle to mitigate irritation resulting from repeated exposure to jet fuel. Cyclophosphamide and anti-asialo GM1 were used as positive controls for immunotoxic effects. In contrast to reported immunotoxic effects of jet fuel in mice, dermal exposure of rats to Jet A did not result in alterations in spleen or thymus weights, splenic lymphocyte subpopulations, immunoglobulin (Ig) M antibody-forming cell response to the T-dependent antigen, sheep red blood cells (sRBC), spleen cell proliferative response to anti-CD3 antibody, or natural killer (NK) cell activity. In each of the immunotoxicological assays conducted, the positive control produced the expected results, demonstrating the assay was capable of detecting an effect if one had occurred. Based on the immunological parameters evaluated under the experimental conditions of the study, Jet A did not adversely affect immune responses of female rats. It remains to be determined whether the observed difference between this study and some other studies reflects a difference in the immunological response of rats and mice or is the result of other factors.

2008: Inman et., Inhibition of jet fuel aliphatic hydrocarbon induced toxicity in human epidermal keratinocytes, Journal of Applied Toxicology, 28 (4), pp. 543-553. ABSTRACT: Jet propellant (JP)-8, the primary jet fuel used by the U.S. military, consists of hydrocarbon-rich kerosene base commercial jet fuel (Jet-A) plus additives DC1-4A, Stadis 450 and diethylene glycol monomethyl ether. Human epidermal keratinocytes (HEK) were exposed to JP-8, aliphatic hydrocarbon (HC) fuel S-8 and aliphatic HC pentadecane (penta), tetradecane (tetra), tridecane (tri) and undecane (un) for 5 min. Additional studies were conducted with signal transduction pathway blockers parthenolide (P; 3.0 μM), isohelenin (I; 3.0 μM), SB 203580 (SB; 13.3 μM), substance P (SP; 3.0 μM) and recombinant human IL-10 (rHIL-10; 10 ng ml-1). In the absence of inhibitors, JP-8 and to a lesser extent un and S-8, had the greatest toxic effect on cell viability and inflammation suggesting, as least in vitro, that synthetic S-8 fuel is less irritating than the currently used JP- 8. Each inhibitor significantly (P < 0.05) decreased HEK viability. DMSO, the vehicle for P, I and SB, had a minimal effect on viability. Overall, IL-8 production was suppressed at least 30% after treatment with each inhibitor. Normalizing data relative to control indicate which inhibitors suppress HC-mediated IL-8 to control levels. P was the most effective inhibitor of IL-8 release; IL-8 was significantly decreased after exposure to un, tri, tetra and penta but significantly increased after JP-8 exposure compared with controls. Inhibitors were not effective in suppressing IL-8 release in JP-8 exposures to control levels. This study shows that inhibiting NFκB,

which appears to play a role in cytokine production in HC-exposed HEK in vitro, may reduce the inflammatory effect of HC in vivo.

2008: Harris et al., JP-8 jet fuel exposure suppresses the immune response to viral infections, Toxicology and Industrial Health, 24 (4), pp. 209-216. ABSTRACT: The US Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Previous work has reported that JP-8 exposure is immunosuppressive. Exposure of mice to JP-8 for 1 /day resulted in immediate secretion of two immunosuppressive agents, namely, interleukin-10 and prostaglandin E2. Thus, it was of interest to determine if jet fuel exposure might alter the immune response to infectious agents. The Hong Kong influenza model was used for these studies. Mice were exposed to 1000 mg/m3 JP-8 (1 /day) for 7 days before influenza viral infection. Animals were infected intra-nasally with virus and followed in terms of overall survival as well as immune responses. All surviving animals were killed 14 days after viral infection. In the present study, JP-8 exposure increased the severity of the viral infection by suppressing the anti-viral immune responses. That is, exposure of mice to JP-8 for 1 /day for 7 days before infection resulted in decreased immune cell viability after exposure and infection, a greater than fourfold decrease in immune proliferative responses to mitogens, as well as an overall loss of CD3+, CD4+, and CD8+ T cells from the lymph nodes, but not the spleens, of infected animals. These changes resulted in decreased survival of the exposed and infected mice, with only 33% of animals surviving as compared with 50% of mice infected but not jet fuel-exposed (and 100% of mice exposed only to JP-8). Thus, short-term, low-concentration JP-8 jet fuel exposures have significant suppressive effects on the immune system which can result in increased severity of viral infections.

2009: Limon-Flores et al., Mast cells mediate the immune suppression induced by dermal exposure to JP-8 jet fuel, Toxicological Sciences, 112 (1), pp. 144-152. ABSTRACT: Applying jet propulsion-8 (JP-8) jet fuel to the skin of mice induces immune suppression. Applying JP-8 to the skin of mice suppresses T-cell-mediated immune reactions including, contact hypersensitivity (CHS) delayed-type hypersensitivity and T-cell proliferation. Because dermal mast cells play an important immune regulatory role in vivo, we tested the hypothesis that mast cells mediate jet fuel - induced immune suppression. When we applied JP-8 to the skin of mast cell deficient mice CHS was not suppressed. Reconstituting mast cell deficient mice with wild-type bone marrow derived mast cells (mast cell "knock-in mice") restored JP-8 - induced immune suppression. When, however, mast cells from prostaglandin E2 (PGE2) - deficient mice were used, the ability of JP-8 to suppress CHS was not restored, indicating that mast cell - derived PGE2 was activating immune suppression. Examining the density of mast cells in the skin and lymph nodes of JP-8-treated mice indicated that jet fuel treatment caused an initial increase in mast cell density in the skin, followed by increased numbers of mast cells in the subcutaneous space and then in draining lymph nodes. Applying JP-8 to the skin increased mast cell expression of CXCR4, and increased the expression of CXCL12 by draining lymph node cells. Because CXCL12 is a chemoattractant for CXCR4+ mast cells, we treated JP-8- treated mice with AMD3100, a CXCR4 antagonist. AMD3100 blocked the mobilization of mast cells to the draining lymph node and inhibited JP-8-induced immune suppression. Our findings demonstrate the importance of mast cells in mediating jet fuel-induced immune suppression.

2009: Ramos et al., JP-8 induces immune suppression via a reactive oxygen species NF- κβ-Dependent mechanism, Toxicological Sciences, 108 (1), pp. 100-109. ABSTRACT: Applying jet fuel (JP-8) to the skin of mice induces immune suppression. JP-8-treated keratinocytes secrete prostaglandin E2, which is essential for activating immune suppressive pathways. The molecular pathway leading to the upregulation of the enzyme that controls prostaglandin synthesis, cyclooxygenase (COX)-2, is unclear. Because JP-8 activates oxidative stress and because reactive oxygen species (ROS) turn on nuclear factor kappa B (NF-κβ), which regulates the activity of COX-2, we asked if JP-8- induced ROS and NF-κβ contributes to COX-2 upregulation and immune suppression in vivo. JP-8 induced the production of ROS in keratinocytes as measured with the ROS indicator dye, aminophenyl fluorescein. Fluorescence was diminished in JP-8-treated keratinocytes overexpressing catalase or superoxide dismutase (SOD) genes. JP-8-induced COX-2 expression was also reduced to background in the catalase and SOD transfected cells, or in cultures treated with N-acetylcysteine (NAC). When NAC was injected into JP-8-treated mice, dermal COX-2 expression, and JP-8-induced immune suppression was inhibited. Because ROS activates NF-κβ, we asked if this transcriptional activator played a role in the enhanced COX-2 expression and JP-8- induced immune suppression. When JP-8-treated mice, or JP-8-treated keratinocytes were treated with a selective NF-κβ inhibitor, parthenolide, COX-2 expression, and immune suppression were abrogated. Similarly, when JP-8-treated keratinocytes were treated with small interfering RNA specific for the p65 subunit of NF-κβ,

COX-2 upregulation was blocked. These data indicate that ROS and NF-κβ are activated by JP-8, and these pathways are involved in COX-2 expression and the induction of immune suppression by jet fuel.

2011: Hilgaertner et al., The influence of hydrocarbon composition and exposure conditions on jet fuel-induced immunotoxicity, Toxicology and Industrial Health, 27 (10), pp. 887-898. ABSTRACT: Chronic jet fuel exposure could be detrimental to the health and well-being of exposed personnel, adversely affect their work performance and predispose these individuals to increased incidences of infectious disease, cancer and autoimmune disorders. Short-term (7 day) JP-8 jet fuel exposure has been shown to cause lung injury and immune dysfunction. Physiological alterations can be influenced not only by jet fuel exposure concentration (absolute amount), but also are dependent on the type of exposure (aerosol versus vapor) and the composition of the jet fuel (hydrocarbon composition). In the current study, these variables were examined with relation to effects of jet fuel exposure on immune function. It was discovered that real-time, in-line monitoring of jet fuel exposure resulted in aerosol exposure concentrations that were approximately one-eighth the concentration of previously reported exposure systems. Further, the effects of a synthetic jet fuel designed to eliminate polycyclic aromatic hydrocarbons were also examined. Both of these changes in exposure reduced but did not eliminate the deleterious effects on the immune system of exposed mice.

6.4 Effects on liver

6.4.1 1996 assessment (COT 1996) The assessment considered three key inhalation studies in which animals were exposed intermittently (occupational-type exposure) to fuel vapors, and which were found no liver histopathological changes Bruner et al. (1993); Wall et al. (1990); Mac Ewen and Vernot (1981). In one study, rats exposed to JP-5 vapors at concentrations of 1,100 or 1,600 mg/m3 for 6 hr per day, 5 days per week for 6 weeks showed no evidence of adverse effects on the liver. In the second study, rats, mice, dogs, and monkeys exposed to JP-4 vapors at 2,500 or 5,000 mg/m3 for 6 hr per day, 5 days per week for 8 months showed no evidence of exposure-related effects except a slight increase in liver weight in the female rats. In the third study, rats and mice exposed to JP-4 vapors at 1,000 or 5,000 mg/m3 for 6 hr per day, 5 days per week for 12 months showed no liver toxicity. No clear evidence of hepatic neoplasia in rats or mice was found. Based on this study, COT (1996) identified a no-observed-adverse- effect-level (NOAEL) of 5,000 mg/m3, which was used to calculate the PEL. By dividing the NOAEL of 5,000 mg/m3 by an uncertainty factor of 10 for interspecies extrapolation, the PEL was calculated to be 500 mg/m3. No uncertainty factor for intraspecies variation was applied because the exposed Navy personnel are considered to be healthy.

6.4.2 2003 assessment (COT 2003)

• In one experimental animal study, F344 rats and C57BL/6 mice continuously exposed to JP-8 vapors at concentrations up to 1,000 mg/m3for up to 90 days did not show significant changes in hepatic function or structure.

• In another study, liver weights in male F344 rats exposed to JP-8 aerosols at up to 1,000 mg/m3 for 1 hr per day for 28 days were not significantly different from liver weights in control animals. There were no significant alterations in serum aspartate aminotransferase and alanine aminotransferase activities, indicators of hepatic function, and there were no marked changes in the liver histopathologic findings and cytochrome P450 content, a measure of xenobiotic metabolism.

COT (2003) recommended that liver toxicity be evaluated in experimental animals exposed to JP-8 vapors and mixtures of vapors and aerosols by the inhalation route. Because inhalation exposures greater than approximately 1,000 mg/m3 for pure JP-8 vapors are difficult to

achieve, the Air Force should consider conducting studies with saturated vapor atmospheres on larger numbers of animals or employ longer exposure durations (i.e., longer than 90 days) to increase the power of the studies for observing adverse responses in various organ systems.

6.4.3 Human data Snawder and Butler (2001) investigated the effects of JP-8 inhalation exposure to humans. Measurements taken in breathing zones of subjects; median concentration of naphthalene, 1.9 μg/m3 (low-exposure group), 447 μg/m3(high-exposure group); median concentration of benzene, 3.1 μg/m3 (low-exposure group), 242 μg/m3(high-exposure group. High-exposure group had persistent exposure to JP-8 (defined as at least 1 hr twice per wk for 9 mo); low- exposure group had no significant exposure to jet fuel or solvents. Concentrations of serum hepatic alpha-GST (Gutathione-S-Transferase) activity in study subjects were found to be within normal range.

Butler et al. (2001) investigated the effects of JP-8 inhalation exposure to humans. Measurements taken in breathing zones of subjects; median concentration of naphthalene, 1.9 μg/m3 (low-exposure group), 10.4 μg/m3(moderate-exposure group), 447 μg/m3 (high- exposure group); median concentration of benzene, 3.1 μg/m3 (low-exposure group), 7.45 μg/m3(moderate-exposure group), 242 μg/m3(high-exposure group). High- and moderate- exposure groups had persistent exposure to JP-8; low exposure group had no significant exposure to jet fuel or solvents. Frequency of CYP2E1 (cytochrome P2E1) and NQOI (quinone oxidoreductase) genotypes was similar in subjects in all exposure groups; no change in enzymatic activity was found.

Gibson et al. (2001a) investigated the effects of JP-8 inhalation exposure to humans. Exposed group (5,706 people) had potential occupational exposure to JP-8. Control group (5,706 people) did not work in occupations in which exposure to JP-8 would occur. Analysis of medical records showed that subjects in all groups had similar health-care visit rates; no differences were noted among groups in digestive ailments.

Gibson et al. (2001b) investigated the effects of JP-8 inhalation exposure to humans. Measurements taken in breathing zones of subjects; median concentration of naphthalene, 1.9 μg/m3 (low-exposure group), 10.4 μg/m3 (moderate-exposure group), 447 μg/m3 (high- exposure group); median concentration of benzene, 3.1 μg/m3 (low-exposure group), 7.45 μg/m3 (moderate-exposure group), 242 μg/m3 (high-exposure group). High- and moderate- exposure groups had persistent exposure to JP-8; low-exposure group had no significant

exposure to jet fuel or solvents. Analysis of self-assessment questionnaire did not report differences among groups in digestive ailments.

6.4.4 In vitro data Grant et al. (2000) examined the in vitro cytotoxic potential of JP-8 in an H4IIE liver cell line. The H4IIE cell line is an established model used to assess hepatic function and responds to polycyclic aromatic hydrocarbons. In 72-hr viability assays, the concentration of JP-8 producing 50% inhibition (IC50) of growth in H4IIE cells was 12.6 ± 0.4 μg/mL. The relevance of the in vitro findings for humans is not known.

6.4.5 Animal data Parton (1994) investigated the effects of JP-8 inhalation exposure to male F344 rats at concentrations in the range of 500 or 1,000 mg/m3(aerosol, nose-only) during 1 hr/day for 7 or 28 days. Body weight gain in rats exposed for 28 days was significantly decreased; final body weights of exposed animals were similar to those of control animals; liver weights not significantly different between groups; relative liver weight increased in high-dose groups; no significant alterations in AST and ALT activity; no marked changes in liver histopathologic findings and CYP450 content.

Mattie et al. (1991) investigated the effects of JP-8 inhalation exposure to male and female F344 rats, C57Bl/6 mice at concentrations in the range of 500 or 1,000 mg/m3 (vapor) during 90 days continuously. Male rats had a statistically significant increase in hepatic basophilic foci. Their presence in the livers of male rats is of uncertain biological significance. No alterations were found in hepatic tissue of female rats or in mice.

Mattie et al. (1995) investigated the effects of JP-8 inhalation exposure to male Sprague- Dawley rats at concentrations in the range of 750, 1,500, or 3,000 mg/kg (gavage) during 90 days consecutively. Serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase) activity increased significantly in all groups, but increase was not dose- related; liver weight similar in all groups; increased relative tissue weight in high-exposure group; liver histologic findings similar in all groups (including control group).

Witzman et al. (2000) investigated the effects of JP-8 inhalation and dermal exposure to male Sprague-Dawley rats at concentrations in the range of 1,000 mg/m3 (vapor, whole-body) during 6 hr/day, 5 days/wk for 6 weeks. Hepatic lamin L83 abundance significantly

decreased; lamin L603 abundance increased; total lamin A abundance not significantly altered by JP-8 exposure.

Dudley et al. (2001) investigated the effects of JP-8 exposure to female B6C3F1, DBA/2 mice at doses in the range of 1 or 2 g/kg per day (oral gavage during 7 days. Significantly increased body weights of B6C3F1 mice, but not DBA/2 mice; increased liver: body weight ratios in both strains; no marked change in expression of CYP1A1.

6.4.6 Recent studies to be considered by SCOEL

2008: Fechter et al., Depletion of liver glutathione levels in rats: A potential confound of nose-only inhalation, Inhalation Toxicology, 20 (9), pp. 885-890. ABSTRACT: Nose-only inhalation exposure chambers offer key advantages to whole-body systems, particularly when aerosol or mixed aerosol-vapor exposures are used. Specifically, nose-only chambers provide enhanced control over the route of exposure and dose by minimizing the deposition of particles either on the subjects skin/fur or on surfaces of a whole-body exposure system. In the current series of experiments, liver, brain, and lung total glutathione (GSH) levels were assessed following either nose-only or whole-body exposures to either jet fuel or to clean, filtered air. The data were compared to untreated control subjects. Acute nose-only inhalation exposures of rats resulted in a significant depletion of liver GSH levels both in subjects that were exposed to clean, filtered air as well as those exposed to JP-8 jet fuel and to a synthetic jet fuel. Glutathione levels were not altered in lung or brain tissue. Whole-body inhalation exposure had no effect on GSH levels in any tissue for any of the treatment groups. A second experiment demonstrated that the loss of GSH did not occur if rats were anaesthetized prior to and during nose-only exposure to clean, filtered air or to mixed hydrocarbons. These data appear to be consistent with studies demonstrating depletion in liver GSH levels among rats subjected to restraint stress. Finally, the depletion of GSH that was observed in liver following a single acute exposure was reduced following five daily exposures to clean, filtered air, suggesting the possibility of habituation to restraint in the nose-only exposure chamber. The finding that placement in a nose-only exposure chamber per se yields liver GSH depletion raises the possibility of an interaction between this mode of toxicant exposure and the toxicological effects of certain inhaled test substances.

6.5 Effects on kidney

6.5.1 1996 assessment (COT 1996) The renal toxicity of military fuels was studied in rats and mice of both sexes. Adverse effects in the kidneys were observed only in male rats after inhalation exposure. Histological sections from the kidneys of affected animals were examined, and the presence of the characteristic hyaline droplets, suggestive of an α2u-globulin pathogenesis, was confirmed. COT (1996) concluded that that these findings are not relevant to humans because this kidney lesion appears to be unique to the male rat.

6.5.2 2003 assessment (COT 2003) COT (2003) considered studies on F344 rats and C57BL/6 mice exposed on a continuous basis by inhalation to JP-8 vapors at concentrations of 500 or 1,000 mg/m3 for 90 days which showed induction of alpha 2u-globulin nephropathy in male rats but not in female rats or in male or female animals of other species. Alpha-2u-globulin-induced nephropathy was found to occur only in male rats and is not relevant to humans. COT (2003) recommended that kidney toxicity be evaluated in experimental animals exposed to JP-8 vapors and mixtures of vapors and aerosols by the inhalation route.

6.5.3 Human data Snawder and Butler (2001) investigated effects of JP-8 exposure on the kidney in humans. Concentrations of urinary neph-alpha GST (glutathione-S-transferase ) and pi-GST in subjects were found in normal range.

Butler et al. (2001) investigated effects of JP-8 exposure on the kidney in humans. Analysis of CYP2EI, GSTT1, and NQO1 (quinone oxidoreductase) genotype data showed no statistically significant interaction between those genotypes, alpha-GST or pi-GST, and JP-8 exposure.

Gibson et al. (2001a) investigated effects of JP-8 exposure on the kidney in humans. Analysis of medical records showed that subjects in all groups had similar health-care visit rates; no differences were found among different exposure groups in kidney-related conditions.

Gibson et al. (2001b) investigated effects of JP-8 exposure on the kidney in humans. Analysis of self-assessment questionnaire did not report differences among groups in kidney-related conditions

6.5.4 Animal data Mattie et al. (1991) investigated the effects of JP-8 exposure on the kidney of male and female F344 rats and male and female C57BL/6 mice at concentrations in the range of 500 or 1,000 mg/m3(vapors, whole-body) during 90 days continuously. It was observed kidney lesions (hyalin droplets, granular casts in outer medulla, nephrosis) in male rats only; no kidney toxicity in female rats or male and female mice.

Parton (1994) investigated the effects of JP-8 exposure on the kidney of male F344 rats at concentrations in the range of 500 or 1,000 mg/m3(aerosol, nose-only) during 1 hr/day for 7 or 28 days. Body weight gain in rats exposed for 28 days significantly decreased; final body weights of exposed animals similar to those of control animals; relative kidney weight increased in animals exposed for 7 days and in animals exposed at high dose for 28 days; changes in relative kidney weight associated with increase in hyalin droplet formation and in alpha-2u-globulin; renal function not compromised.

Mattie et al. (1995) investigated the effects of JP-8 exposure on the kidney of male Sprague- Dawley rats at concentrations in the range of 750, 1,500, or 3,000 mg/kg (by gavage) during 90 days consecutively. Serum sodium and chloride concentrations increased in highest-dose group; serum creatinine concentrations increased in low-and middle-dose groups (but not in high-dose group); urinary creatinine and protein concentrations not significantly altered by exposure; urinary pH significantly lower in the middle- and high-dose groups; exposure did not alter absolute renal weights but produced significant increase in kidney: body weight ratio in the middle- and high-dose groups; increased renal weight caused by accumulation of hyalin droplets.

Witzmann et al. (2000a) investigated the effects of JP-8 exposure on the kidney of male Swiss-Webster mice at concentrations in the range of 1,000 mg/m3(aerosol, nose-only) during 1 hr/day for 5 days. Exposure significantly altered abundance of 56 proteins; concentrations of 21 proteins increased, concentrations of 35 proteins decreased, compared with controls.

Witzmann et al. (2000) investigated the effects of JP-8 exposure on the kidney of male Sprague-Dawley rats at concentrations in the range of 1,000 mg/m3(vapor, whole-body) during 6 hr/day, 5 days/wk for 6 wk. Renal GST homolog and 10-formyltetrahydrofate dehydrogenase increased in charge modification index; no change in abundance.

6.5.5 Recent studies to be considered by SCOEL

6.6 Effects on reproduction and development

6.6.1 1996 assessment (COT 1996) COT (2003) did not review reproductive and developmental effects of exposure to jet fuels.

6.6.2 2001 assessment (COT 2001) COT (2001) report focused on the evaluation of exposures of chemicals and of other agents for reproductive and developmental toxicity, reviewed the potential toxicity of JP-8 for these endpoints. In that report, a dosage that is unlikely to cause toxicity (only for effects that are observed at birth and only for short-term exposure) was calculated to be 1 mg/kg per day (equivalent to 0.8 ppm for humans, assuming 8-hr/day exposure, 100% absorption, 69-kg body weight, and respiratory minute volume of 0.42 mL/min per kilogram of body weight).

6.6.3 2003 assessment (COT 2003) Male and female Sprague-Dawley rats exposed to JP-8 by oral gavage at concentrations up to 1,500 (females) or 3,000 (males) mg/kg per day prior to and during mating and, in the case of the females, during gestation and lactation, showed a decrease in body weight, but no adverse effects on fertility were observed in either sex. Dermal exposure of rats to HDS kerosene at doses up to 494 mg/kg per day did not affect fertility in males or females exposed prior to and during mating and, in the case of the females, during gestation and lactation.

Maternal-gestational weight gain and fetal body weights were reduced in Sprague-Dawley rats exposed to JP-8 by oral gavage at 1,500 or 2,000 mg/kg per day on days 6-15 of pregnancy; the types of fetal abnormalities did not differ significantly between JP-8 dose groups and the unexposed animals, and there was a progressive increase in the overall incidence of abnormalities with increasing dose from 500 to 1,500 mg/kg per day, but not at 2,000 mg/kg per day. No developmental toxicity was reported in the offspring of Sprague- Dawley rats dermally exposed to HDS kerosene at doses up to 494 mg/kg per day. There are no developmental-toxicity studies that evaluate postnatal and long-term effects (such as neurologic effects) of in utero exposures. Because of the paucity of data and because military personnel are occupationally exposed to JP-8, the subcommittee recommends that experimental-animal studies be conducted to determine the reproductive and developmental toxicity potential of JP-8.

6.6.4 Human data No studies were found in the literature that examined potential female reproductive effects or developmental effects of JP-8 or other jet fuels in humans. (LeMasters et al. 1999) assessed male reproductive effects of inhalation of jet fuel (type not specified) and hydrocarbon solvents after 15 and 30 wk of exposure. In that study, exposure to jet fuel increased sperm concentration in workers who fuelled jets and decreased sperm linearity in flight-line workers; exposure to jet fuels did not appear to affect semen quality in aircraft-maintenance workers.

6.6.5 Animal data Mattie et al. (1995), (2000) investigated the effects of JP-8 exposure on reproduction in male and female Sprague-Dawley rats at concentrations in the range of 750, 1,500, 3,000 mg/kg per day for the males; and for females, 325, 750, 1,500 mg/kg per day (gavage) during 70 days before mating and during mating (up to 90 days) for the males; and for females during 90 days before mating and during mating, gestation, delivery, lactation. The effects observed on males included: no differences between exposed and control groups in sperm concentration, motile sperm concentration, percentage motility, velocity, linearity, maximal ALH, mean ALH, beat/cross frequency, mean radius, number of circular cells, percentage circular cells/motile cells, and percentage circular cells/all cells; no effect on fertility of unexposed female mating partners. The effects observed on females included:: no significant differences between exposed and control groups in pregnancy rates, gestation lengths, number of pups/litter, litter size, viability and survival of pups; pups from dams exposed at 1,500 mg/kg per day had significantly reduced body weight compared with controls. Price et al. (2001) investigated the effects of JP-8 exposure on reproduction in male rats at concentrations in the range of 250, 500, 1,000 mg/m3 during 6 hr/day, 7 days/wk for 90 days. No significant differences between exposed and control groups on sperm count and concentration; no pathologic findings in testes of treated animals; significant difference between the treated and control animals in sperm motility.

Schreiner et al. (1997) investigated the effects of HDS (HydroDeSulfurized) Kerosene exposure on reproduction and development in male and female Dawley rats at concentrations in the range of 165, 330, 494 mg/kg (dermal) during 8 wk starting 14 days for males before mating; and for females, 7 wk starting 14 days before mating, sacrificed on days 4-6 of lactation. No treatment-related effect on fertility; no treatment-related microscopic changes in

testes or epididymides of adult male rats or in ovaries of adult female rats. No treatment- related developmental toxicity was found.

Cooper and Mattie (1996) investigated the effects of JP-8 exposure on development in female Sprague-Dawley rats at concentrations in the range of 500, 1,000, 1,500, 2,000 mg/kg per day (oral) during days 6-15 of pregnancy. Maternal and fetal body weights were markedly reduced in 1,000-, 1,500-, and 2,000-mg/kg per day groups; number and type of fetal malformations and variations did not differ significantly between groups; progressive increase in overall incidence of fetal alterations with increasing dose between 500- and 1,500-mg/kg per day groups, but not for 2,000-mg/kg per day groups.

6.6.6 Recent studies to be considered by SCOEL

2003: Witzmann et al., Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor, Proteomics, 3 (6), pp. 1016-1027. ABSTRACT: We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m3 ± 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.

6.7 Effects on cardiovascular system

6.7.1 1996 Assessment (COT 1996) COT (1996) concluded that the animal data are not useful for determining permissible exposure levels (PELs), because the oral route of exposure was not directly relevant and the chemical composition of the liquid JP-5 differs from that of the vapours.

6.7.2 2003 Assessment (COT 2003)

• A comparison of medical records of occupationally exposed personnel showed no increase in medical visits related to cardiovascular events.

• Results of a health survey of Air Force personnel that used a self-assessment questionnaire showed that the total number of medical visits and the number of visits for specific reasons, including palpitations and chest tightness, were higher among high- and moderate-exposure groups than in the low-exposure group. The reported effects were not dose-related; the moderate-exposure group showed greater incidence of adverse effects than the high-exposure group.

COT (2003) concluded on the basis of the studies listed in chapters 6.7.1 and 6.7.2 that:

• many potential, uncontrolled biases are associated with the investigations on humans, and the lack of adequate exposure data makes interpretation of the results difficult.

• that when exposure-assessment data become available, the cardiovascular effects data in humans be re-evaluated.

• cardiovascular toxicity in experimental animals exposed to JP-8 vapors and mixtures of vapors and aerosols needs to be evaluated by the inhalation route

6.7.3 Human data Gibson et al. 2001a investigated the effects of JP-8 Exposure on the Cardiovascular System in Humans. The study covered 5,706 exposed subjects and 5,706 unexposed subjects. Exposed group had potential occupational exposure to JP-8; control group did not work in occupations in which exposure to JP-8 would occur. Medical records showed no increase in medical visits related to cardiovascular events.

Gibson et al. (2001b) investigated the effects of JP-8 Exposure on the Cardiovascular System in Humans. The study covered 328 individuals. JP-8 concentration measurements were taken in the breathing zone of the exposed subjects. The High- and moderate- exposure groups had persistent exposure to JP-8 (defined as at least 1 hr twice per wk for at least 9 mo); low- exposure group had no significant exposure to jet fuel or solvents. Data collected from self- assessment questionnaire; subjects in moderate- and high-exposure groups reported more heart palpitations and chest tightness than subjects in low-exposure group; odds ratios for subjects in moderate-exposure group, but not high-exposure group, were significantly greater than for low-exposure group.

6.7.4 Animal data Mattie et al. (1991) investigated the effects of JP-8 Exposure on the Cardiovascular system in F344 rat, C57BL/6 mouse at concentrations in the range of 500 and 1,000 mg/m3(via inhalation) during 90 days continuously. No histopathologic changes to cardiovascular system were observed.

Parker et al. (1981) investigated the effects of JP-5 Exposure on the cardiovascular system in Sprague-Dawley rats at doses in the range of 24 mL/kg (via oral gavage) during 3 days. No increase in serum creatinine phosphokinase concentrations was observed.

Carpenter et al. (1976) investigated the effects of Deodorized kerosene vapour exposure on the Cardiovascular system in Beagle rats at concentrations in the range of 20, 48, and 100 mg/m3(via inhalation) during 6 hr/day, 5 days/wk for up to 67 day. No treatment-related changes in clinical pathologic and histopathologic measures of cardiovascular system were observed; no electrocardiographic changes related to treatment were observed in dogs.

6.7.5 Recent studies to be considered by SCOEL No studies were found.

6.8 Genotoxic effects

6.8.1 1996 Assessment (COT 1996) COT (1996) considered the literature published before 1996 and concluded that the vapours of the fuels JP-5, JP-8, and DFM (Diesel Fuel Marine) do not constitute an important genotoxic hazard.

6.8.2 2003 Assessment (COT 2003) COT (2003) considered the studies outlined in chapters 8.2.1 and 8.2.2 and concluded

• that the available data were 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.

• that 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.

COT (2003) recommended further that the Air Force conducts 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.

6.8.3 Human data Pitarque et al. (1999) investigated the genotoxic effects of Hydro-carbons, jet-fuel, and derivatives in humans. The study covered 34 male airport workers and 11 unexposed controls. Exposure concentrations in the range of 0.10 ± 0.05 mg/m3 Benzene,; 0.13 ± 0.01 mg/m3 toluene; 0.13±0.02 mg/m3 xylenes were measured at the Barcelona airport. The exposure duration considered was 9.77 yr (mean). No increases in SCE (sister chromatid exchange), MN (micronucleus), 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.

Lemasters et al. (1997), (1999) investigated the genotoxic effects of JP-4 and solvents in humans. The study covered 58 aircraft-maintenance workers, 8 unexposed controls. Exposure levels were reported to be well below threshold limit values. Exposure duration was at least 30 weeks. Small but statistically significant increase in frequency of SCE (sister chromatid exchange) occurred after 30 wk of exposure in sheet-metal workers and painters; MN (micronucleus), frequency in sheet-metal workers initially showed statistically significant increase but had decreased by 30 weeks.

6.8.5 Animal data Grant et al. 2001 investigated the genotoxic effects of JP-8 in H4IIE rat hepatoma cells. The exposure concentration was 1-20 μg/mL and the duration 4 hours. At these conditions 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.

6.8.6 Recent studies to be considered by SCOEL

2003: Espinosa et al., Macroarray analysis of the effects of JP-8 jet fuel on gene expression in Jurkat cells, Toxicology, 189 (3), pp. 181-190. ABSTRACT: The jet fuel JP-8 is widely used and a large number of military and civilian personnel is, therefore, exposed to it. Treatment of several cell lines, including human Jurkat cells, with JP-8 induces cell death that exhibits various biochemical and morphological characteristics of apoptosis. The molecular mechanism of JP-8 cytotoxicity, however, has remained unclear. The effects of exposure of Jurkat cells to JP-8 (1/10 000 dilution) for 4 h on gene expression have now been examined by cDNA macroarray analysis. We had previously shown in these cells that under the above conditions, JP-8 causes significant apoptosis, based upon the observation that caspase-3 activation occurs at approximately 4 h and consequently most of the other classical apoptotic biochemical and morphological alterations progress until apoptotic cell death at 24 h. Of the 439 apoptosis- or stress response-related genes examined, the expression of 16 genes was up-regulated and that of ten genes was down-regulated by a factor of ≥2. The changes in the expression of 11 of these 26 genes were confirmed by reverse transcription and polymerase chain reaction analysis. These results provide insight into the mechanism of JP-8 toxicity and the associated induction of apoptosis.

2004: Vijayalaxmi et al., Cytogenetic studies in mice treated with the jet fuels, Jet-A and JP-8, Cytogenetic and Genome Research, 104 (1-4), pp. 371-375. ABSTRACT: The genotoxic potential of the jet fuels, Jet-A and JP-8, were examined in mice treated on the skin with a single dose of 240 mg/mouse. Peripheral blood smears were prepared at the start of the experiment (t = 0), and at 24, 48 and 72 h following treatment with jet fuels. Femoral bone marrow smears were made when all animals were sacrificed at 72 h. In both tissues, the extent of genotoxicity was determined from the incidence of micronuclei (MN) in polychromatic erythrocytes. The frequency of MN in the peripheral blood of mice treated with Jet-A and JP-8 increased over time and reached statistical significance at 72 h, as compared with concurrent control animals. The incidence of MN was also higher in bone marrow cells of mice exposed to Jet-A and JP-8 as compared with controls. Thus, at the dose tested, a small but significant genotoxic effect of jet fuels was

2004: Cavallo et al., Assessment of genotoxic and oxidative effects in aircraft maintenance workers occupationally exposed to polycyclic aromatic hydrocarbons (PAH), Giornale Italiano di Medicina del Lavoro ed Ergonomia, 26 (4 SUPPL.), pp. 44-45. ABSTRACT: Airport personnel are occupationally exposed, on flight lines and during aircraft routine maintenance procedures, to several polycyclic aromatic hydrocarbons (PAH) produced by jet fuel, diesel fuel or kerosene combustion. The aim of this study was to evaluate early genotoxic and oxidative effects in airport personnel (No.=16) occupationally exposed to complex mixtures of PAH in comparison to a selected control group (No.=12). We used a micronucleus test and an Fpg modified Comet assay to study early genotoxicity and oxidative DNA damage on exfoliated buccal cells. The exposed group showed a higher mean value of micronuclei frequency (%) with respect to controls (0.080 vs 0.071). For the exposed group, the Fpg modified Comet test revealed a higher value of mean tail moment TM (the product of comet relative tail intensity and length, calculated on 50 randomly selected comets) both for enzyme treated cells TMenz (114.52 vs 89.81), which provide a parameter of oxidative DNA damage, and for cells untreated with enzyme TM (90.22 vs 81.85), which provide a parameter of direct DNA damage. The presence of oxidative DNA damage was evaluated in each subject using the TMenz/TM ratio. When this ratio was higher than 2.0, the subject was estimated to have oxidative DNA damage. We found the presence of oxidative DNA damage in 12.5% of those exposed with respect to the absence of oxidative damage of controls. These results demonstrate the high degree of sensitivity of exfoliated buccal cell for indicating early genotoxic effects of PAH exposure and confirm the high sensitivity of the Comet assay for assessing early direct and oxidative DNA damage. The results obtained on exfoliated buccal cells suggest the use of this sampling, obtained using a non-invasive procedure, for assessing the occupational exposure to a mixture of chemicals at low doses since they represent the target tissue for this exposure and appear to be a useful tool in the study of populations chronically exposed to PAH.

2006: Cavallo et al., Occupational exposure in airport personnel: Characterization and evaluation of genotoxic and oxidative effects, Toxicology, 223 (1-2), pp. 26-35. ABSTRACT: Airport personnel can be exposed to several polycyclic aromatic hydrocarbons (PAHs) from jet fuel vapours, jet fuel combustion products and diesel exhaust. The aim of this study was to characterize the exposure and to evaluate genotoxic and oxidative effects in airport personnel (n = 41) in comparison with a selected control group (n = 31). Environmental monitoring of exposure was carried out analysing 23 PAHs on air samples collected from airport apron, airport building and terminal/office area during 5 working days. The urinary 1-hydroxy-pyrene (1-OHP) following 5 working days, was used as biomarker of exposure. Genotoxic effects and early direct-oxidative DNA damage were evaluated by micronucleus (MN) and Fpg-modified comet assay on lymphocytes and exfoliated buccal cells, and by chromosomal aberrations (CA) and sister chromatid exchange (SCE) analyses. For comet assay, tail moment (the product of comet relative tail intensity and length) values from Fpg-enzyme treated cells (TMenz) and from untreated cells (TM) were used as parameters of oxidative and direct DNA damage, respectively. We found 27,703 μg/m3 total PAHs in airport apron, 17,275 μg/m3 in airport building and 9,494 μg/m3 in terminal/office area. Urinary OH-pyrene did not show differences between exposed and controls. The exposed group showed a higher mean value of SCE frequency in respect to controls (4.6 versus 3.8) and an increase (1.3-fold) of total structural CA in particular breaks (up to 2.0-fold) and fragments (0.32% versus 0.00%), whereas there were no differences of MN frequency in both cellular types. Comet assay evidenced in the exposed group a higher value in respect to controls of mean TM and TMenz in both exfoliated buccal cells (TM 118.87 versus 68.20, p = 0.001; TMenz 146.11 versus 78.32, p < 0.001) and lymphocytes (TM 43.01 versus 36.01, p = 0.136; TMenz 55.86 versus 43.98, p = 0.003). An oxidative DNA damage was found, for exfoliated buccal cells in the 9.7% and for lymphocytes in the 14.6% of exposed in respect to the absence in controls. Our findings furnish a useful contribution to the characterization of civil airport exposure and suggest the use of comet assay on exfoliated buccal cells to assess the occupational exposure to mixtures of inhalable pollutants at low doses since these cells represent the target tissue for this exposure and are obtained by non-invasive procedure.

2006: Vijayalaxmi et al., Micronucleus studies in the peripheral blood and bone marrow of mice treated with jet fuels, JP-8 and Jet-A, Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 608 (1), pp. 82-87. ABSTRACT: The potential adverse effects of dermal and inhalation exposure of jet fuels are important for health hazard evaluation in humans. The genotoxic potential of jet fuels, JP-8 and Jet-A, was investigated in an animal model. Mice were treated dermally with either a single or multiple applications of these jet fuels. Peripheral blood and bone marrow smears were prepared to examine the incidence of micronuclei (MN) in

polychromatic erythrocytes (PCEs). In all experiments, using several different exposure regimens, no statistically significant increase in the incidence of MN was observed in the bone marrow and/or peripheral blood of mice treated with JP-8 or Jet-A when compared with those of untreated control animals. The data in mice treated with a single dose of JP-8 or Jet-A did not confirm the small but statistically significant increase in micronuclei reported in our previous study.

2011: Erdem et al., Evaluation of genotoxic and oxidative effects in workers exposed to jet propulsion fuel, International Archives of Occupational and Environmental Health, pp. 1- 9. ABSTRACT: Purpose: Jet fuel is a common occupational exposure risk among military and civilian populations. The purpose of this study was to evaluate genotoxic and oxidative effects in workers occupational exposure to jet propulsion fuel (JP-8). Methods: In this study, sister-chromatid exchange (SCE), high frequency of SCE cells (HFCs), and micronuclei (MN) were determined for 43 workers exposed to JP-8 and 38 control subjects. We measured the antioxidant enzyme activities including that of superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). The levels of thiobarbituric acid-reactive substances (TBARS) were also studied. Urinary 1- and 2-naphthol excretion was used as a biomarker of occupational exposure to JP-8. Results: The results obtained from cytogenetic analysis show a statistically significant increase in frequency of SCE in the exposed workers when compared to controls (P < 0.05). Interestingly, the mean value of the frequency (%o) of MN and HFCs for workers and controls did not show any statistical differences (P > 0.05). Oxidative stress parameters were not statistically different between exposed and control groups except for TBARS levels. Conclusion: Urinary 1-and 2-naphthol levels of exposed workers were found to be significantly higher than those of control subjects. Occupational exposure to JP-8 resulted in no significant genotoxic and oxidative effects, while smoking is the principal confounding factor for the some parameters. To understand the genotoxic and oxidative effects of JP-8 exposure, further studies should be planned to find out whether human populations may be at increased risk for cancer because of the exposures related to occupation and lifestyle.

6.9 Carcinogenic effects

6.9.1 1996 Assessment (COT 1996) COT (1996) considered as a key study an epidemiological study by Selden and Ahlborg (1991) of approximately 2,200 Swedish military personnel exposed to jet-fuel vapors at concentrations greater than 350 mg/m3 for several years did not show increased incidence of cancer. COT 1996 stated that this study was only capable of detecting high risks of cancer because there were few cancer deaths, the sample was small, and the follow-up was short.

COT (1996) considered further

• studies of petroleum workers, ranging from refinery workers to service-station attendants, reported increases in cancer, but few studies reported on persons exposed only to jet-fuel vapours. Exposure to benzene appeared to be of consequence in many of the excesses found.

• long-term animal studies involving inhalation exposure to unleaded gasoline, kidney cancers were observed only in male rats. That finding raises the question of whether longer exposure of male rats to JP-5, JP-8, or DFM might also result in increased kidney cancers. However, the increased incidence of kidney cancer in male rats exposed to the gasoline was due to an α2u-globulin nephropathy—a lesion that apparently does not occur in humans, in other animals, or in female rats.

Based on the available human and animal data, COT (1996) concluded that inhalation of JP-5, JP-8, and DFM vapors does not present a carcinogenic risk to humans. That conclusion is supported by studies that show that these military fuels are not genotoxic. However, laboratory studies provided evidence of potential carcinogenicity of DFM (Diesel Fuel Marine) via the dermal route. Epidemiological studies show skin-cancer excesses in certain industrial workers, such as machine operators, whose skin might come into contact with lubricating oils derived from coal tar or petroleum. Exposure conditions in the studies that resulted in excessive skin damage are unlikely to occur on Navy ships.

6.9.2 2003 Assessment (COT 2003) Key considerations of COT (2003) include

• The carcinogenicity of JP-8 has not been investigated in epidemiologic studies.

• Chronic lifetime inhalation-exposure studies have not been conducted in experimental animals to determine the carcinogenicity of JP-8 or related jet fuels.

• No increase in the incidence of tumors was observed in 90-day continuous inhalation-exposure studies of JP-5 conducted in F344 rats and C57BL/6 mice (with a 19- or 21-mo observation period after cessation of exposure).

• Positive results of in vitro genotoxicity tests in cultured human and rat cell lines suggest that JP-8 has the potential to induce DNA damage; however, the genotoxicity of JP-8 has not been evaluated adequately in vivo.

• Among the FP-8 components on which carcinogenicity data are available, three chemicals (benzene, ethylbenzene, and naphthalene), which together make up 1% or less (volume/volume) of the fuel, are known to be carcinogenic. The carcinogenicity data available on mixtures similar to JP-8 (such as other jet fuels and MDFs) indicate that most of these materials induce skin tumors in mice when topically applied in excessive amounts. The mixtures have also been shown to have tumour-promoting but not tumour-initiating activity in the two-stage mouse skin tumour model. However, those carcinogenic effects are observed only under conditions of excessive skin irritation.

COT (2003) concluded that the available data at the time of the assessment were insufficient to draw a conclusion regarding the carcinogenicity of inhaled JP-8. However, because some studies showed that chronic dermal exposure to high doses of jet fuels or other petroleum products produces skin tumors, the subcommittee recommends that the Department of Defence (DOD) conduct lifetime carcinogenicity bioassays by the inhalation route in two animal species to determine whether JP-8 is carcinogenic via inhalation.

COT (2003) also recommended:

• that DOD follow a cohort of military personnel (including obtaining their exposure history) to determine whether exposure to JP-8 is associated with an increased incidence of various types of cancers.

• Air Force personnel engaged in particular jobs (such as fuel-cell workers) s dermally exposed to substantial amounts of JP-8 to wear appropriate protective clothing in order to avoid dermal exposures to JP-8.

6.9.3 Human data Parent et al. (2000) investigated carcinogenic effects of Jet Fuels in Humans. This population-based case-control study covered 142 male patients with renal cancer, 1,900 controls with other types of cancers, 533 population-based controls. The study reported an indication of excess risk of renal-cell carcinoma among aircraft mechanics and others with workplace exposures to jet fuel.

6.9.4 Animal data Nessel et al. (1998); Nessel et al. (1999) investigated carcinogenic effects of Jet Fuels (several middle distillates, including kerosene) in C3H, CD-1 mouse. Exposure to fuels was carried out using undiluted fuel or 50% or 28.6% dilutions in mineral oil. Exposure duration was 2 times/wk (undiluted), 7 times/wk (28.6%), and 4 times/wk (50%) for 52 wk or 2 yr following initiation with DMBA (dimethylbenzanthracene). Mice exposed to undiluted materials had significant increases in skin tumors, with incidence of 23-57%; exposure to diluted materials did not lead to increases in numbers of skin tumors.

6.9.5 Studies to be considered by SCOEL

2007: Harris et al., JP-8 jet fuel exposure potentiates tumor development in two experimental model systems, Toxicology and Industrial Health, 23 (9), pp. 545-552. Toxicology and Industrial Health, 23 (9), pp. 545-552. ABSTRACT: The US Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Previous work has reported that JP-8 exposure is immunosuppressive. Exposure of mice to JP-8 for 1 h/day resulted in immediate secretion of two immunosuppressive agents; namely, interleukin-10 (IL-10) and prostaglandin E2 (PGE2). Thus, it was of interest to determine if jet fuel exposure might promote tumor growth and metastasis. The syngeneic B16 tumor model was used for these studies. Animals were injected intravenously with tumor cells, and lung colonies were enumerated. Animals were also examined for metastatic spread of the tumor. Mice were either exposed to 1000 mg/m 3 JP-8 (1 h/day) for 7 days before tumor injection or were exposed to JP-8 at the time of tumor injection. All animals were killed 17 days after tumor injection. In the present study, JP8 exposure potentiated the growth and metastases of B16 tumors in an animal model. Exposure of mice to JP-8 for 1 h/day before tumor induction resulted in an approximately 8.7-fold increase in tumors, whereas those mice exposed to JP8 at the time of tumor induction had a 5.6-fold increase in tumor numbers. Thus, low concentration JP-8 jet fuel exposures have significant immune suppressive effects on the immune system that can result in increased tumor formation and metastases. We have now extended the observations to an experimental subcutaneous tumor model. JP8 exposure at the time of tumor induction in this model did not affect the growth of the tumor. However, JP8- exposed, tumor-bearing animals died at an accelerated rate as compared with air-exposed, tumor-bearing mice. © 2007 SAGE Publications.

6.10 Dermal effects and toxicity

Recent studies claim evidence of pronounced dermal effects. Such studies have not been considered systematically by COT (1996), (2003). It is proposed further consideration of that issue by the present scoping to SCOEL.

2003: Singh, S. et al., In vivo percutaneous absorption, skin barrier perturbation, and irritation from JP-8 jet fuel components, Drug and Chemical Toxicology, 26 (2), pp. 135- 146. ABSTRACT: JP-8 jet fuel has been reported to cause systemic and dermal toxicities in animal models and humans. There is a great potential for human exposure to JP-8. In this study, we determined percutaneous absorption and dermal toxicity of three components of JP-8 (i.e., xylene, heptane, and hexadecane) in vivo in weanling pigs. In vivo percutaneous absorption results suggest a greater absorption of hexadecane (0.43%) than xylene (0.17%) or heptane (0.14%) of the applied dose after 30 min exposure. Transepidermal water loss (TEWL) provides a robust method for assessing damage to the stratum corneum. Heptane showed greater increase in TEWL than the other two chemicals. No significant (p < 0.05) increase in temperature was observed at the chemically treated site than the control site. Heptane showed greater TEWL values and erythema score than other two chemicals (xylene and hexadecane). We did not observe any skin reactions or edema from these chemicals. Erythema was completely resolved after 24 h of the patch removal in case of xylene and hexadecane.

2004: Monteiro-Riviere et al., Skin toxicity of jet fuels: Ultrastructural studies and the effects of substance P, Toxicology and Applied Pharmacology, 195 (3), pp. 339-347. ABSTRACT: Topical exposure to jet fuel is a significant occupational hazard. Recent studies have focused on dermal absorption of fuel and its components, or alternatively, on the biochemical or immunotoxicological sequelae to exposure. Surprisingly, morphological and ultrastructural analyses have not been systematically conducted. Similarly, few studies have compared responses in skin to that of the primary target organ, the lung. The focus of the present investigation was 2-fold: first, to characterize the ultrastructural changes seen after topical exposure to moderate doses (335 or 67 μl/cm2) of jet fuels [Jet A, Jet Propellant (JP)-8, JP-8+100] for up to 4 days in pigs, and secondly, to determine if co-administration of substance P (SP) with JP-8 jet fuel in human epidermal keratinocyte cell cultures modulates toxicity as it does to pulmonary toxicity in laboratory animal studies. The primary change seen after exposure to all fuels was low-level inflammation accompanied by formation of lipid droplets in various skin layers, mitochondrial and nucleolar changes, cleft formation in the intercellular lipid lamellar bilayers, as well as disorganization in the stratum granulosum-stratum corneum interface. An increased number of Langerhans cells were also noted in jet fuel-treated skin. These changes suggest that the primary effect of jet fuel exposure is damage to the stratum corneum barrier. SP administration decreased the release of interleukin (IL)-8 normally seen in keratinocytes after JP-8 exposure, a response similar to that reported for SP's effect on JP-8 pulmonary toxicity. These studies provide a base upon which biochemical and immunological data collected in other model systems can be compared.

2004: Gallucci et al., JP-8 jet fuel exposure induces inflammatory cytokines in rat skin, International Immunopharmacology, 4 (9), pp. 1159-1169. ABSTRACT: The Department of Defense (DoD) has identified that one of the main complaints of personnel exposed to JP-8 jet fuel is irritant dermatitis. The purpose of this investigation is to describe the JP-8-induced inflammatory cytokine response in skin. JP-8 jet fuel or acetone control (300 μl) was applied to the denuded skin of rats once a day for 7 days. Skin samples from the exposed area were collected 2 and 24 h after the final exposure. Histological examination of skin biopsies showed neutrophilic inflammatory infiltrate. Reverse transcription-polymerase chain reaction (RT-PCR) was performed utilizing skin total RNA to examine the expression of various inflammatory cytokines. The CXC chemokine GROα was significantly upregulated at both time points, whereas GROβ was only increased 2 h post final exposure. The CC chemokines MCP-1, Mip-1α, and eotaxin were induced at both time points, whereas Mip-1β was induced only 24 h post exposure. Interleukins-1β and -6 (IL-1β and IL-6) mRNAs were significantly induced at both time points, while TNFα was not significantly different from control. Enzyme-linked immunosorbent assay (ELISA) of skin protein confirmed that MCP-1, TNFα, and IL-1β were modulated as indicated by PCR analysis. However, skin IL-6 protein content was not increased 2 h post exposure, whereas it was significantly upregulated by jet fuel after 24 h. Data from the present study indicate that repeated (7 days) JP-8 exposure induces numerous proinflammatory cytokines in

skin. The increased expression of these cytokines and chemokines may lead to increased inflammatory infiltrate in exposed skin, resulting in JP-8-induced irritant dermatitis.

2004: McDougal and Rogers, The cytotoxicity of volatile JP-8 jet fuel components in keratinocytes, Toxicology, 197 (2), pp. 113-121. ABSTRACT: Jet propellant-8 (JP-8) jet fuel is a version of commercial jet fuel, Jet A, and is a complex mixture of primarily aliphatic (but also aromatic) hydrocarbons that varies in composition from batch to batch. There is potential for dermal exposure to jet fuels with personnel involved in aircraft refueling and maintenance operations as well as ground personnel. Cutaneous exposures have the potential to cause skin irritation, sensitization or skin cancer. JP-8 has been shown to be irritating and causes molecular changes in the skin of laboratory animals. The mechanisms of some of these effects have been investigated in intact skin and cultured skin cells. Hydrocarbons have also been shown to cause skin cancer with repeated application to the skin. Additionally, there is concern about systemic toxicity from dermal exposures to jet fuels, such as JP-8. Assessing risks from systemic absorption of hydrocarbon components is complex because most of the components are present in the mixture in small quantities (less than 1%). The effect of the fuel as a vehicle, different rates of penetration through the skin and different target organ toxicities all complicate the assessment of the hazards of cutaneous exposures. The purpose of this manuscript is to review studies of local and systemic toxicity of JP-8.

2006: Chatterjee et al., In vitro and in vivo comparison of dermal irritancy of jet fuel exposure using EpiDerm™ (EPI-200) cultured human skin and hairless rats, Toxicology Letters, 167 (2), pp. 85-94. ABSTRACT: The purpose of this study was to evaluate an in vitro EpiDerm™ human skin model (EPI-200) to study the irritation potential of jet fuels (JP-8 and JP-8+100). Parallel in vivo studies on hairless rats on the dermal irritancy of jet fuels were also conducted. Cytokines are an important part of an irritation and inflammatory cascade, which are expressed in upon dermal exposures of irritant chemicals even when there are no obvious visible marks of irritation on the skin. We have chosen two primary cytokines (IL-1α and TNF-1α) as markers of irritation response of jet fuels. Initially, the EPI-200 was treated with different quantities of JP-8 and JP-8+100 to determine quantities which did not cause significant cytotoxicity, as monitored using the MTT assay and paraffin embedded histological cross-sections. Volumes of 2.5-50 μl/tissue ( 4.0-78 μl/cm2) of JP-8 and JP- 8+100 showed a dose dependent loss of tissue viability and morphological alterations of the tissue. At a quantity of 1.25 μl/tissue ( 2.0 μl/cm2), no significant change in tissue viability or morphology was observed for exposure time extending to 48 h. Nonetheless, this dose induced significant increase in IL-1α and TNF-α release versus non-treated controls after 24 and 48 h. In addition, IL-1α release for JP-8+100 was significantly higher than that observed for JP-8, but TNF-α release after 48 h exposure to these two jet fuels was the same. These findings parallel in vivo studies on hairless rats, which indicated higher irritation levels due to JP-8+100 versus JP-8. In vivo, transepidermal water loss (TEWL) and IL-1α expression levels followed the order JP-8+100 > JP-8 > control. Further, in vivo TNF-α levels for JP-8 and JP-8+100 were also elevated but not significantly different from one another. In aggregate, these findings indicate that EPI-200 tissue model can be utilized as an alternative to the use of animals in evaluating dermal irritation.

2006: Leggat and Smith, Dermatitis and aircrew, Contact Dermatitis, 54 (1), pp. 1-4. ABSTRACT: Dermatitis is a common problem both in the workplace and in the general community. Airline personnel represent a novel occupational group as they are also exposed to a wide range of potential chemical irritants and other aggravating factors, such as low relative humidity and airborne pollutants. Common skin

irritants include dielectric fluids from electrodischarge machining, 'prepreg' materials and sealants in aircraft manufacture, kerosene and various jet-fuel components. Commercial jet fuel is a complex mixture of aliphatic and aromatic compounds, and there is potential for dermal exposure among refueling and maintenance crew. Low relative humidity appears to exacerbate dermatitis amongst aircrew, especially on longer flight durations. Pilots may also be exposed to additional skin irritants outside of the cabin environment, such as ethylene glycol, hydraulic fluid or jet fuel, all of which may be encountered during routine inspections of aircraft before and after flight. Given these factors, preventive measures must carefully consider the undoubted potential for contact with irritants and allergens, which may lead to dermatitis in airline personnel.

2007: McDougal et al., Gene expression and target tissue dose in the rat epidermis after brief JP-8 and JP-8 aromatic and aliphatic component exposures, Toxicological Sciences, 97 (2), pp. 569-581. ABSTRACT: The jet fuel jet propulsion fuel 8 (JP-8) has been shown to cause an inflammatory response in the skin, which is characterized histologically by erythema, edema, and hyperplasia. Studies in laboratory animal skin and cultured keratinocytes have identified a variety of changes in protein levels related to inflammation, oxidative damage, apoptosis, and cellular growth. Most of these studies have focused on prolonged exposures and subsequent effects. In an attempt to understand the earliest responses of the skin to JP-8, we have investigated changes in gene expression in the epidermis for up to 8 h after a 1-h cutaneous exposure in rats. After exposure, we separated the epidermis from the rest of the skin with a cryotome and isolated total mRNA. Gene expression was studied with microarray techniques, and changes from sham treatments were analyzed and characterized. We found consistent twofold increases in gene expression of 27 transcripts at 1, 4, and 8 h after the beginning of the 1-h exposure that were related primarily to structural proteins, cell signaling, inflammatory mediators, growth factors, and enzymes. Analysis of pathways changed showed that several signaling pathways were increased at 1 h and that the most significant changes at 8 h were in metabolic pathways, many of which were downregulated. These results confirm and expand many of the previous molecular studies with JP-8. Based on the 1-h changes in gene expression, we hypothesize that the trigger of the JP-8-induced, epidermal stress response is a physical disruption of osmotic, oxidative, and membrane stability which activates gene expression in the signaling pathways and results in the inflammatory, apoptotic, and growth responses that have been previously identified.

2010: Mallamapati et al., Evaluation of EpiDerm full thickness-300 (EFT-300) as an in vitro model for skin irritation: Studies on aliphatic hydrocarbons, Toxicology in Vitro, 24 (2), pp. 669-676. ABSTRACT: The aim of this study was to understand the skin irritation effects of saturated aliphatic hydrocarbons (HCs), C9-C16, found jet fuels using in vitro 3-dimensional EpiDerm full thickness-300 (EFT- 300) skin cultures. The EFT-300 cultures were treated with 2.5 μl of HCs and the culture medium and skin samples were collected at 24 and 48 h to measure the release of various inflammatory biomarkers (IL-1α, IL-6 and IL-8). To validate the in vitro results, in vivo skin irritation studies were carried out in hairless rats by measuring trans epidermal water loss (TEWL) and erythema following un-occlusive dermal exposure of HCs for 72 h. The MTT tissue viability assay results with the EFT-300 tissue show that 2.5 μl/tissue (≈4.1 μl/cm2) of the HCs did not induce any significant changes in the tissue viability for exposure times up to 48 h of exposure. Microscopic observation of the EFT-300 cross-sections indicated that there were no obvious changes in the tissue morphology of the samples at 24 h, but after 48 h of exposure, tridecane, tetradecane and hexadecane produced a slight thickening and disruption of stratum corneum. Dermal exposures of C12-C16 HCs for 24 h significantly increased the expression of IL-1α in the skin as well as in the culture medium. Similarly, dermal exposure of all HCs for 24 h significantly increased the expression of interleukin-6 (IL-6) and IL-8 in the skin as well as in the culture medium in proportion to the HC chain length. As the exposure time increased to 48 h, IL-6 concentrations increased 2-fold compared to the IL-6 values at 24 h. The in vivo skin irritation data also showed that both TEWL and erythema scores increased with increased HCs chain length (C9-C16). In conclusion, the EFT-300 showed that the skin irritation profile of HCs was in the order of C9 ≤ C10 ≤ C11 ≤ C12 < C13 ≈ C14 ≈ C16 and that the tissue was an excellent in vitro model to predict in vivo irritation and to understand the structural activity relationship of HCs.

2010: Sharma and Locke, Jet fuel toxicity: Skin damage measured by 900-MHz MRI skin microscopy and visualization by 3D MR image processing, Magnetic Resonance Imaging, 28 (7), pp. 1030-1048. ABSTRACT: The toxicity of jet fuels was measured using noninvasive magnetic resonance microimaging (MRM) at 900-MHz magnetic field. The hypothesis was that MRM can visualize and measure the epidermis

exfoliation and hair follicle size of rat skin tissue due to toxic skin irritation after skin exposure to jet fuels. High- resolution 900-MHz MRM was used to measure the change in size of hair follicle, epidermis thickening and dermis in the skin after jet fuel exposure. A number of imaging techniques utilized included magnetization transfer contrast (MTC), spin-lattice relaxation constant (T1-weighting), combination of T2-weighting with magnetic field inhomogeneity (T2*-weighting), magnetization transfer weighting, diffusion tensor weighting and chemical shift weighting. These techniques were used to obtain 2D slices and 3D multislice-multiecho images with high-contrast resolution and high magnetic resonance signal with better skin details. The segmented color- coded feature spaces after image processing of the epidermis and hair follicle structures were used to compare the toxic exposure to tetradecane, dodecane, hexadecane and JP-8 jet fuels. Jet fuel exposure caused skin damage (erythema) at high temperature in addition to chemical intoxication. Erythema scores of the skin were distinct for jet fuels. The multicontrast enhancement at optimized TE and TR parameters generated high MRM signal of different skin structures. The multiple contrast approach made visible details of skin structures by combining specific information achieved from each of the microimaging techniques. At short echo time, MRM images and digitized histological sections confirmed exfoliated epidermis, dermis thickening and hair follicle atrophy after exposure to jet fuels. MRM data showed correlation with the histopathology data for epidermis thickness (R2=0.9052, P<0002) and hair root area (R2=0.88, P<0002). The toxicity of jet fuels on skin structures was in the order of tetradecane>hexadecane>dodecane. The method showed a sensitivity of 87.5% and a specificity of 75%. By MR image processing, different color-coded skin structures were extracted and 3D shapes of the epidermis and hair follicle size were compared. In conclusion, high-resolution MRM measured the change in skin epidermis and hair follicle size due to toxicity of jet fuels. MRM offers a three-dimensional spatial visualization of the change in skin structures as a method of toxicity evaluation and for comparison of jet fuels.

2010: Kezic et al., Review of dermal effects and uptake of petroleum hydrocarbons, CONCAWE Reports, (5), 166. ABSTRACT: The extent of dermal absorption of petroleum hydrocarbons was studied. The experimentally determined absorption from aqueous solutions were several orders of magnitude higher than the absorption after dermal exposure to either a neat chemical or a petroleum product such as a jet fuel. Dermal exposure to petroleum hydrocarbons, even following long-term exposures, e.g., in occupational settings, will not cause systemic toxicity under normal working conditions and assuming an intact skin barrier. Skin contact with some petroleum products may cause skin irritation, resulting in dermatitis, particularly after repeated or prolonged exposure. The skin barrier function may be affected following repeated contact with petroleum hydrocarbons, making the skin potentially more susceptible to other irritants, sensitizing agents, and bacteria. The impaired skin barrier may lead to increased dermal penetration of hydrocarbons and other substances.

7. CONCLUSIONS

• Naphthalene • Benzene, toluene, ethylbenzene, m-lp xylene, o-xylene (BTEX). • N-hexane • Aerosols and combustion products

Based on the analysis of the OELs set in the USA by COT (1996), COT (2003) and the preliminary consideration of the literature that has emerged since 2003, the following options and/or alternative scenarios are proposed for further action by SCOEL:

• Adopt a similar approach as COT (1996), (2003) and update the OELs' level according to the findings of the recent literature. • Establish health based biological limit values (BLVs) in the light of recent breakthroughs in biomonitoring outlined in Chapter 5.5.3. • Assign a skin notation, in the light of recent breakthroughs in dermal exposure assessment and evaluation of related health effects. • To distinguish OELs related to vapours and aerosols. • To consider particular action for individual chemicals, of major concern, such as naphthalenes.

Furthermore, SCOEL and DG EMPL decided to deal with combustion products from aviation fuels in the frame of a separate scoping study.

REFERENCES

ACGIH, 2002. ‘Threshold Limit Values and Biological Exposure Indices’, American Conference of Governmental Industrial Hygienists, Cincinnati, Ohio.

Allen, D.C., J.E. Riviere, and N.A. Monteiro-Riviere. 2000. 'Identification of early biomarkers of inflammation produced by keratinocytes exposed to jet fuels Jet A, JP-8, and JP-8(100)'. J. Biochem. Mol. Toxicol. 14(5):231-237.

Anger, W.K., and D. Storzbach. 2001. 'Results and discussion -neurobehavioral -interim report', JP8 Final Risk Assessment. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX pp. 65-67.

ATSDR, 1995.'Hazardous Substances Emergency Events Surveillance (HSEES)', US Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA.

ATSDR, 1997. 'Hazardous Substances Emergency Events Surveillance (HSEES)', US Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA.

ATSDR, 1999, 2000. 'Toxicological Profile for Total Petroleum Hydrocarbons', US Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA.

ATSDR, 1998. ‘Toxicological Profile for Jet Fuels (JP-5 and JP-8)’, U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA.

ATSDR, 2004. ‘Interaction Profile for: Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX)’, U.S. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry, Atlanta, GA.

ASTM D1655, 'Standard Specification for Aviation Turbine Fuels'.

B'Hymer, C., Mathias, P., Krieg Jr., E., Cheever, K.L., Toennis, C.A., Clark, J.C., Kesner, J.S., Gibson, R.L., Butler, M.A., '(2-Methoxyethoxy)acetic acid: a urinary biomarker of exposure for jet fuel JP-8', International Archives of Occupational and Environmental Health, pp. 1-8.

Baldwin, C.M., F.P. Houston, M.N. Podgornik, R.S. Young, C.A. Barnes, and M.L. Witten. 2001. 'Effects of aerosol-vapor JP-8 jet fuel on the functional observational battery, and learning and memory in the rat', Arch. Environ. Health 56(3):216-226.

Bhattacharya, A. 2001. 'Postural balance measurements. Risk assessment of acute exposure to jet fuel'. JP8 Final Risk Assessment. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX. pp. 72-75

Bekkedal, M.Y.V., S.M. McInturf, G.D. Ritchie, and J. Rossi III. 2001. 'Eyeblink conditioning response test used to assess performance in JP-8 exposed air force personnel', JP8 Final Risk Assessment. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX, pp. 69-71

Bruner, R.H., E.R. Kinkead, T.P. O’Neill, C.D. Fleming, D.R. Mattie, C.A. Russell, and H.G. Wall. 1993. 'The toxicologic and oncogenic potential of JP-4 jet fuel vapors in rats and mice: 12-month intermittent inhalation exposures', Fundam. Appl. Toxicol. 20(1):97-110.

Butler, M.A., C.A. Flugel, E.F. Krieg, J.E. Snawder, and J.S. Kesner. 2001. 'Gene- environment interactions and exposure to JP8 jet fuel', JP8 Final Risk Assessment. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX, pp. 76- 80.

Carlton, G.N., Smith, L.B., 2000. ‘Exposures to jet fuel and benzene during aircraft fuel tank repair in the U.S. Air Force’, Applied Occupational and Environmental Hygiene, 15 (6), pp. 485-491.

Carpenter, C.P., D.L. Geary Jr., R.C. Myers, D.J. Nachreiner, L.J. Sullivan, and J.M. King. 1976. 'Petroleum hydrocarbon toxicity studies'. XI. Animal and human response to vapors of deodorized kerosene. Toxicol. Appl. Pharmacol. 36(3):443-456.

Chao, Y.-C.E. , Gibson, R.L., Nylander-French, L.A., 2005. 'Dermal exposure to jet fuel (JP- 8) in US Air Force personnel', Annals of Occupational Hygiene, 49 (7), pp. 639- 645.

Chao, Y.-C.E., Nylander-French, L.A., 2004. 'Determination of Keratin Protein in a Tape- stripped Skin Sample from Jet Fuel Exposed Skin', Annals of Occupational Hygiene, 48 (1), pp. 65-73.

Chao, Y.-C.E., Gibson, R.L., Nylander-French, L.A., 2006. 'Dermal exposure to jet fuel (JP- 8) in US Air Force personnel', Annals of Occupational Hygiene, 49 (7), pp. 639- 645.

Chou, C.-C., Yang, J.-H., Chen, S.-D., Monteiro-Riviere, N.A., Li, H.-N.e , Chen, J.J.W., 2006. 'Expression profiling of human epidermal keratinocyte response following 1- minute JP-8 exposure', Cutaneous and Ocular Toxicology, 25 (2), pp. 141-153.

Cooper, J.R., and D.R. Mattie. 1996. 'Developmental toxicity of JP-8 jet fuel in the rat'. J. Appl. Toxicol. 16(3):197-200.

COT, 1996. ‘Permissible Exposure Levels for Selected Military Fuel Vapors’, National Research Council, Committee on Toxicology, Washington, DC: National Academy Press.

COT, 2001. 'Evaluating Chemical and Other Agent Exposures for Reproductive and Developmental Toxicity'. Washington, DC: National Academy Press.

COT 2003. The National Academies, Subcommittee of Jet-Propulsion Fuel 8, Committee on Toxicology, National Research Council (NRC), 'Toxicologic Assessment of Jet- Propulsion Fuel 8, The National Academies Press, Washigton D.C., 2003.

CRC, 2006. 'World Sampling Program', Coordinating Research Council, Inc., CRC Report No. 647.

DEF STAN 91-91, 2008. 'Turbine Fuel, Aviation Kerosine Type, Jet A-1', Ministry of Defence, Defence Standard 91-91, CAA.

Dudley, A.C., M.M. Peden-Adams, J. EuDaly, R.S. Pollenz, and D.E. Keil. 2001. 'An aryl hydrocarbon receptor independent mechanism of JP-8 jet fuel immunotoxicity in Ah-responsive and Ah-nonresponsive mice', Toxicol. Sci. 59(2):251-259.

Egeghy, P.P., Hauf-Cabalo, L., Gibson, R., Rappaport, S.M., 2003. 'Benzene and naphthalene in air and breath as indicators of exposure to jet fuel', Occupational and Environmental Medicine, 60 (12), pp. 969-976.

EPA US, 'Screening-level hazard characterization', Hazard Characterization Document, 2011.

EUROSTAT. http://epp.eurostat.ec.europa.eu/portal/page/portal/eurostat/home

ExxonMobil BS, 2001. ‘ExxonMobil Occupational Exposure Limits for Chemical Contaminants’. ExxonMobile Biomedical Sciences Inc., Annandale, New Jersey.

Fanning E., Yu R.C., Lu R., Froines J., 2007. Monitoring and modelling of ultrafine particles and black carbon at the Los Angeles international airport. Final report ARB Contract #04-325, University of California

Gibson, R.L., S. Shanklin, and R.L. Warner. 2001a. 'Health effects comparisons', JP-8 Final Risk Assessment Report. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX Pp. 125-129.

Gibson, R.L., S. Shanklin, and R.L. Warner. 2001b. 'Self-reported health status', JP-8 Final Risk Assessment Report. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX, pp. 132-139.

Grant, G.M., K.M. Shaffer, W.Y. Kao, D.A. Stenger, and J.J. Pancrazio. 2000. Investigation of in vitro toxicity of jet fuels JP-8 and jet A. Drug Chem. Toxicol. 23(1):279-291.

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.

Gregg S. D., Campbell J. L, Fisher J.W and Barlett M. G., 'Methods for the characterization of Jet Propellent-8: vapor and aerosol', Biomed. Chromatogr. 21: 463-472, 2007.

Hays, A.M., G. Parliman, J.K. Pfaff, R.C. Lantz, J. Tinajero, B. Tollinger, J.N. Hall, and M.L. Witten. 1995. Changes in lung permeability correlate with lung histology in a chronic exposure model. Toxicol. Ind. Health 11(3):325-336.

Harris, D.T., D. Sakiestewa, R.F. Robledo, and M. Witten. 1997a. Immunotoxicological effects of JP-8 jet fuel exposure. Toxicol. Ind. Health 13(1):43-55.

Harris, D.T., D. Sakiestewa, R.F. Robledo, and M. Witten. 1997b. Short-term exposure to JP- 8 jet fuel results in long-term immunotoxicity. Toxicol. Ind. Health 13(5): 559-570.

Harris, D.T., D. Sakiestewa, R.F. Robledo, and M. Witten. 1997c. Protection from JP-8 jet fuel induced immunotoxicity by administration of aerosolized substance P. Toxicol. Ind. Health 13(5):571-588.

Harris, D.T., D. Sakiestewa, R.F. Robledo, and M. Witten. 2000. Effects of short-term JP-8 jet fuel exposure on cell-mediated immunity. Toxicol. Ind. Health 16(2):78-84.

Hsu, H.H., Adamkiewicz, G., Houseman E.A., Vallarino J., Melly J:S., The Relationship between Aviation Activities and Ultrafine Particulate Matter Concentrations Near a Mid-Sized Airport. Atmospheric Environment 50, pp. 328 – 337.

IARC, 1987. ‘IARC Monographs on the Evaluation of Carcinogenic Risks to Humans’, Vol. 1-42. Lyon: International Agency for Research on Cancer, World Health Organization.

IARC, 1989. ‘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.

IARC, 2000. ‘IARC Monographs on the Evaluation of Carcinogenic Risks to Humans’, Vol. 77. Lyon: International Agency for Research on Cancer, World Health Organization.

IARC, 2002. ‘IARC Monographs on the Evaluation of Carcinogenic Risks to Humans’, Vol. 82. Lyon: International Agency for Research on Cancer, World Health Organization.

IRIS, 2001. Integrated Risk Information System. http://www.epa.gov/iris/.

Kabbur, M.B., J.V. Rogers, P.G. Gunasekar, C.M. Garrett, K.T. Geiss, W.W. Brinkley, and J.N. McDougal. 2001. Effect of JP-8 jet fuel on molecular and biological parameters related to acute skin irritation. Toxicol. Appl. Pharmacol. 175(1):83-88.

Kang-Sickel, J.-C.C., Fox, D.D., Nam, T.-G., Jayaraj, K., Ball, L.M., French, J.E., Klapper, D.G., Gold, A., 'Nylander-French, L.A., S-arylcysteine-keratin adducts as biomarkers of human dermal exposure to aromatic hydrocarbons', Chemical Research in Toxicology (21), Issue 4, pp 852-858.

Kang-Sickel, J.-C.C., Stober, V.P., French, J.E., Nylander-French, L.A., 2010. 'Exposure to naphthalene induces naphthyl-keratin adducts in human epidermis in vitro and in vivo', Biomarkers, 15 (6), pp. 488-497.

Kang-Sickel, J.-C.C., Butler, M.A., Frame, L., Serdar, B., Chao, Y.-C.E., Egeghy, P., Rappaport, S.M., Toennis, C.A., Li, W., Borisova, T., French, J.E., Nylander- French, L.A., 2010 'The utility of naphthyl-keratin adducts as biomarkers for jet- fuel exposure', Biomarkers, 16 (7), pp. 590-599.

Keil, D.E., D.A. Warren, M.M. Peden-Adams, and J. EuDaly. 2001. The effects of JP-8 on immune function and thyroid hormone levels in B6C3F1 mice exposed in utero. Toxicologist 60(1):218.

Kim, D., Andersen, M.E., Nylander-French, L.A., 2006. 'Dermal absorption and penetration of jet fuel components in humans', Toxicology Letters, 165 (1), pp. 11-21.

Kim, D.a , Andersen, M.E., Chao, Y.-C.E., Egeghy, P.P., Rappaport, S.M., Nylander-French, L.A., 2007. 'PBTK modeling demonstrates contribution of dermal and inhalation exposure components to end-exhaled breath concentrations of naphthalene', Environmental Health Perspectives, 115 (6), pp. 894-901.

Knave, B., H.E. Persson, J.M. Goldberg, and P. Westerholm. 1976. Long-term Exposure to jet fuel: An investigation on occupationally exposed workers with special reference to the nervous system. Scand. J. Work Environ. Health 2(3):152-164.

Knave, B., B.A. Olson, S. Elofsson, F. Gamberale, A. Isaksson, P. Mindus, H.E. Persson, G. Struwe, A. Wennberg, and P. Westerholm. 1978. Long-term exposure to jet fuel. II. A cross-sectional epidemiologic investigation on occupationally exposed industrial workers with special reference to the nervous system. Scand. J. Work Environ. Health 4(1):19-45.

Knave, B., P. Mindus, and G. Struwe. 1979. Neurasthenic symptoms in workers occupationally exposed to jet fuel . Acta Psychiatr. Scand. 60(1):39-49.Mattie, D.R., C.L. Alden, T.K. Newell, C.L. Gaworski, and C.D. Flemming. 1991. A 90- day continuous vapor inhalation toxicity study of JP-8 jet fuel followed by 20 or 21 months of recovery in Fischer 344 rats and C57BL/6 mice. Toxicol. Pathol. 19(2):77-87.

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., D.M. Olsen, J.H. Yiin, J.E. Lockey, R. Shukla, S.G. Selevan, S.M. Schrader, G.P. Toth, D.P. Evenson, and G.B. Huszar. 1999. Male reproductive effects of solvent and fuel exposure during aircraft maintenance. Reprod. Toxicol. 13(3):155- 166.

McDougal, J.N., Pollard, D.L., Weisman, W., Garrett, C.M., Miller, T.E., 2000. 'Assessment of skin absorption and penetration of JP-8 jet fuel and its components', Toxicological Sciences, 55 (2), pp. 247-255.

McEwen, J.D., and E.H. Vernot. 1981. Toxic Hazards Research Unit Annual Technical Report. AMRL-TR-81-126. Aerospace Medical Research Laboratory, Wright- Patterson Air Force Base, Dayton, OH.

Mattie, D.R., C.L. Alden, T.K. Newell, C.L. Gaworski, and C.D. Flemming. 1991. A 90-day continuous vapor inhalation toxicity study of JP-8 jet fuel followed by 20 or 21 months of recovery in Fischer 344 rats and C57BL/6 mice. Toxicol. Pathol. 19(2):77-87.

Mattie, D.R., G.B. Marit, C.D. Flemming, and J.R. Cooper. 1995. The effects of JP-8 jet fuel on male Sprague-Dawley rats after a 90-day exposure by oral gavage. Toxicol. Ind. Health 11(4):423-435.

Mattie, D.R., G.B. Marit, J.R. Cooper, T.R. Sterner, and C.D. Flemming. 2000. Reproductive Effects of JP-8 Jet Fuel on Male and Female Sprague-Dawley Rats After Exposure by Oral Gavage. AFRL-HE-WP-TR-2000-0067. Human Effectiveness Directorate, Air Force Research Laboratory, Wright Patterson AFB, OH. March.

Mattorano, D.A., Kupper, L.L., Nylander-French, L.A., 2004. 'Estimating Dermal Exposure to Jet Fuel (Naphthalene) Using Adhesive Tape Strip Samples', Annals of Occupational Hygiene, 48 (2), pp. 139-146.

Morata, T.C., Dunn, D.E., Kretschmer, L.W., Lemasters, G.K., Keith, R.W, 1993. 'Effects of Occupational Exposure to Organic Solvents and Noise on Hearing', Scandinavian Journal of Work, Environment and Health 19 (4), pp. 245-254.

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., R.A. Priston, R.H. McKee, G. Cruzan, A.J. Riley, R. Hagemann, R.T. Plutnick, and B.J. Simpson. 1998. A comprehensive evaluation of the mechanism of skin tumorigenesis by straight-run and cracked petroleum middle distillates. Toxicol. Sci. 44(1):22-31.

Newton, P.E., S.V. Becker, and C.J. Hixon. 1991. Pulmonary function and particle deposition and clearance in rats after a 90-day exposure to shale-oil-derived jet fuel JP-4. Inhal. Toxicol. 3(2):195-210.

NIOSH, 1997. NIOSHPocket Guide to Chemical Hazards. DHHS (NIOSH) 97-140. U.S. Dept. of Health and Human Services, Public Health Service, Centres for Disease Control and Prevention, National Institute for Occupational Safety and Health, Cincinnati, OH.

Nordholm, A.F., J. Rossi III, G.D. Ritchie, S. McInturf, M.E. Hulme, C. McCool, L. Narayanan, K.L. MacMahon, J. Eggers, H.F. Leahy, and R.E. Wolfe. 1999. Repeated exposure of rats to JP-4 vapor induces changes in neurobehavioral capacity and 5HT/5-HIAA levels. J. Toxicol. Environ. Health 56(7):471-499.

Nygren E., Aleklett K., Hook M. 2009. Aviation fuel and future oil production scenarios. Energy Policy 37(10): 4003–4010.

Odkvist, L., Larsby, B., Tham, R., Hyden, D., 1986. 'Vestibulo-oculomotor disturbances caused by industrial solvents', Otolaryngology - Head and Neck Surgery, (91), 5,, Pages 537-539.

Olsen, D.M., Mattie, D.R., Gould, William D., Witzmann, F., Ledbetter, M., 1998. ‘A Pilot Study of Occupational Assessment of Air Force Personnel Exposure to Jet Fuel Before and After Conversion to JP-8’, Air Force Research Lab Wright-Patterson AFB OH Human Effectiveness Directorate.

OSHA 29 CFR 1910.1000, 1997. ‘Title 29 – Labor’, (Revision 1999) Code of Federal Regulations, US Government Printing Office.

Parent, M.E., Y. Hua, and J. Siemiatycki. 2000. Occupational risk factors for renal cell carcinoma in Montreal. Am. J. Ind. Med. 38(6):609-618.

Parker, G.A., V. Bogo, and R.W. Young. 1981. Acute toxicity of conventional versus shale- derived JP5 jet fuel: Light microscopic, hematologic, and serum chemistry studies. Toxicol. Appl. Pharmacol. 57(3):302-317.

Parton, K.H. 1994. The Effects of JP-8 Jet Fuel Inhalation on Liver and Kidney Function in Male F-344 Rats. M.S. Thesis, University of Arizona. 76pp.

Pfaff, J., K. Parton, R.C. Lantz, H. Chen, A.M. Hays, and M.L. Witten. 1995. Inhalation exposure to JP-8 jet fuel alters pulmonary function and substance P levels in Fischer 344 rats. J. Appl. Toxicol. 15(4):249-256.

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.

Pleil, J.D., Smith, L.B., Zelnick, S.D., 2000. ‘Personal exposure to JP-8 jet fuel vapors and exhaust at air force bases’, Environmental Health Perspectives, 108 (3), pp. 183- 192.

Price, W.A., G.B. Briggs, K.A. Grasman, and K.R. Still. 2001. Evaluation of reproductive toxicity from exposure of male rats to jet propulsion fuel JP-8 vapor. Toxicologist 60(1):251(1194).

Proctor, P.S., Heaton, J.K., Smith, W.K., Rodrigues, R.E., Widing, E.D., Herrick. R, Vasterling, J.J., McClean, D.M., 'The Occupational JP8 Neuroepidemiology Study (OJENES): Repeated workday exposure and central nervous system functioning among US Air Force personnel', Neurotoxicology 32, 799-808, 2011.

Puhala , E., Lemasters, G., Smith, L., Talaska, G., Simpson, S., Joyce, J., Trinh, K., Lu, J., 1997. 'Jet fuel exposure in the United States Air Force', Applied Occupational and Environmental Hygiene (12), 9, pp. 606-610.

Rhodes, A.G., G.K. LeMasters, J.E. Lockey, J.W. Smith, J.H. Yiin, R. Gibson, and S. Rappaport. 2001. The effects of JP8 jet fuel on immune cell counts of tank entry workers. Pp. 100-120 in JP-8 Final Risk Assessment Report. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX. August 2001.

Ritchie, G.D., K.R. Still, W.K. Alexander, A.F. Nordholm, C.L. Wilson, J. Rossi III and D.R. Mattie. 2001a. A review of the neurotoxicity risk of selected hydrocarbon fuels. J. Toxicol. Environ. Health Part B Crit. Rev. 4(3):223-312.

Ritchie, G.D., J. Rossi III, A.F. Nordholm, K.R. Still, R.L. Carpenter, G.R. Wenger, and D.W. Wright. 2001b. Effects of repeated exposure to JP-8 jet fuel vapor on learning of simple and difficult operant tasks by rats. J. Toxicol. Environ. Health Part A 64(5):385-415.

Ritchie, G.D., G.R. Wenger, M.Y.V. Bekkedal, R.L. Carpenter, D. Wright, A.F. Nordholm, and J. Rossi III. 2000. Long-term effects of repeated exposure to JP-8 fuel vapor on higher cognitive capacity in rats. Soc. Neurosci. Abstr. 26:263.

Ritchie, G.D., Still, K., Rossi, J. 3rd , 2003. Biological and Health Effects of Exposure to Kerosene-Based Jet Fuels and Performance Additives. J. Environ. Health B. Crit. Rev., pp. 357-451.

Robledo, R.F., and M.L. Witten. 1998. Acute pulmonary response to inhaled JP-8 jet fuel aerosol in mice. Inhal. Toxicol. 10(5):531-553.

Robledo, R.F., R.S. Young, R.C. Lantz, and M.L. Witten. 2000. Short-term pulmonary response to inhaled JP-8 jet fuel aerosol in mice. Toxicol. Pathol. 28(5):656-663.

Rosenthal, D.S., C.M. Simbulan-Rosenthal, W.F. Liu, B.A. Stoica, and M.E. Smulson. 2001. Mechanisms of JP-8 jet fuel cell toxicity. II. Induction of necrosis in skin fibroblasts and keratinocytes and modulation of levels of Bcl-2 family members. Toxicol. Appl. Pharmcol. 171(2):107-116.

Rossi, J., A.F. Nordholm, R.L. Carpenter, G.D. Ritchie, and W. Malcolm. 2001. Effects of repeated exposure of rats to JP-5 or JP-8 jet fuel vapor on neurobehavioral capacity and neurotransmitter levels . J. Toxicol. Environ. Health A 63(6):397-428.

Schreiner, C., Q. Bui, R. Breglia, D. Burnett, F. Koschier, P. Podhasky, L. Lapadula, R. White, M. Feuston, A. Kruegger, and S. Rodriquez. 1997. Toxicity evaluation of petroleum blending streams: Reproductive and developmental effects of hydrodesulfurized kerosene. J. Toxicol. Environ. Health 52(3):211-229.

Serdar, B., Egeghy, P.P., Waidyanatha, S., Gibson, R., Rappaport, S.M., 2003. 'Urinary biomarkers of exposure to jet fuel (JP-8)', Environmental Health Perspectives, 111 (14), pp. 1760-1764.

Serdar, B., Egeghy, P.P., Gibson, R., Rappaport, S.M., 2004. ‘Dose-dependent production of urinary naphthols among workers exposed to jet fuel (JP-8)’, American Journal of Industrial Medicine, 46 (3), pp. 234-244.

Shell Material Safety Data Sheet, 'AVGAS', 'JetA-1', 2010.

Smith, K.W., Proctor, S.P., Ozonoff, A., McClean, M.D., 'Inhalation exposure to jet fuel (JP8) among U.S. Air Force personnel', Journal of occupational and environmental hygiene, 7 (10), pp. 563-572.

Smith, K. W., Proctor, S.P., Ozonoff, A., McClean, M.D., 2012. 'Urinary Biomarkers of Occupational Jet Duel Exposure among Air Force Personnel', Journal of Exposure Science and Environmental Epidemiology 22, pp. 35-45.

Snawder, J.E., and M.A. Butler. 2001. Sensitive early indicators of hepatic and kidney damage in workers exposed to jet fuel. Pp. 81-86 in JP-8 Final Risk Assessment Report. The Institute of Environmental and Human Health (TIEHH), Lubbock, TX . August 2001.

The Danish Ecocouncil, 2012. Air pollution in airports: Ultrafine particles, solutions and successful cooperation. http://www.google.com/url?sa=t&rct=j&q=the%20danish%20ecocouncil%20air%2 0pollution%20in%20airports&source=web&cd=1&ved=0CFEQFjAA&url=http%3 A%2F%2Fwww.ecocouncil.dk%2Findex.php%3Foption%3Dcom_docman%26task %3Ddoc_download%26gid%3D990%26Itemid%3D990&ei=hyIIUL6jCMWA4gS- 6KTJBA&usg=AFQjCNGpwsvVQYv4pDU7nS29l8ucYms99g&cad=rja

Todd, G.R.G., and B. Buick. 2000. Asthma due to kerosine exposure: Three case reports. Int. J. Occup. Med. Environ. Health 13(1):23-25.

Tu, R.H., Mitchell, C.S.a , Kay, G.G., Risby, T.H., 2004. 'Human Exposure to the Jet Fuel, JP-8' Aviation Space and Environmental Medicine, 75 (1), pp. 49-59.

Tunnicliffe, W.S., S.P. O’Hickey, T.J. Fletcher, J.F. Miles, P.S. Burges, and J.G. Ayres. 1999. Pulmonary function and respiratory symptoms in a population of airport workers. Occup. Environ. Med. 56(2):118-123.

Ullrich, S.E. 1999. Dermal application of JP-8 jet fuel induces immune suppression. Toxicol. Sci. 52(1):61-67.

Ullrich, S.E., and H.J. Lyons. 2000. Mechanisms involved in the immunotoxicity induced by dermal application of JP-8 jet fuel. Toxicol. Sci. 58(2):290-298.

U.S. Department of the Air Force. 2001. Sensory Irritation Study in Mice. Final Report. Project Number 162951. Test Substance: JP-4( MRD-00-629), JP-8(MRD-00-630), JP-8+100 (MRD-00-631). Prepared by ExxonMobil Biomedical Sciences, Inc., Annandale, NJ, for the U.S. Department of the Air Force, Brooks Air Force Base, TX.

Wall, H.G., A. Vingegar, and E.R. Kinkead. 1990. Evaluation of Toxic Effects in Rats and Mice Exposed to JP-4 Vapor for One Year. Toxic Hazards Research Unit Annual Technical Report. AMRL-TR-90-063. Aerospace Medical Research Laboratory, Wright-Patterson Air Force Base, Dayton, OH.

Wang, S.J., R.S. Young, and R.L. Witten. 2001. Age-related differences in pulmonary inflammatory responses to JP-8 jet fuel aerosol inhalation. Toxicol. Ind. Health 17(1):23-29.

Westerdahl, D., 2008. The Los Angeles international airport as a source of ultrafine particles and other pollutants to nearby communities. Atmospheric Environment 42(13), pp. 3143-3155.

Witten L. M., Zeiger E., Ritchie G. D., 'Jet Fuel Toxicology', CRC Press, Taylor & Francis Group, New York, 2011.

Witzmann, F.A., M.D. Bauer, A.M. Fieno, R.A. Grant, T.W. Keough, S.E. Kornguth, M.P. Lacey, F.L. Siegel, Y. Sun, L.S. Wright, R.S. Young, and M.L. Witten. 1999. Proteomic analysis of simulated occupational jet fuel exposure in the lung. Electrophoresis 20(18):3659-3669.

Witzmann, F. A., M.D. Bauer, A.M. Fieno, R.A. Grant, T.W. Keough, M.P. Lacey, Y. Sun, M.L. Whiten, and R.S. Young. 2000a. Proteomic analysis of the renal effects of simulated occupational jet fuel exposure. Electrophoresis 21(5):976-984.

Witzmann, F.A., R.L. Carpenter, G.D. Ritchie, C.L. Wilson, A.F. Nordholm, and J. Rossi III. 2000. Toxicity of chemical mixtures: Proteomic analysis of persisting liver and kidney protein alterations induced by repeated exposure of rats to JP-8 jet fuel vapor. Electrophoresis 21(11):2138-2147.

Yeung, P., Rogers, A., Davies, B., 1997. 'Safe working in aircraft fuel tanks: An Australian experience', Applied Occupational and Environmental Hygiene (12), 9, Pages 587- 594.