Executive Summary

Overview

The main objective of this project was to investigate the toxicity of JP-8 fuel and the solvents used in the Deseal/Reseal programme using a systems biology approach. In the exposure environment (fuel tanks, aircraft hangers etc), workers were typically exposed either by inhalation of vapours or by absorption through the skin. There were occasionally reports of direct ingestion through the mouth. Health studies of exposed workers and other research reports show premature death for some individuals, an increased risk of unusual malignancies in internal organs such as small bowel, erectile dysfunction, and behavioural disturbances. These findings may manifest years after exposure suggesting changes to the cells and tissues not directly exposed to the fuel and solvents. Changes to the systems biology was investigated by proteomic and genomic studies.

Laboratory cell studies of DeSeal/ReSeal compounds

Development of Cell exposure model

Previous methods for studying cellular responses to JP8 and solvents involved direct addition of these compounds to cells in laboratory growth plates using other solvents such as Ethanol. These methods were considered to be inadequate because they did not recognise the role of circulating blood plasma in distributing these compounds to internal organs. The JFES project team developed a method of studying cells by exposing them to blood plasma, which they believe is a better model of the inhalation and skin exposure routes for distributing solvents to internal organs. This method has been published in a peer reviewed journal. (See Appendix 1)

Distribution of JP8 and DeSeal/ReSeal solvents

The studies of plasma exposed to JP8 and solvents showed that the compounds are not distributed by plasma in the same proportions as found in the fuel and solvent mixtures. This means that higher levels of some compounds are actually presented to cells and organs than those proportions in the fuel solvent mixtures. The study showed that the majority of the compounds are distributed by binding to plasma lipids rather than simply dissolved in the plasma water. This raises the possibility that individuals with higher bloods lipids may distribute more of the compounds to internal organs.

The effects of the JP8 and solvents on cells

The study then tested the effects of the JP8 and solvents on cells. The JP8
and solvents were tested as a mixture and individually. The key findings
were:-

• Plasma exposed to JP8 alone is directly toxic to cells
• Plasma exposed to a mixture of JP8 combined with solvents has greater
  toxicity to cells with 40% cells showing changes before 4 hours, and 90%
  cells affected at 12 hours.

The following individual components were found to have the highest cellular toxicity:-

• Kerosene
• Benzene and butylbenzene
• All Alkanes including iso-octane, decane, dodecane, tetradecane and
  hexadecane
• Diegme
• N, N Dimethyl acetimide
• Naptha
• Thiophenol

The solvents used in the Deseal/Reseal programme demonstrated either low cell toxicity or manifest toxicity to a lesser extent than the JP8 fuel components.

Effects on gene expression

Gene expression in cells was altered following exposure. Changes greater then 5 fold were considered significant. The genes altered are shown in table (3). The function of these genes involved mostly cell survival/death, metabolism, cell cycle, DNA maintenance (housekeeping), and cell regulation. These genes have been implicated in pathological processes including cancer, neurodegeneration, and immune suppression.

Effects on proteins

Cellular proteins were altered after exposure. The changes to cellular proteins reflected the changes in gene expression involving cell survival/death, metabolism, cell division, and roles in cellular gene transcription/translation.

Cell Death

Cell death occurred by two mechanisms. A number of cells appeared more vulnerable with death occurring by disruption of cellular membranes and by lysis (bursting) of the cells. The more common mechanism of cell death was by apoptosis, which is a programmed response of cells to injury. Not all injured cells undergo complete apoptosis indicating persistence of injured cells. This may suggest a survival of injured cells with malignant potential. The cell culture methods could not determine the long term effects.

Study of exposed workers

The study of exposed workers showed differences from the matched control group in health indices, and in some genomic studies. The changes were not as significant as those seen in the acute cell exposure model in the laboratory.

Rating of exposure

Because of the unavailability of accurate exposure data (degree and duration), a problem also encountered in other studies, the workers were classified into 3 groups.

1. Definite high exposure who worked inside the fuel tanks
2. Significant contact such as by dosing of skin or accidental ingestion
3. Minimal contact in the general area such as collection of rags or
   cleaning of the area.

Health Assessment Scores

The Health assessment scores showed exposed workers to have a lower health rating than controls. There did not appear to be a decrease in the health scores (dose response) related to the degree of exposure. Workers with mild exposure had the same decrease in their health scores as those with high exposure. This suggests that other factors beyond the Deseal/ Reseal contact have decreased the health scores.

Genetic studies of blood cells from exposed workers

All studies were undertaken on plasma and white blood cells as these were
the only tissues for which it was possible to obtain samples. The genetic studies of blood cells examined two types of changes in gene expression, the presence of chromosomal changes, and for appearance of mutations in
the mitochondrial DNA. There were no chromosomal changes detected at a
level of 50Kb using a high resolution SNP ARRAY.

There were no changes in the mitochondrial DNA mutation load between exposed workers and age matched controls (Mitochondrial DNA changes can accumulate with age).

There were no changes in the amount or type of protein coding mRNA expression, which is an index of cell activity. In disease states , these are usually tissue specific and may not appear in blood cells unless they are directly involved in the disease process.

There were small but significant and consistent changes in the expression of regulatory microRNAs that control activity of other genes. The regulatory functions of the altered genes have been linked to neurological changes and neurodegenerative disorders. It must be emphasised that interpretation of the function of regulatory genes is an evolving science with much uncertainty at present. The regulatory genes, which compose 98% of our genome, have a major role in human development, adaptation and response to disease. The function is only known for ~40% of these at present. Disease causing associations, with some early exceptions, are still unmapped.

Protein studies of plasma and blood cells

No significant changes were seen in the levels and types of protein expressed in the plasma and blood cells of exposed workers. A few small changes were seen consistently, but these did not reach a level that the researchers considered significant.

Discussion and Conclusions

Confounders and sensitivity

Dose response not detected

A dose response would have been expected but was not observed in the workers with different exposure histories. The unexpected similarity in the health scores and genomic studies within the exposed groups (low, medium, high) raises several hypotheses:-

Confounders

There are other factors independent of Deseal/Reseal exposure which could produce the changes seen. Confounders could include:-

• An ascertainment bias whereby only those workers affected by any exposure volunteered to participate in the study.
• An ascertainment bias whereby only those workers NOT affected by the exposure (i.e. Survivors) volunteered to participate in the study.
• The workers were stratified by their exposure to Deseal/Reseal materials. The effects seen may NOT be due to the Deseal/Reseal materials but to some other experience of the workers. The cellular studies suggest that exposure to fuel alone could be responsible.
• It was not possible to examine other possible shared confounding events in the work careers or in the lifestyle of the personnel. (e.g. other occupational exposure not related to Deseal/Reseal such as medications, substance abuse, nutrition)
• This study was conducted on individuals between 10 and 30 years after their exposure. If significant changes occurred at the time of exposure, normal cellular repair and selection mechanisms may have lessened the biological signal that could be observed in this study. The small but consistent changes observed suggest this possibility. Either the effect at the time was minimal but has persisted, or the effect was larger but has diminished over-time.
• The cellular studies show that the compounds are mostly distributed by plasma lipids. The exposure to organs within the body would likely depend on the concentration of plasma lipids at the time of fuel exposure. Plasma lipids vary genetically between individuals, with lifestyle and alcohol intake, with composition of their diet, as well as the time after meals when the exposure occurred. The lack of a dose effect could be explained if workers in the lower exposure group had higher plasma lipids at the time of exposure. Individuals in the high exposure group worked within the fuel tanks and were selected because they were leaner and smaller, possibly protected to some extent by lower plasma lipids.

Significance of findings

The cellular findings, supported by other recently published genomic studies, indicate a definite toxicity from JP8 to exposed cells. The components of JP8 tested are commonly found in most (aviation) fuels. The results indicate that there is a need for concern about exposure to fuels in general. The study was not designed to determine the degree of occupational exposure necessary to produce cellular changes. However, the results show that cells grown in a nutrient containing as little as 5% exposed plasma are affected. In the body, blood cells have 100% exposure to plasma while other organs will have less exposure depending on the net blood flow and cellular membrane barriers. Organs such as brain, liver and bowel have very high blood flow. Cellular membranes generally have greater permeability to substances dissolved in lipids.

The study was also not designed to determine the most toxic routes of exposure (inhalation, ingestion, skin contact), but did demonstrate that fuel components can be distributed to organs through blood plasma, i.e. organs such as brain or liver, not directly exposed in the contact, may undergo secondary exposure. The implication is that all body systems must be considered in assessing/monitoring the health of exposed workers.

While the changes seen many years after exposure were small, they were consistent. The changes are most apparent in gene regulation and had some association to the health problems (e.g., malignancy) identified in other studies.

There were no chromosomal changes or mutations linked to the exposure. The genes changes seen can be described as Epigenetic, which is a mechanism of cellular adaptation to some environmental influence. Epigenetic changes are less clearly linked (at the present knowledge) to disease. Epigenetic changes occur through a variety of cellular mechanisms and these were not investigated in this study. Some epigenetic changes can be transferred down through successive generations but currently have not been shown to cause birth defects or mutation in off-spring.

Recommendations

The cell results show a definite cellular toxicity from JP8 fuel. The components of the fuel exhibiting toxicity are common to most fuels. Consideration should be given to further studies of workers exposed to fuel of any type.

Newer genomic and bioinformatic technologies have been developed during the time of this study and have been employed in other studies of occupational fuel exposure. These technologies can be applied to other exposure risks (including PTSD) in defence (veteran) health risk assessment. An expert committee should be constituted to advise on research and clinical application of these technologies.

Plasma free DNA sequencing can now be used to assess (from blood samples), the cellular death associated with tumours, transplant rejection, miscarriage and infections. Targeted RNA expression studies can reveal immediate changes in gene activity following fuel exposure. A study of workers with recent or past fuel exposure is recommended.

The best time to study cellular changes would be immediately after direct exposure. A protocol should be established for assessment of an exposed individual to include sample collection immediately after the exposure for quantification of plasma lipids, plasma fuel components, free DNA sequencing, and targeted RNA expression.

Exposed veterans should be reassured that while small and consistent changes were observed in this study, there were no changes detected known to have immediate or severe health consequences. The changes support the findings from other studies that there is a possible increased risk of developing health problems. As the changes observed are in gene regulation, it is also possible that healthy lifestyle changes may ameliorate the risk.

31st JULY 2014

Download the full report on the Royal Australian Air Force website below.

• http://www.airforce.gov.au/docs/123_1415_Report.pdf

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Difference between Jet A1 & JP8

Jet fuel, aviation turbine fuel (ATF), or avtur, is a type of aviation fuel designed for use in aircraft powered by gas-turbine engines. It is colorless to straw-colored in appearance. The most commonly used fuels for commercial aviation are Jet A and Jet A-1, which are produced to a standardized international specification. The only other jet fuel commonly used in civilian turbine-engine powered aviation is Jet B, which is used for its enhanced cold-weather performance.

Jet fuel is a mixture of a large number of different hydrocarbons. The range of their sizes (molecular weights or carbon numbers) is defined by the requirements for the product, such as the freezing or smoke point. Kerosene-type jet fuel (including Jet A and Jet A-1) has a carbon number distribution between about 8 and 16 (carbon atoms per molecule); wide-cut or naphtha-type jet fuel (including Jet B), between about 5 and 15.[1]

Additives

The DEF STAN 91-91 (UK) and ASTM D1655 (international) specifications allow for certain additives to be added to jet fuel, including:[13][14]

• 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 a component, 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.[15]
• 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.

As the aviation industry’s jet kerosene demands have increased to more than 5% of all refined products derived from crude, it has been necessary for the refiner to optimize the yield of jet kerosene, a high value product, by varying process techniques. New processes have allowed flexibility in the choice of crudes, the use of coal tar sands as a source of molecules and the manufacture of synthetic blend stocks. Due to the number and severity of the processes used, it is often necessary and sometimes mandatory to use additives. These additives may, for example, prevent the formation of harmful chemical species or improve a property of a fuel to prevent further engine wear.

• https://en.wikipedia.org/wiki/Jet_fuel

JP-8, or JP8 (for “Jet Propellant 8”) is a jet fuel, specified and used widely by the US military. It is specified by MIL-DTL-83133 and British Defence Standard 91-87, and similar to commercial aviation’s Jet A-1, but with the addition of corrosion inhibitor and anti-icing additives.

A kerosene-based fuel, JP-8 is projected to remain in use at least until 2025. It was first introduced at NATO bases in 1978. Its NATO code is F-34.

• https://en.wikipedia.org/wiki/JP-8