Appendices of the Draft Screening Assessment Petroleum Sector Stream Approach Aviation Fuels [Fuels] Chemical Abstracts Service Registry Numbers 64741-87-3 64741-86-2 68527-27-5 Environment Canada Health Canada April 2013

LOAEC (inhalation; mouse) = 50 mg/m³. Male mice (C57BL/6 and B6.A.D.; 12/strain/concentration group) were nose-only exposed to 0, 5, 12, 28, 50 and 113 mg/m³ JP-8 aerosols/vapours (generated using a nebulizer) for 1 hour. At 24–30 hours post-exposure, measurements of respiratory function, permeability and cellular injury were taken. Significantly increased respiratory permeability, concentration-dependent alveolar macrophage hyperplasia and infiltration, and significant mild to moderate microscopic and ultrastructural injury to the terminal bronchioles were noted at 50 mg/m³. The authors hypothesize that these are reversible effects (Robledo and Whitten 1998).

Other studies

Female C57Bl/6 mice were nose-only exposed to 1000 mg/m³ JP-8 aerosols for 1 hour. An immediate loss of immune function, accompanied by significant loss of viable immune cells and significant decreases in immune organ weights, were observed (Harris et al. 2002).

A 28-day unoccluded dermal study was conducted in female SD rats (10/dose). Groups were exposed to 0, 165, 330 or 495 mg/kg-bw/day of Jet A in mineral oil (positive control groups received cyclophosphamide and anti-asialo GM1). No immunotoxicity was identified in the test substance groups that included screening for spleen and thymus weights, IgM antibody-forming cell response to T-dependent antigen, splenic lymphocyte subpopulations and cell proliferative response to anti-CD3 antibody, natural killer cell activity and immune response to sheep red blood cells (Mann et al. 2008).

A 14-day dermal study was conducted using rabbits. Test substance was applied 5 times/week at 6400 mg/kg-bw/day. Depression, weight loss and severe skin damage at the application site was noted. Considered secondary to the skin damage was liver necrosis and kidney and bladder hyperplasia (API 1985a, 1985c).

JP-8

LOAEC (inhalation; mouse): 45 mg/m³.MaleC57BL/6 mice (12/exposure level; 6/control group) were nose-only exposed to JP-8 aerosols (5–15% of total) and vapours (85–95% of total) at an average concentration of 45, 267 and 406 mg/m³ for 1 hour/day for 7 days (daily exposures were within 10% of the listed averages). At all concentrations, generalized sloughing of the bronchiolar epithelium was seen, and various cellular changes were observed in alveolar type II epithelial cells, including increased number and size of surfactant-producing lamellar bodies; however, at the lower concentrations, lung function was not affected. At the highest concentration, a statistically significant 20% decrease in inspiratory dynamic lung compliance was observed (Herrin et al 2006).

Other inhalation studies

Groups of male B6.A.D. mice (12/concentration level) were nose-only exposed to JP-8 aerosols (5–15% of total) and vapours (85–95% of total) (generated with a nebulizer) at average concentrations of 0, 7, 12, 26, 48 and 118 mg/m³ for 1 hour/day for 7 days. Mice exposed to 48 mg/m³ exhibited increased respiratory permeability (as measured by the pulmonary clearance of intratracheally instilled ^99mTc-labelled diethylenetriaminepentaacetic acid), increased total protein in the bronchoalveolar lavage fluid and concentration-dependent morphological lung and alveolar injury (Robledo et al. 2000).

Male and female mice (C57BL/6 and B6.A.D.; 3–21/group) were nose-only exposed to 0, 100, 250, 500, 1000 and 2500 mg/m³ JP-8 vapours/aerosols (generated using a nebulizer) for 1 hour/day for 7 days. A concentration-dependent, significant loss of total viable cells from the thymus was seen for the group(s) exposed to 100 mg/m³. A statistically significant (p < 0.05) suppressive effect on splenic immune cell proliferation was also seen at this concentration. A statistically significant, concentration-dependent decrease in spleen and thymus weights was noted at the three highest concentrations. The authors reported that male and female mice were equally affected by exposure to JP-8, but they did not provide gender- or strain-specific data (Harris et al. 1997).

Female C57B1/6 mice were exposed by nose-only inhalation to 0 or 1000 mg/m³ aerosolized JP-8 for 1 hour/day for 7 days. A significant change in thymus cell subpopulations was reported in the exposed mice, as was a suppression of splenic cell immune function (Harris et al. 2000).

C57Bl/6 mice exhibited significant immunosuppression after exposure to 1000 mg/m³ JP-8 for 1 hour/day for 1 (Harris et al. 2002) to 7 days (Harris et al. 2008), and during gestation (Harris et al. 2007a). JP-8 exposure was shown to reduce the immune response to influenza viral infection, including decreased immune cell viability, and resulted in a greater than four-fold decrease in immune cell proliferative responses to mitogens and a loss of T cells from the lymph nodes (Harris et al. 2008). Immunotoxicity of JP-8 has been implicated as a mechanism for increasing the incidence and metastatsis of lung tumours, and decreased survival, in a melanoma B16 mouse tumour model (Harris et al. 2007b).

53 mg/m³: significant increase in inspiratory and expiratory lung resistance compared to controls in male C57BL/6 mice exposed via nose-only inhalation to vapour/aerosol of 0 and 53 mg/m³ JP-8 daily for 1 hour for 7 days. In addition, cell injury was noted in the Clara cells of the terminal bronchioles, and changes to type II epithelial cells were reported (Wong et al. 2008).

1000 mg/m³: significant differences in inflammatory response of young (3.5 month old) and adult (12 month old) male C57BL/6 mice to inhalation of 0 or 1000 mg/m³ aerosolized JP-8 daily for 1 hour for 7 days. Broncho alveolar lavage fluid (BALF) cell differential, tumour necrosis factor-α (TNF-α), 8-isoPGF2 levels were different between young and adult mice, where increased lung compliance, respiratory permeability, MIP-2 levels, as well as decreased PGE2 levels were reported similarities (Wang et al. 2001).

1023 mg/m³: significant increase in pulmonary vascular permeability, BALF SP levels in female C57BL/6 mice exposed via inhalation to 0 or 1023 mg/m³ aerosolized JP-8 for 1 hour for 7 days, compared to controls. Dilation of respiratory bronchioles and alveoli were also observed (Wong et al. 2004).

Male Long-Evans Rats were exposed via inhalation (nose-only) to 0, 500, 1000 or 2000 mg/m³ aerosolized JP-8 for 4 hours/day for 5 days. Following exposure, groups were exposed to 1 hour of noise, or to no noise. No ototoxicity was noted in rats exposed to JP-8 without subsequent noise (Fechter et al. 2010).

Male Long-Evans rats were exposed through inhalation (nose-only) to 1000 mg/m³ aerosolized JP-8 for 4 hours/day for 1 or 5 days. In addition, noise or no noise followed JP-8 treatment. No ototoxicity was noted after a single JP-8 exposure. Repeated exposure was reported to have an effect on outer hair cell function (decrease in distortion product otoacoustic emissions [DPOAE] amplitude); however, some recovery was noted 4 weeks after exposure. A significant decrease in liver glutathione levels was reported immediately after, and 1 hour following, exposure (Fechter et al. 2007).

JP-5 / JP-8

JP-8

SD rats were exposed to 0 or 1000 mg/m³ JP-8 vapour or 1200 mg/m³ JP-5 vapour for 6 hours/day, 5 days/week for 6 weeks. Significant changes in neurobehavioural capacity were noted, including significant changes to neurotransmitter levels, and testing results (appetitive reinforcer approach sensitization [ARAS], forelimb grip strength) from the Neurobehavioural Toxicity Assessment Battery (NTAB) (Rossi et al. 2001).

Male F344 rats were exposed via inhalation (nose-only) to control or the mean aerosolized JP-8 level of 1236.8 mg/m³ for 1 hour/day, 5 days/week for 28 days. Exposed mice were reported to have significant differences in spontaneous activity and central nervous system (CNS) excitability compared to controls, as well as more locomotive behaviour and faster swim speeds when conducting the functional observational battery (FOB) (Baldwin et al. 2001).

Male SD rats were exposed via whole-body inhalation to 0, 500 or 1000 mg/m³ JP-8 vapour for 6 hours/day, 5 days/week for 6 weeks. At the low concentration, treated rats exceeded control animals when learning and performing complex tasks. At the high-concentration level, deficits in learning and performance at moderate or difficult tasks were reported. Treated rats were also noted to have significantly higher neurotransmitter levels compared to control animals (Ritchie et al. 2001b).

In another study, SD rats were exposed to 1100 mg/m³ test substance vapours for 30 days. Significant polydipsia was noted in the exposed group relative to the control group
(Bogo et al 1984).

Other inhalation studies

Markedly increased hyaline droplets in kidney proximal tubular cells and dilated corticomedullary tubules (that were plugged with necrotic debris) were seen in almost all male Fischer 344 rats exposed to 150 or 750 mg/m³ petroleum and shale-derived JP-5 vapours (generated by heating the fuels to 50–57oC) for 24 hours/day for 90 days. Other effects noted included decreased growth rate of male rats, and statistically significant increases in blood urea nitrogen (BUN) and serum creatinine levels in high-concentration male and female rats. Animals were followed for 19 months post-exposure; exposure-related effects included concentration-dependent medullary intratubular mineralization, concentration-related focal hyperplasia of the renal pelvis, and increased severity of progressive renal nephropathy (tubule degeneration). The kidney effects may be mediated through a male rat-specific protein, α-2-microglobulin, and therefore the relevance of these effects to humans is questionable (Bruner 1984; Gaworski et al. 1984; MacNaughton and Uddin 1984).

Other inhalation studies

Male SD rats were whole-body exposed to heated JP-8 vapours at 0, 250, 500 and 1000 mg/m³ for 6 hours/day for 91 days. At the lowest concentration, concentration-dependent effects included mild damage to kidney proximal convoluted tubules, a 10% reduction in bone marrow fat cells/globules, and a low level of cell proliferation in the bone marrow. At the two highest concentrations, these effects were enhanced, and histological changes to the liver, bone marrow and heart damage, as well as enlargement of lung capillaries were noted (Hanas et al. 2010).

Male and female Fischer 344 rats (7–15/sex/group) and C57BL/6 mice (100/sex/group) were exposed continuously to 0, 500 or 1000 mg/m³ JP-8 vapour for 90 days. In mice, no effects were observed apart from necrotizing dermatitis due to fighting, which caused increased mortality, especially in males. In male rats, a significant decrease in body weight, increased absolute and relative kidney weight and increased basophilic foci in livers were noted at both concentration levels. In addition, renal effects consistent with chronic progressive nephrosis due to α-2-microglobulin were observed in male rats. This is a mechanism that may not be relevant to humans.
LOAEC: 500 mg/m³ as identified by U.S. EPA (2011) for decreased body weight and increased absolute and relative kidney weight in male rats (Mattie et al. 1991).

NOAEC (inhalation; rat): 400 ppm (2945 mg/m³)^[d]. Charles River CD rat dams were exposed to 100 and 400 ppm (736 and 2945 mg/m³) Jet-A for 6 hours/day on days 6–15 of gestation. No embryotoxic, fetotoxic or teratogenic effects were observed (Beliles and Mecler 1982).

NOAEC (inhalation; rat): 400 ppm (2945 mg/m³)^[d]. Groups of 20 SD rat dams wereexposed to 100 or 400 ppm (736 and 2945 mg/m³) of test substance vapour for 6 hours/day on gestation days 6–15. There was a slight increase in fetuses with retarded bone ossification in the high-concentration group, but these effects were not considered by the authors to be adverse. No other effects were noted (API 1979b).

In a different study (dominant lethal assay), exposure of male mice to Jet-A vapours at 100 or 400 ppm (736 and 2945 mg/m³)^[d] for 6 hours/day, 5 days/week for 8 weeks did not affect female reproductive parameters after mating, such as fertility index, number of implants and proportion of dead implantations (API 1980b).

Chronic health effects (non-carcinogenicity studies)

LOAEL (dermal; mouse): 250 mg/kg-bw/day. Male and female B6C3F1 mice (50/group) were exposed to JP-5 navy fuel* at 0, 250 or 500 mg/kg-bw/day in 0.1 mL acetone for 5 days/week for 103 weeks (90 weeks for high-dose females). A marked increase in the incidence of dermal ulceration, inflammation and epithelial hyperplasia were observed. High-dose males and females exhibited multiple organ amyloidosis, and high-dose females had approximately 50% decreased survival to 90 weeks relative to low-dose females at 105 weeks (17/50 vs. 33/50, respectively) (NTP 1986). *also referred to as CAS RN 8008-20-6 (kerosene) in the study

50 µL (1170 mg/kg-bw)^{[e], [i], [j]} of undiluted test substance (straight-run kerosene; sample API 83-09) was applied twice weekly to mice for periods ranging from 3–24 months. Some animals showed skin ulceration and one squamous cell carcinoma was found at 12 months. Also, chronic skin irritation, and increases in absolute and relative kidney, liver and lung weights, were reported (API 1986c).

Skin painting study
Undiluted test substance (API 81-08; 50 µL [970 mg/kg-bw])^{[e], [f], [g]} was applied to the shaved intrascapular skin of male C3H/HeJ mice (group of 50) twice/week for life. A non-statistically- significant increase in the incidence of squamous cell papillomas (4%) and carcinomas (2%) was noted (3/50 mice in the test substance group developed tumours). The toluene-only-exposed group had 4 mice with tumours with a squamous cell carcinoma incidence of 6% and fibrosarcoma incidence of 2%, and all mice (49/49) in the positive control group (0.05% w/v benzo[a]pyrene in toluene) developed tumours. Mean latency to tumour formation was 113 weeks in the test group, 111 weeks in the toluene-exposed group and 49 weeks in the positive control group (Skisak et al. 1994).

Skin painting study
Undiluted test substance (API 81-08; 50 µL [970 mg/kg-bw])^{[e], [f], [g]} was applied over at least 1cm₂ to the clipped intrascapular region of the backs of male C3H/HeJ mice (50/group) twice per week for life. After 31 months, 4 mice in the test group each had a benign tumour, while the negative control mice had no tumours, and 33 mice in the positive control group had tumours (14 benign and 19 malignant). Mean latency to tumour formation was 112 weeks for the test group and 84.5 weeks for the positive control group (API 1986b, 1986d).

Skin painting study
C3H male mice (a group of 47) were exposed twice weekly for 139 weeks to 50 µL (970 mg/kg-bw)^{[e], [f], [g]} test substance API 81-08. Benign skin tumours developed in 4% of test group mice (0% incidence in both the negative and solvent control groups). Malignant skin tumours developed in 2% of test group mice (0% and 8% for the control groups as above, respectively). Regarding benign and malignant tumours at other sites, 2% of mice in the test substance group had benign tumours (0% and 2% for control groups, respectively) and 4% had malignant tumours (2% and 0% for control groups, respectively). Using a Chi square test, it was determined that the test substance did not cause a statistically significant increase in tumours above that seen in the negative and solvent control groups (API 1989a).

Initiation study
Male CD-1 mice (30/group) were exposed to 50 µL (970 mg/kg-bw/day)^{[e], [f], [g]} of undiluted test substance for 5 consecutive days. After a 2-week rest period, 50 µL of the tumour promoter phorbol-12-myristate-13-acetate (PMA) was administered twice/week for 25 weeks. Both substances were applied to shaved dorsal intrascapular skin. There was no increased incidence of tumour formation in the test group (3/29 mice in the test group developed tumours (squamous cell papillomas) compared with 3/30 mice in the negative control group and 30/30 in the positive control group). Mean latency to tumour formation was 20 weeks (Skisak et al. 1994).

Promotion study
Male CD-1 mice (30/group) were exposed once to 50 µL of tumour initiator 7,12-dimethylbenzanthracene (DMBA). After 2 weeks, 50 µL (970 mg/kg-bw per day)^{[e], [f], [g]} of undiluted test substance was applied twice/week for 25 weeks. Both DMBA and test substance were applied to shaved dorsal intrascapular skin. No tumours formed in the test and negative control groups, whereas 30/30 mice in the positive control group developed tumours (Skisak et al. 1994).

Skin painting study
Male C3H/HeJ mice (50/group) were exposed to 50 mg test substance (1430 mg/kg-bw)^{[e], [h]} twice weekly for 80 weeks or until a papilloma larger than 1 mm³ appeared. Test substance was applied to the shaved interscapular region. In 2 test substance groups, 9 of 30 and 4 of 27 mice developed tumours with average latency periods of 70 and 62 weeks, respectively. The negative control groups consisted of shaved-only (four groups) or toluene-treated (7 groups) and, combined, 0 and 3 mice developed tumours within these groups, respectively (Blackburn et al. 1986).

Skin painting study
Male C3H/HeJ mice (50/group) were exposed to 100% test substance (1170 mg-kg/bw^{[e], [i], [j]} MD-3 twice/week, or 50% (580 mg-kg/bw) 4 times/week, or 28.5% (330 mg-kg/bw) 7 times/week (in 50 mL) for 104 weeks. A negative control group received 35 mL mineral oil 7 times/week. Substances were applied to the shorn dorsal skin. Skin tumours did not form in the groups receiving 0%, 28.5% or 50% test substance. However, 12 of 50 mice developed skin tumours (squamous cell carcinomas, papillomas, fibrosarcomas) in the group exposed to 100% test substance. Dermal irritation was highest in this group and was suspected of playing a role in tumour development (CONCAWE 1991).

Skin painting study
Exposure of 50 mice twice weekly for life (> 2 years) to 50 mL test substance (1170 mg-kg/bw)^{[e], [i], [j]} API 83-09 resulted in 1 benign and 19 malignant skin tumours. Mean latency to tumour development was 76 weeks (API 1989b).

Skin painting study
Male and female C3H/HeN mice (25/sex/group) were exposed to 25 mg test substance (710 mg/kg-bw)^{[e], [h]} 3 times/week for 105 weeks. Skin tumours (squamous cell carcinomas and fibrosarcomas) formed in 11 of 43 mice after exposure to petroleum-derived Jet-A, with a mean latency to tumour development of 79 weeks (Clark et al. 1988).

In another study, the role of dermal irritation in skin tumourigenicity was investigated. One group of mice received test substance thrice weekly, whereas another group received test substance intermittently, and only when signs of dermal irritation were diminished. In the former group, 44% of the mice had skin tumours whereas in the latter only 2% had tumours. The authors concluded that chronic skin irritation may play a role in skin tumourigenicity of this substance (Freeman et al. 1993).

Chromosomal aberration
Bone marrow cytogenetic tests in SD rats were negative with four samples of kerosene (API 1977, 1979, 1984, 1985c). One study administered test substance API 83-09 via intraperitoneal injection at 300, 1000 and 3000 mg/kg-bw.

Sister chromatid exchange (SCE)
A positive result was seen in male mice and a negative result in female mice in a sister chromatid exchange assay (API 1988).

Chromosomal aberration
Hydrodesulfurized kerosene in corn oil was applied intraperitoneally to B6C3F1 mice (5/sex/dose) at levels of 0, 400, 2000 or 4000 mg/kg-bw (U.S. EPA 2011). Significant increases in chromosomal aberrations were induced in male mice at all doses.

No structural/chromosomal aberrations were observed after intraperitoneal administration of 0, 0.3, 1 or 3 g/kg hydrodesulfurized kerosene to male and female SD rats (15/sex/dose) (U.S. EPA 2011; API 1984).

Micronuclei induction
There was a significant difference in micronuclei incidence in peripheral blood of female mice 72 hours after dermal exposure to JP-8 (240 mg/mouse or 300 µl) compared to negative controls (Vijayalaxmi et al. 2004).

Female C3H/H3NCR mice were dermally exposed to 50, 100 or 300 µl of undiluted JP-8 for 3 consecutive days. Application weekly for 3 weeks or a single exposure did not increase micronuclei incidence in bone marrow and peripheral blood (Vijayalaxmi et al. 2006).

Chromosomal aberration
Test substance induced chromosomal aberrations in the bone marrow of male and female SD rats exposed via inhalation for 20 days to 100 ppm (736 mg/m³)^[d] or 5 days to 400 ppm (2945 mg/m³)^[d] (API 1979c; Conaway et al. 1984). Nasal irritation, sneezing and respiratory distress were noted in the animals.

Mutagenicity
Test substance was negative in a dominant lethal assay after administration to male CD-1 mice at 100 and 400 ppm via inhalation for 6 hours/day, 5 days/week for 8 weeks (API 1973, 1980b).

Micronuclei induction
There was a significant difference for micronuclei incidence in peripheral blood of female mice 72 hours after dermal exposure to Jet-A (240 mg/mouse or 300 µl) compared to negative controls (Vijayalaxmi et al. 2004).

Female mice were dermally exposed to 50, 100 or 300 µl of undiluted Jet-A for 3 consecutive days. Application weekly for 3 weeks or a single exposure did not increase micronuclei incidence in bone marrow and peripheral blood (Vijayalaxmi et al. 2006).

Mouse lymphoma
No increase in mutation frequency with or without activation in mouse lymphoma L5178Y cells. Cells were exposed to 0, 6.25, 12.5, 25 and 37.5 nl/ml hydrodesulfurized kerosene (API sample 81-07) in ethanol for 4 hours with or without metabolic activation (U.S. EPA 2011; API 1984).

Sister chromatid exchange
No increased incidence of sister chromatid exchange in Chinese hamster ovary cells with and without activation. Cells were exposed to 0.007–0.05 µl/ml hydrodesulfurized kerosene (sample API 81-07) in acetone (U.S. EPA 2011; API 1988).

DNA damage
Increase in strand breaks and DNA lesions with increasing concentration of JP-8 (3-20 µg/ml) in rat hepatoma (H4IIE) cells compared to ethanol controls, where the cell strain is noted to be metabolically active (Grant et al. 2001).

Significant difference for 1:300 to 1:75 JP-8 dilutions compared to control for mean tail moment and mean percent DNA when JP-8 (dilutions from 1:500 to 1:75) was added to peripheral lymphocytes and monocytes from whole peripheral blood of human volunteers (Jackman et al. 2002).

Mutagenicity
Test substance was not mutagenic in the Ames assay at 0.1–10 mg per plate with or without Aroclor 1254-induced rat or hamster liver S9. Salmonella typhimurium strains TA97, TA98, TA100 and TA1535 were used (NTP 1986).

Test substance was negative in the mouse lymphoma assay at 10 mg/plate, with and without activation. L5178 TK+/- cells were used (NTP 1986).

DNA damage
There was a significant difference for 1:300 to 1:75 JP-5 dilutions compared to control for mean tail moment and mean percent DNA when JP-5 (dilutions from 1:500 to 1:75) was added to peripheral lymphocytes and monocytes from whole peripheral blood of human volunteers (Jackman et al. 2002).