Registration Dossier

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles.
Reason / purpose:
read-across: supporting information
Objective of study:
metabolism
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 417 (Toxicokinetics)
GLP compliance:
not specified
Radiolabelling:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Fourteen Fischer-344 male rats weighing 307+/-10g. Feed (Purina Rat Chow, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum.
Route of administration:
oral: gavage
Vehicle:
not specified
Duration and frequency of treatment / exposure:
2 weeks
Remarks:
Doses / Concentrations:
0.8 g/kg
No. of animals per sex per dose:
8 treated, 6 control
Control animals:
yes, concurrent no treatment
Details on study design:
Fourteen Fischer-344 male rats weighing 307±10 g were randomly divided into 2 groups (8 treated, 6 control). Doses (0.8 g/kg) of t-butylcyclohexane or water (0.8 g/kg) were administered by gavage on an every other day regimen for 2 weeks. Feed (Purina Rat Chow, Ralston Purina Co., St. Louis, MO) and water were provided ad libitum and animals were weighed daily. Following the 14-day exposure period, the rats were sacrificed by halothane.
Details on dosing and sampling:
During the first 48 h of the initial dosing period, the rats were placed in metabolism cages and the urine was collected. A 5.0 ml aliquot of each urine sample was adjusted to a pH of 4.0, and 0.2 ml glucuronidase/sulfatase was added. The sample was shaken for 16 h at 37°C, then cooled to room temperature and filtered through a diatomaceous earth column using methylene chloride as the eluent. The methylene chloride extracts of the hydrolyzed rat urine were analyzed on a gas-liquid chromatograph (GC) equipped with a flame ionization detector. A 25 m x 0.2 mm ID carbowax 20 M fused silica column was used with injection port and detector temperatures of 200 and 250°C, respectively. The oven temperature was programmed to rise from 60 to 170° C at a rate of 5°C/mm and helium was used as the carrier gas. Additional metabolite identification was accomplished using a Hewlett-Packard 5985 gas chromatography/mass spectrometer (GS/MS) system. The GC was equipped with a 15 m x 0.2 mm ID DX-4 capillary column and the injection port temperature and the oven temperature were the same as reported above. Helium was the carrier gas. The MS was a quadrupole instrument operated in the electron impact mode with a voltage of 70 eV and an ion source temperature of 200°C.

The relative abundance of the urinary metabolites was found by preparing a standard solution utilizing a weighed amount of each pure compound and dodecane as an internal standard and then comparing the integrated areas of the GC curve with the areas of the CC urinary metabolite curve containing added dodecane.
Metabolites identified:
yes
Details on metabolites:
The majority of the oxidative metabolism of t-butylcyclohexane occurred on the cyclohexane structure. Of the 2 products which were the result of metabolic oxidation of the t-butyl side chain, 2-methyl-2-cyclohexylpropanoic acid was formed in greater abundance. The formation of the other alkyl side chain, metabolite 2-methyl-2-cyclohexyl-1,3-propanediol can be envisioned as 2 separate monooxidation steps involving the t-butyl group. Each of the branched chain products could hypothetically originate from a common precursor: 2-methyl-2-cyclohexylpropanol. The GC showed no trace of this propanol precursor, suggesting that, if formed, it was rapidly metabolized.

The metabolic oxidation of the cyclohexane ring of t-butylcyclohexane preferentially occurred at the 4-position. The bulkiness of the t-butyl group apparently inhibited monooxidation at the 2- and 3-positions. Both cis- and trans-4-t-butyl- cyclohexanol are formed with the latter being the overall principle metabolic product. The remaining minor metabolites identified were the result of dioxidation on the cyclohexane ring at the 3-position. The least formed cyclohexanediol was the isomer in which all the ring substituents exist in the equatorial position of the cyclohexane chair conformation of 2- hydroxy-4-t-butylcyclohexanol. 2-Hydroxy-4-t-butylcyclohexanol has the t-butyl and the OH at C1 groups in the equatorial position, while the OH at C2 is in the axial position. The most abundant cyclohexanediol, 2-hydroxy-4-t-butylcyclohexanol, has the OH at C2 and the t-butyl groups in the equatorial positions while the OH at C1 is in the axial position. If one envisions that the starting materials for the cyclohexanediols were the corresponding 4-t-butylcyclohexanols, the small amount of cis-4-t-butylcyclohexanol found may be due to its facile conversion into the 2 most abundant t-butyl-1,2-cyclohexanediols isolated. The large amount of trans-4-t-butylcyclohexadiol found may be a reflection of its easier conjugation with glucuronic acid relative to cis-4-t-butylcyclohexanol. The difficulty in glucuronidation of the cis-4-t-butylcyclohexanol could then, conceivably, result in further hydroxylation to facilitate urinary excretion by creating more polar metabolites (diols).
Conclusions:
Interpretation of results:
The major metabolites of t-butylcyclohexane were found to be: trans-4-t-butylcyclohexanol, 2c-hydroxy-4t-t-butylcyclohexanol, 2-methyl-2-cyclohexylpropanoic acid, 2c-hydroxy-4c-t-butylcyclohexanol, 2-methyl-2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-t-butylcyclohexanol, and cis -4-t-butylcyclohexanol.
Executive summary:

The major metabolites of t-butylcyclohexane were found to be: trans-4-t-butylcyclohexanol, 2c-hydroxy-4t-t-butylcyclohexanol, 2-methyl-2-cyclohexylpropanoic acid, 2c-hydroxy-4c-t-butylcyclohexanol, 2-methyl-2-cyclohexyl-1,3-propanediol, 2t-hydroxy-4t-t-butylcyclohexanol, and cis -4-t-butylcyclohexanol.

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles: non-GLP. Source of data is from secondary literature.
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
The purpose of this study was to investigate the absorption and penetration of aromatic and aliphatic components of JP-8 in humans. A surface area of 20 cm2 was delineated on the forearms of human volunteers and 1 mL of JP-8 was applied to the skin. Tape-strip samples were collected 30 min after application. Blood samples were taken before exposure (t = 0 h), after exposure (t = 0.5 h), and every 0.5 h for up to 4 h past exposure.
GLP compliance:
no
Species:
human
Sex:
male/female
Details on test animals and environmental conditions:
Study volunteers
Ten healthy adult volunteers (five males and five nonpregnant females) with no occupational exposure to jet fuel were recruited for participation. No restrictions on age, race, gender, or skin type were applied other than that the group was to be equally divided between males and females. If volunteers had a history of cardiovascular disease or atopic dermatitis, were current smokers, or were on prescription medication for a current or chronic illness, they were excluded from the study. Volunteers were not permitted to drink any alcoholic beverages 24 h before or during the experiment. Individuals occupationally exposed to compounds chosen to represent JP-8 were also excluded (e.g., auto mechanics). Approval for this study was obtained from the Office of Human Research Ethics (School of Public Health, The University of North Carolina at Chapel Hill, Chapel Hill, NC). Informed consent was received from all study volunteers.
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
0.5 h exposure period
Doses:
1 mL JP-8
No. of animals per group:
10 subjects; 5 males, 5 nonpregnant females
Details on study design:
The volunteer’s forearms were examined for obvious skin defects (abrasions, inflammation) that could enhance or impair the penetration of JP-8. After the volunteer was seated comfortably, one forearm was placed palm up inside the exposure chamber , and two aluminum application wells (10 cm2 per well) were pressed against the skin to prevent JP-8 from spreading during the experiment. The exposure chamber was sealed for the duration of the experiment (0.5 h).

The volume of JP-8 to be applied to the skin in order to have sufficient concentrations in blood was estimated using the limit of detection (LOD) of a published analytical method and estimates of permeability coefficients from an in vitro study (McDougal et al., 2000; Waidyanatha et al., 2003). Although the method by Waidyanatha et al. (2003) was developed for the analysis of naphthalene in urine, a similar LOD (5.0×l0−4 ng/ml) was assumed to apply for blood samples. Three times the LOD was assumed to be adequate for detection in blood. It was determined, using a permeability coefficient of 5.1×10−4 cm/h, that 1ml of JP-8 should produce measurable blood concentrations. Neat JP-8 was applied to the volar forearm using a 0.5 ml gas-tight syringe through two openings on top of the exposure chamber; 0.5 ml was applied to each of two wells for a total of 1.0 ml JP-8 on an area of 20 cm2. Upon application, the openings were sealed to prevent loss from the chamber.

At the end of the 0.5 h exposure period, the two exposed skin sites were wiped with a gauze pad and tape-stripped as many as 10 times. Tape-stripping has also been used in dermatopharmacokinetic studies of therapeutic agents. Tape strips were placed in 10 ml of acetone containing 1 µg/ml of internal standards (naphthalene-d8). All tape-strip samples were stored in 20 ml vials and refrigerated at 4 ◦C. Blood samples were drawn from the unexposed arm at baseline, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, and 3.5 h and collected in 6ml test tubes containing sodium heparin. The blood samples were stored at −80 ◦C until analysis.


Tape-strip samples were analyzed by gas chromatography mass spectrometry (GC–MS). Blood samples were analyzed using head-space solid-phase microextraction (HS-SPME) and the GC-MS system used to analyze the tape-strip samples.

Data Analysis
Exploratory analyses of skin and blood concentrations of JP-8 components were conducted using descriptive statistics. The skin and blood concentrations were plotted as functions of time. The first tape strip was not included in these plots because of potential residual contamination from the dose applied to the skin (Shah et al., 1998). The volume of blood was estimated using allometric relationships (Davies and Morris, 1993). The equation is Volume of blood (Vb) = 72.447×(body weight in kg)^1.007. Vb was used to estimate the total mass of naphthalene, 1-methyl naphthalene, 2-methyl naphthalene, decane, undecane, and dodecane in the blood of each volunteer. The steady state flux (J, µg/cm2/h) was estimated from the slope of the linear portion of the cumulative mass per cm2 versus time curve. The slope of the curve during the uptake period (i.e., exposure duration) was estimated for each subject. The permeability coefficient (Kp, cm/h) was estimated by dividing the flux by the concentration of the chemical (CJP-8, µg/cm3) in the 1ml of JP-8 that was applied to the skin (McDougal and Boeniger, 2002): Kp = J/CJP-8.
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Remarks on result:
other:
Conclusions:
The permeability coefficients (cm/h) of aromatic and aliphatic hydrocarbons were determined to be: Naphthalene 5.3E−05; 1-Methyl naphthalene 2.9E−05; 2-Methyl naphthalene 3.2E−05; Decane 6.5E−06; Undecane 4.5E−07; Dodecane 1.6E−06.
Executive summary:

Chemicals placed on the skin undergo absorption into the stratum corneum and evaporation from the surface of the skin. After absorption, the chemicals may be stored in deeper layers of the stratum corneum or in the viable epidermis, or they may penetrate into the dermis for eventual movement into the systemic circulation. Some absorbed compound may also transfer back to the skin surface and evaporate into the surrounding air.

 

The results are similar to in vitro studies that use diffusion cells and pig skin. The tape-strip data showed evidence of absorption of naphthalene, 1-methyl naphthalene, 2-methyl naphthalene, decane, undecane, and dodecane, although decane seemed to disappear faster from the surface of the stratum corneum. It is estimated that aromatic components of JP-8 penetrate faster than the aliphatic components. The flux of the aliphatic components is greater than the flux of the aromatic components because the concentration of the aliphatics in JP-8 is more than an order of magnitude greater than the concentration of the aromatics. The overall estimates of the apparent Kp were smaller than the in vitro estimates.

 

Consequently, the study shows that permeability coefficients estimated in vitro may overestimate the internal dose of various components of JP-8. The results of the study need to be interpreted with caution because in vitro systems do not account for distribution and clearance mechanisms, i.e., processes such as uptake into peripheral tissues, binding to proteins, metabolism, and exhalation are not incorporated in diffusion-cell experiments.

 

The permeability coefficients (cm/h) of aromatic and aliphatic hydrocarbons were determined to be: Naphthalene 5.3E−05; 1-Methyl naphthalene 2.9E−05; 2-Methyl naphthalene 3.2E−05; Decane 6.5E−06; Undecane 4.5E−07; Dodecane 1.6E−06.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles: non-GLP. Source of data is from secondary literature.
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
Three aliphatic (dodecane, tridecane, and tetradecane) chemicals, major components of JP-8, were investigated for changes in skin lipid and protein biophysics, and macroscopic barrier perturbation from dermal exposure. Percutaneous absorption was examined in vitro using porcine ears (Yorkshire marine pigs, male). Fourier transform infrared (FTIR) spectroscopy was employed to investigate the biophysical changes in stratum corneum (SC) lipid and protein. FTIR results showed that all of the above five components of JP-8 significantly (P< 0.05) extracted SC lipid and protein. Macroscopic barrier perturbation was determined by measuring the rate of transepidermal water loss (TEWL).
GLP compliance:
not specified
Radiolabelling:
yes
Species:
pig
Strain:
other: in vitro: from Yorkshire marine pigs
Sex:
male
Type of coverage:
other: in vitro
Vehicle:
unchanged (no vehicle)
Control animals:
no
Details on study design:
Model System
Porcine ears (Yorkshire marine pigs, male) were obtained. The external/dorsal skin was dermatomed to 0.5 mm thickness and used in the in vitro percutaneous absorption and TEWL studies. The method of Kligman and Christophers was used to separate epidermis from whole skin to produce the stratum corneum (SC).

In vitro percutaneous absorption
Franz diffusion cells were used in the in vitro percutaneous absorption studies. The dermatomed skin was sandwiched between the cells with the epidermis facing the donor compartment. The maximum capacities of the donor and receiver compartments were 1 and 5 ml, respectively, and the effective diffusion area was 0.785 cm2. The donor compartment contained 4 mCi of radiolabeled test chemicals in 1 ml of JP-8 and the receiver compartment was filled with 5 ml of PBS, pH 7.4 containing 0.1% formaldehyde and 0.2% Tween 80 to act as preservative and solubilizer, respectively. The donor compartment was fitted to minimize evaporation of volatile test chemical. The cells were maintained at 37oC. At appropriate times, 1 ml samples were withdrawn from the receiver compartment and transferred to scintillation vials. The samples were assayed by liquid scintillation counting. The instrument was programmed to give counts for 10 min. Net dpm was obtained by subtracting background dpm measured in the control samples. All experiments were performed in replicates of six, and the results were expressed as the mean +/-S.D. (n = 6).

Binding of chemicals
SC was pulverized in a mortar with a pestle. Ten milligrams of pulverized SC was mixed by vortexing for 5 min with 1 ml of JP-8 containing 4 mCi of the test chemical. The mixture was shaken for 10 h at 37 8C. Since the lag time of these chemicals for attaining steady state transport was well below 2 h, 10 h contact time was considered adequate for reaching equilibrium. After 10 h of contact time, the mixture was separated by centrifugation, and the supernatant was removed. The sediment was resuspended three times in JP-8 to remove chemical adsorbed on the surface. The amount of radioactivity in the supernatants was determined by liquid scintillation counting. The amount of chemical that bound to the SC was obtained by subtracting the amount of chemical recovered in supernatants from the amount of chemical originally added. Six sets of experiments were performed for each chemical.

Biophysical properties of SC lipids and proteins by FTIR
The SC samples were treated for 24 h by applying 500ml of chemical on 10 cm2 area of SC in a closed petridishes. The samples were vacuum-dried (650 mmHg) at 21oC for 3 days and stored in a desiccator to evaporate JP-8 (Yamane et al., 1995). The treated SC was then subjected to FTIR spectroscopy. Attention was focused on characterizing the occurrence of peaks near 2850 and 2920 per cm, which were due to the symmetric and asymmetric C-H stretching, respectively. Strong amide absorbance occurred in the region of 1500-1700 per cm due to C-O stretching and N-H bending. The decrease in peak heights and areas of methylene and amide absorbances is related to the SC lipid and protein extraction, respectively. For each SC sample, peak height and area were measured before and after the chemical treatment. This experimental strategy allowed each sample to serve as its own control.

In vitro transepidermal water loss (TEWL) through skin
Franz diffusion cells were used for in vitro TEWL studies. The dermatomed skin was treated with chemical in a manner similar to the SC for FTIR studies. The treated dermatomed skin was then sandwiched between the diffusion cells with the SC side up and the dermal side exposed to the receiver compartment containing isotonic saline (0.9% sodium chloride solution). Holding the probe over the donor cell opening until a stable TEWL value was achieved performed TEWL measurement. The experiments were performed in a room with an ambient temperature between 20 and 26 oC and relative humidity between 30 and 45%. In all the cases, six replicates of experiments were performed and the results expressed as the mean +/- S.D. (n=6). Experiments were performed in the same manner without chemical treatment of the dermatomed skin to serve as control.

Data analysis
The chemical concentration was corrected for sampling effects (Hayton and Chen, 1982): The permeability coefficient (Kp) was calculated as (Scheuplein, 1978): The binding of chemicals to the SC (P) was calculated as (Zhao and Singh, 2000). Statistical comparisons were made using the Student’s t -test and analysis of variance (ANOVA). The level of significance was taken as P< 0.05.
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Remarks on result:
other:

RESULTS

Binding to the stratum corneum can be determined by calculating the Log PC (octanol/water) values. There is increased binding of the aliphatic JP-8 components with increased Log PC value. Log PC values are 8.76 +/- 0.74, 13.15 +/- 1.05, 15.85 +/- 1.36 for dodecane (DOD), tridecane (TRI), and tetradecane (TET), respectively.

The flux (JSS), values were determined to be (mean) 1.94, 13.80, and 1.40 (nmol/cm2 per h)*10E-2 for DOD, TRI, and TET, respectively. The diffusion coefficient values were determined to be (0.21 +/- 0.02)E-6, (6.84 +/- 0.57)E-6,  and (0.20 +/- 0.04)E-6 cm2/h for DOD, TRI, TET, respectively. The lag time, values were determined to be (mean) 1.33, 0.89, and 1.63 hours for DOD, TRI, and TET, respectively. FTIR results suggest that all of the test chemicals significantly (P<0.05) extracted SC lipid and protein in comparison to control. TRI exhibited greater extraction of the SC lipid and protein as well as greater transport through the skin than other chemicals.

 

The TEWL values through control and chemically treated porcine skin. All of the test chemicals caused significant (P<0.05) increase in TEWL in comparison to control. TRI produced larger increase in TEWL (29.22 +/- 0.99 g/m2 per h) followed by DOD (15.15 +/- 1.34 g/m2 per h), and TET (11.64 +/- 1.42 g/m2 per h).

Conclusions:
Binding to the stratum corneum can be determined by calculating the Log PC (octanol/water) values. There is an increase in binding of the aliphatic JP-8 components to with increasing Log PC value. Log PC values are 8.76 +/- 0.74, 13.15 +/- 1.05, 15.85 +/- 1.36 for dodecane (DOD), tridecane (TRI), and tetradecane (TET), respectively.

The flux, JSS (nmol/cm2 per h)*10E-2, values were determined to be 1.94 +/- 0.39, 13.80 +/- 0.82, and 1.40 +/- 0.20 for DOD, TRI, and TET, respectively. The permeability coefficients, Kp (cm/h)*10E-4, were 0.37 +/- 0.13, 18.46 +/- 1.50, 0.64 +/- 0.20 for DOD, TRI, and TET, respectively. The diffusion coefficient values, D (cm/h)*10E-6, were determined to be 0.21 +/- 0.02, 6.84 +/- 0.57, and 0.20 +/- 0.04 for DOD, TRI, TET, respectively. The lag time (hours) was determined to be 1.33 +/- 0.07, 0.89 +/- 0.17, and 1.62 +/- 0.34 hours for DOD, TRI, and TET, respectively. FTIR results suggest that all of the test chemicals significantly (P<0.05) extracted SC lipid and protein in comparison to control. TRI exhibited greater extraction of the SC lipid and protein as well as greater transport through the skin than other chemicals.
Executive summary:

Three aliphatic (dodecane, tridecane, and tetradecane) chemicals, major components of JP-8, were investigated for changes in skin lipid and protein biophysics, and macroscopic barrier perturbation from dermal exposure. Percutaneous absorption was examined in vitro using porcine ears (marine pigs, male).  Fourier transform infrared (FTIR) spectroscopy was employed to investigate the biophysical changes in stratum corneum (SC) lipid and protein. FTIR results showed that all of the tested components of JP-8 significantly (P < 0.05) extracted SC lipid and protein.

Binding to the stratum corneum can be determined by calculating the Log PC (octanol/water) values. There is an increase in binding of the aliphatic JP-8 components to with increasing Log PC value. Log PC values are 8.76 +/- 0.74, 13.15 +/- 1.05, 15.85 +/- 1.36 for dodecane (DOD), tridecane (TRI), and tetradecane (TET), respectively.

 

The flux, JSS (nmol/cm2 per h)*10E-2, values were determined to be 1.94 +/- 0.39, 13.80 +/- 0.82, and 1.40 +/- 0.20 for DOD, TRI, and TET, respectively. The permeability coefficients, Kp (cm/h)*10E-4, were 0.37 +/- 0.13, 18.46 +/- 1.50, 0.64 +/- 0.20 for DOD, TRI, and TET, respectively. The diffusion coefficient values, D (cm/h)*10E-6, were determined to be 0.21 +/- 0.02, 6.84 +/- 0.57, and 0.20 +/- 0.04 for DOD, TRI, TET, respectively. The lag time (hours) was determined to be 1.33 +/- 0.07, 0.89 +/- 0.17, and 1.62 +/- 0.34 hours for DOD, TRI, and TET, respectively. FTIR results suggest that all of the test chemicals significantly (P<0.05) extracted SC lipid and protein in comparison to control. TRI exhibited greater extraction of the SC lipid and protein as well as greater transport through the skin than other chemicals.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable well-documented study report which meets basic scientific principles.
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline available
Principles of method if other than guideline:
In vitro isolated perfused porcine skin flap (IPPSF) Studies.
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
14C-naphthalene, 3H-dodecane, 14C- hexadecane
Species:
pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
in vitro experiment
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Duration of exposure:
5 hours; 4 trials
Doses:
25 uL of Jet fuel with radio labeled tracers
Control animals:
no
Details on study design:
In vitro isolated perfused porcine skin flap (IPPSF) Studies.
In these studies, jet fuel mixtures were applied non-occluded to mimic field exposure conditions, and experiments were conducted for a total of 5 h in IPPSFs with 4 replicates per treatment condition. A 1 x 5 cm dosing area was drawn on the surface of the skin flap with a surgery marker. A dose, containing 25 uLof the specified jet fuel containing approximately 2 uCi of 14C-naphthalene plus 10 uCi of 3H-dodecane and 14C- hexadecane was applied directly to the surface of the skin flap. The specific activities of the marker compounds were sufficient that the added radiolabeled compounds had little effect on the final concentration of naphthalene (1.21% instead of 1.1%) and dodecane (4.701% instead of 4.7%). Single label studies were initially conducted and compared to the dual-label results to test whether using this dual-label experimental design had any effect on marker absorption. No effect was detected.
Perfusate samples (3 ml) were collected every 5 mm for the first 40 mm. then every 10 mm until 1.5 h. and then every 15 mm until termination at 5 h. At termination, several samples were taken for mass balance of the marker compounds. The surface of the dose area was swabbed twice with a 1% soap solution and gauze, and then 12 stratum corneum tape strips were collected using cellophane tape (3M Corporation, Minneapolis. MN). The entire dose area was removed. A 1x 1 cm core of the dose area was removed and frozen for subsequent depth of penetration studies. This consisted of laying the core sample epidermal side down in an aluminum foil boat and embedding in Tissue-Tek OCT compound (Miles. Inc., Elkhart, IN), snap freezing in liquid nitrogen, followed by sectioning (40 zm) on a Reichart-Jung Model 1800 Cryocut (Warner Lambert, Buffalo, NY). The remaining dosed area as well as the surrounding skin was separated from the fat and held for analysis. All samples (including swabs, tape strips, core sections, skin, fat, mass balance samples, etc.) were dissolved separately in Soluene. A representative volume of each sample was oxidized completely via a Packard Model 307 Tissue Oxidizer. The 3H and 14C samples were counted separately on a Packard Model 1900TR TriCarb Scintillation Counter.

Data analysis. Data was entered into a custom IPPSF database and the resulting analysis reported. Since all experiments were conducted using the identical marker doses across all fuels, and the absolute concentrations of these marker compounds were similar, these results are expressed as percentage applied dose to give a representative assessment of the absorption and cutaneous penetration of a complex mixture such as jet fuel. This is appropriate since the absolute concentrations of jet fuel hydrocarbons are not fixed across all fuels due to differences that arise from the natural sources of the petroleum and different refining processes. Area under the curve (AUC) in the perfusate was calculated using the trapezoidal method. Peak flux was the maximum flux (% dose/mm) observed at any one time point.

The experimental compartments which were analyzed in these studies used the following definitions: (1) Surface is the residue removed by washing the surface of the IPPSF at termination of the experiment plus the residues remaining in the dosing template. (2) Stratum corneum is the residue extracted from the outermost stratum corneum via 12 tape strips at the termination of the experiment. (3) Dosed skin is the residue that remained in the dosed skin plus the depth of penetration core taken at termination. (4) Absorption is the cumulative amount of the marker compound collected in the effluent over the course of the 5-h experiment. (5) Fat is the residue remaining in the fat when it was separated from the dermis at the end of the experiment. (6) Penetration is the summation of the label in the effluent plus skin plus fat, but not stratum corneum nor surface. (7) Evaporative loss is that label which was lost to evaporation. Our previous studies in the IPPSF indicated that the penetration estimate is the best empirical correlate to predict eventual in vivo absorption in humans.

Statistical significance of absorption and penetration parameters were determined using ANOVA or by a priori-defined orthogonal contrasts where appropriate at the 0.05 level of significance. A least significance difference (LSD) procedure was used for multiple comparisons on overall tissue disposition.
Details on in vitro test system (if applicable):
A 1 x 5 cm dosing area was drawn on the surface of the skin flap with a surgery marker. A dose, containing 25 uL of the specified jet fuel containing approximately 2 uCi of 14C-naphthalene plus 10 uCi of 3H-dodecane and 14C- hexadecane was applied directly to the surface of the skin flap. Perfusate samples (3 ml) were collected every 5 mm for the first 40 mm. then every 10 mm until 1.5 h. and then every 15 mm until termination at 5 h. At termination, several samples were taken for mass balance of the marker compounds. The surface of the dose area was swabbed twice with a 1% soap solution and gauze, and then 12 stratum corneum tape strips were collected using cellophane tape (3M Corporation, Minneapolis. MN). The entire dose area was removed. A 1x 1 cm core of the dose area was removed and frozen for subsequent depth of penetration studies. This consisted of laying the core sample epidermal side down in an aluminum foil boat and embedding in Tissue-Tek OCT compound (Miles. Inc., Elkhart, IN), snap freezing in liquid nitrogen, followed by sectioning (40 zm) on a Reichart-Jung Model 1800 Cryocut (Warner Lambert, Buffalo, NY). The remaining dosed area as well as the surrounding skin was separated from the fat and held for analysis.
Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Conclusions:
Within JP-8, the rank order of absorption for all marker components was (mean +/- SEM; % dose) naphthalene (1.17 +/- 0.07)> dodecane (0.63 +/- 0.04) > hexadecane (0.18 +/- 0.08).  The area under the curve (AUC) was determined to be (mean +/- SEM; % dose-h/mL): naphthalene (0.0199 +/- 0.0020)> dodecane (0.0107 +/- 0.0009) > hexadecane (0.0017 +/- 0.0003). In contrast, deposition within dosed skin showed the reverse pattern.
Executive summary:

The purpose of these studies was to assess the percutaneous absorption and cutaneous disposition of topically applied (25 uL/5 cm2) neat Jet-A, JP-8, and JP-8(100) jet fuels by monitoring the absorptive flux of the marker components 14C naphthalene and 4H dodecane simultaneously applied non-occluded to isolated perfused porcine skin flaps (IPPSF) (a = 4). Absorption of 14C hexadecane was estimated from JP-8 fuel. Absorption and disposition of naphthalene and dodecane were also monitored using a nonvolatile JP-8 fraction reflecting exposure to residual fuel that might occur 24 h after a jet fuel spill. In all studies, perfusate, stratum corneum, and skin concentrations were measured over 5 h. Naphthalene absorption had a clear peak absorptive flux at less than 1 h, while dodecane and hexadecane had prolonged, albeit significantly lower, absorption flux profiles. Within JP-8, the rank order of absorption for all marker components was (mean +/- SEM; % dose) naphthalene (1.17 +/- 0.07)> dodecane (0.63 +/- 0.04) > hexadecane (0.18 +/- 0.08).  The area under the curve (AUC) was determined to be (mean +/- SEM; % dose-h/mL): naphthalene (0.0199 +/- 0.0020)> dodecane (0.0107 +/- 0.0009) > hexadecane (0.0017 +/- 0.0003). In contrast, deposition within dosed skin showed the reverse pattern.

Description of key information

Text was truncated. The full text is available in the migration report.

Key value for chemical safety assessment

Additional information

Toxicokinetic data is available for constituents of the C9-C14 aliphatic, 2-25% aromatic hydrocarbon fluids; principally the C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids and the C9 aromatic fluids. Based on the data from the constituents, C9-C14 aliphatic, 2-25% aromatic hydrocarbon fluids, can be absorbed when inhaled, ingested, or when dermally exposed. C9-C14 aliphatic, 2-25% aromatic hydrocarbon fluids are expected to be rapidly metabolized and eliminated with little possibility of bioaccumulation.

 

C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids

C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids are readily absorbed when inhaled or ingested.  C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids can be dermally absorbed. Regardless of exposure route, C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids are rapidly metabolized and eliminated. Bioaccumulation of C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids is not expected.

 

C9 aromatic fluids

C9 aromatic fluids are readily absorbed when inhaled or ingested.  C9 Aromatic fluids can be dermally absorbed. Bioaccumulation of C9 Aromatic fluids is not expected.

Discussion on bioaccumulation potential result:

Toxicokinetic data is available for constituents of the C9-C14 aliphatic, 2-25% aromatic hydrocarbon fluids; principally the C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids and the C9 aromatic fluids. 

C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids

C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids are apparently well absorbed if ingested or inhaled. C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids undergo metabolism and rapid excretion; bioaccumulation of the test substance in the tissues is not likely to occur. 

 

C9 to C14 isoalkanes are taken up into the blood and distributed to the internal organs including brain, liver, kidney and fat.  Twelve hours after the exposure, levels in blood, brain, liver and kidney were below detection levels; levels in fat were about half those found at the end of the exposure period.  These data demonstrate that isoalkanes are rapidly eliminated and do not accumulate. The concentration of isoalkanes in blood, brain, liver and fat increased with increasing number of carbon atoms. The C9 and C10 isoalkanes showed increasing concentrations in fat during the exposure period and high concentrations 12 hr after cessation of exposure.

 

Inhaled n-alkanes, nC9-nC13, are taken up into the blood and distributed to the internal organs including the brain.  There is also a corresponding reduction in the blood/air and brain/air ratios with increasing carbon numbers up to C10.  At high carbon numbers the ratios decrease suggesting blood/brain barrier effects for high molecular weight hydrocabons (Nilsen et. al 1988). Thus the efficiency of uptake into both blood and brain also decreases with increasing carbon number. A brain/blood ratio of 11.4 and a fat/blood ratio of 113 was determined for n-nonane. n-Decane was found to have a half-life of 2 hours. The percentage retention of n-alkanes showed an inverse linear relationship to chain length that was describable by the regression line: (percentage retained) = 115.9-3.94 * (number of carbon atoms). This line had a correlation coefficient of -0.995, standard error of estimate Sy * x= 3.30, t = 30.85 and P < 0.001.  The absorption of paraffins with 9-13 carbons ranges from 65-80%.  

 

Based on a study of jet propellant 8 (JP-8) jet fuel components, the in vitro (rat liver microsomal oxidation) nonlinear kinetic constants for n-nonane and n-decane were V(max) (nmol/mg protein/min) = 7.26 +/- 0.20 and 2.80 +/- 0.35, respectively, and K(M) (micro M) = 294.83 +/- 68.67 and 398.70 +/- 42.70, respectively. Metabolic capacity as assessed by intrinsic clearance, V(max)/K(M), was approximately four-fold higher for nonane (0.03 +/- 0.005) than for decane (0.007 +/- 0.001).

 

The blood/brain ratio and the fat/blood ratios for trimethylcyclohexane were determined to be 11.4 and 135, respectively. A marked decrease in biological concentrations of trimethylcyclohexane during the initial phase of exposure indicates that this hydrocarbon is capable of inducing its own metabolic conversion resulting in lower steady state levels.

 

The tissue disposition after 3 weeks of exposure to dearomatised white spirit, mixed aliphatic, and cycloaliphatic constituents was determined. After 3 weeks of exposure the concentration of total white spirit was 1.5 and 5.6 mg/kg in blood; 7.1 and 17.1 mg/kg in brain; 432 and 1452 mg/kg in fat tissue at the exposure levels of 400 and 800 ppm, respectively. The concentrations of n-nonane, n-decane, n-undecane, and total white spirit in blood and brain were not affected by the duration of exposure. Two hours after the end of exposure the n-decane concentration decreased to about 25% in blood and 50% in brain. A similar pattern of elimination was also observed for n-nonane, n-undecane and total white spirit in blood and brain. In fat tissue the concentrations of n-nonane, n-decane, n-undecane, and total white spirit increased during the 3 weeks of exposure. The time to reach steady-state concentrations is longer than 3 weeks. Post-exposure decay in blood could be separated into two phases with half-lives of approximately 1 and 8 hr for n-nonane, n-decane, and n-undecane. In brain tissue two slopes with half-lives of 2 and 15 hr were identified. In fat tissue, only one slope with half-life of about 30 hr was identified. In conclusion, after 3 weeks of exposure the fat:brain:blood concentration coefficients for total white spirit were approximately 250:3:1.

 

Nilsen, O., Haugen, O., Zahlsen, K. Halgunset, J., Helseth, A., Aarset, H., and Eide,1988. Toxicity of n-C9 to n-C13 alkanes in the rat on short term inhalation. Pharmacology and Toxicology 62:259-266.

C9 aromatics

DISTRIBUTION

Human Data: 1,3,5-trimethylbenzene has a very large volume of distribution (30-30 L/kg), implying wide tissue distribution and substantial partitioning in the tissues (2).  One hour post exposure blood levels of 1,3,5-trimethylbenzene in human volunteers exposed to 25 ppm for 4 hours were similar to the steady-state level that occurred during the exposure period (2, 3).  These data, combined with the very high oil:air partition coefficients of trimethylbenzenes (9620-11300) imply substantial redistribution of inhaled trimethylbenzenes to fatty tissues (2, 3, 6)

Animal Data: Tissue levels of 1,2,4-trimethylbenzene following exposure to 100 ppm for 12 hours per day for 3 days are summarized in Table 1.

 

Table 1.  Distribution of 1,2,4-trimethylbenzene in rat tissues (mean value from four animals)following exposure to 100 ppm of the substances 12 h daily for 3 days. Values in parentheses are from animals that had a 12-h recovery period after the last exposure (10).                                                         

                                            

Tissue

Concentration (µmol/kg)

Blood  

17.1

Brain

36.5

Liver

35.4

Kidney

103.6

Fat

1070 (120)

 

Following inhalation exposure in rats, the fat:blood partition coefficient of trimethylbenzenes is around 63 (10, 13). The data in Table 1 is consistent with this partition coefficient given the selective redistribution of 1,2,4-trimethylbenzene to adipose tissue.  Twelve hours following the cessation of exposure, adipose tissue levels of 1,2,4-trimethylbenzene were decreased by a factor of approximately 9, demonstrating rapid mobilization of this substance from body adipose tissue.  Thus normally, long-term accumulation of this material in fat does not occur. Trimethylbenzene does not selectively redistribute to other body tissues other than adipose tissues.

Oral dosing of rats with 14C-1,2,4-trimethylbenzene was associated with a rapid and wide tissue distribution of radioactivity throughout the body, with selective and preferential re-distribution to adipose tissue (14).  There was no preferential uptake of radioactivity into any other organ or tissue (14).

 

Summary: Collectively, these data demonstrate that during inhalation exposure, C9 aromatics undergo substantial partitioning into adipose tissues.  Following cessation of exposure, the level of C9 aromatics in body fats rapidly declines.  Thus, the C9 aromatics are unlikely to bioaccumulate in the body.  Selective partitioning of the C9 aromatics into the non-adipose tissues is unlikely. No data is available regarding distribution following dermal absorption.  However, distribution following this route of exposure is likely to resemble the pattern occurring with inhalation exposure.

 

METABOLIC TRANSFORMATION

 

Human Data: The major metabolites of trimethylbenzenes most commonly identified in urine are the dimethylbenzoic acid and dimethylhippuric acid derivatives of the parent molecule (1-3, 5, 15-20).  Both the dimethylbenzoic and hippuric acid metabolites of the trimethylbenzenes are commonly used for biomonitoring of human exposures.

 

Animal Data: In rats, urinary metabolites of 1,2,4-trimethylbenzene consist of a complex mixture of isomeric triphenols, the sulphate, glucuronide and mercapturic conjugates of dimethylbenzyl alcohols, dimethylbenzoic acids and dimethylhippuric acids (14).  The major metabolites are 3,4-dimethylhippuric acid (30.2% of the dose), sulphate and glucuronide conjugates of 2,4-dimethylbenzyl alcohol (12.7% of the dose), and sulphate and glucuronide conjugates of 2,5-dimethylbenzyl alcohol (11.7% of the dose) (14).

 

In rats, approximately 78% of an oral dose of 1,3,5-trimethylbenzene is excreted as 3,5-dimethylhippuric acid; an additional 7.6 and 8.2% were excreted as glucuronic and sulphuric acid conjugates (21). The corresponding values for the glycine, glucuronic and sulphuric acid conjugates of 1,2,4-trimethylbenzene and 1,2,3-trimethylbenzene were 43.2, 6.6, and 12.9% and 17.3, 19.4, and 19.9%, respectively (21). In rabbits, the major urinary metabolites of 1,2,4-trimethylbenzene following oral dosing are 2,4-dimethylbenzoic acid and 3,4-dimethylhippuric acid (22).

 

Biotransformation of 1,2,4-trimethylbenzene to dimethylbenzoic acids in the rat follows the Lineweaver-Burk equation with Km ranging from 7-28 mg/l and Vmax ranging from 23-96 mg/h/kg depending on the particular species of dimethylbenzoic acid formed (7). Biotransformation to 3,4-dimethylbenzoic acid is favoured, given its Km of 28 mg/l and Vmax of 96 mg/h/kg.  Notably, in rats, the biotransformation of trimethylbenzenes is inhibited by pre-treatment with ethanol and enhanced by ethyl acetate (8, 23). In rats, 1,3,5-trimethylbenzene is an inducer of cytochrome p450, cytochrome b5, aminopyrine N-demethylase, aryl hydrocarbon hydroxylase, aniline hydroxylase,and NADPH-cytochrome c reductase in the rodent liver (24).  In the kidney, 1,3,5-trimethylbenzene induces cytochrome P-450 and cytochrome b5 (24).  Thus, trimethylbenzenes are inducers of their own metabolism in the rat.  These data are consistent with the low propensity for bioaccumulation of trimethylbenzenes in mammals.

 

Summary: Collectively these data demonstrate that C9 aromatics may undergo several different Phase I dealkylation, hydroxylation and oxidation reactions which may or may not be followed by Phase II conjugation to glycine, sulphation or glucuronidation.  However, the major predominant biotransformation pathway is typical of that of the alkylbenzenes and consists of: (1) oxidation of one of the alkyl groups to an alcohol moiety; (2)oxidation of the hydroxyl group to a carboxylic acid; (3) the carboxylic acid is then conjugated with glycine to form a hippuric acid. The minor metabolites can be expected to consist of a complex mixture of isomeric triphenols, the sulphate and glucuronide conjugates of dimethylbenzyl alcohols, dimethylbenzoic acids and dimethylhippuric acids.  Consistent with the low propensity for bioaccumulation of the C9 aromatics, these substances are likely to be significant inducers of their own metabolism. 

 

ELIMINATION AND EXCRETION

Human Data: The identified routes of excretion of the trimethylbenzenes following inhalation exposure in humans include: (1) exhalation of the unchanged volatile parent substance (3); (2) urinary excretion of the unchanged volatile parent substance(25); and (3) urinary excretion of metabolites (1-3, 5, 15-20).  Post-exposure exhalation of unmetabolized trimethylbenzenes accounts for 20-37% of the absorbed amount (2).  Urinary excretion of unmetabolized trimethylbenzenes is low (≤ 0.002%) (2).  Overall urinary excretion of metabolites (predominantly 3,4-dimethylhippuric acid) of 1,2,4-trimethylbenzene in the first 24 hours post-exposure accounts for 22% of the inhaled dose (18). Urinary excretion of unconjugated dimethylbenzoic acid metabolites accounts for only small percent of the inhaled dose of trimethylbenzenes (18). The bulk of the absorbed dose of trimethylbenzenes is metabolized and excreted in urine, predominantly as their dimethylhippuric acids or conjugated (sulphated or glucuronidated) dimethylbenzoic acid derivatives (1-3, 5, 15-20).  The urinary excretion of dimethylhippuric acids is well-correlated with exposure to trimethylbenzenes and has been commonly used for biomonitoring purposes. 

 

The initial blood clearance of trimethybenzenes in man is 0.6-1 l/hr/kg (2). However, trimethylbenzenes have longer terminal half-lives in blood (T½ 78-120 hr) due to their extensive partitioning into adipose tissues (2).  The kinetics of urinary elimination of unmetabolized 1,3,5-trimethylbenzene follows a biphasic pattern with a T½ for Phase I of 0.45-0.88 hr and a T½ for Phase II of 6.7-19.2 hr.25  Elimination of unmetabolized 1,3,5-trimethylbenzene in exhaled air is biphasic with an initial T½ of 1 hr (3).  Urinary elimination of dimethylbenzoic acids following inhalation exposure to 1,3,5-trimethylbenzene is biphasic with a T½ for Phase I of 13 hr and a T½ for Phase II of 60 hr (3).  Notably, co-exposure to white spirits interferes with the metabolic elimination of 1,2,4-trimethylbenzene (5, 16).

 

Animal Data: Following oral administration of 14C-1,2,4-trimethylbenzene, > 99% of the administered radioactivity was excreted in urine within 24 hours (14).  The predominant urinary species being the 3,4-dimethylhippuric acid metabolite (14).  Likewise, urine is the major route of excretion of trimethylbenzene metabolites in rats following inhalation exposure. As in humans, there is a strong correlation between the level of trimethylbenzene inhalation exposure and the concentration of metabolites in urine.

 

Summary: Collectively these data demonstrate that the predominant route of excretion of C9 aromatics following inhalation exposure involves either exhalation of the unmetabolized parent compound, or urinary excretion of its metabolites.  When oral administration occurs, there is little exhalation of unmetabolized C9 aromatics, presumably due to the first pass effect in the liver.  Under these circumstances, urinary excretion of metabolites is the dominant route of excretion.

 

References

1.            Kostrzewski P, Wiaderna-Brycht A, Czerski B. Biological monitoring of experimental human exposure to trimethylbenzene. Sci Total Environ 1997;199:73-81.

2.            Jarnberg J, Johanson G, Lof A. Toxicokinetics of inhaled trimethylbenzenes in man. Toxicol Appl Pharmacol 1996;140:281-288.

3.            Jones K, Meldrum M, Baird E, et al. Biological monitoring for trimethylbenzene exposure: a human volunteer study and a practical example in the workplace. Ann Occup Hyg 2006;50:593-598.

4.            Jarnberg J, Johanson G. Physiologically based modeling of 1,2,4-trimethylbenzene inhalation toxicokinetics. Toxicol Appl Pharmacol1999;155:203-214.

5.            Jarnberg J, Johanson G, Lof A, Stahlbom B. Inhalation toxicokinetics of 1,2,4-trimethylbenzene in volunteers: comparison between exposure to white spirit and 1,2,4-trimethylbenzene alone. Sci Total Environ 1997;199:65-71.

6.            Jarnberg J, Johanson G. Liquid/air partition coefficients of the trimethylbenzenes.Health 1995;11:81-88.

7.            Swiercz R, Rydzynski K, Wasowicz W, Majcherek W, Wesolowski W. Toxicokinetics and metabolism of pseudocumene (1,2,4-trimethylbenzene) after inhalation exposure in rats. Int J Occup Med Environ Health2002;15:37-42.

8.            Romer KG, Federsel RJ, Freundt KJ. Rise of inhaled toluene, ethyl benzene, m-xylene, or mesitylene in rat blood after treatment with ethanol. Bull Environ Contam Toxicol 1986;37:874-876.

9.            Dahl AR, Damon EG, Mauderly JL, et al. Uptake of 19 hydrocarbon vapors inhaled by F344 rats. Fundam Appl Toxicol 1988;10:262-269.

10.          Zahlsen K, Nilsen AM, Eide I, Nilsen OG. Accumulation and distribution of aliphatic (n-nonane), aromatic (1,2,4-trimethylbenzene) and naphthenic (1,2,4-trimethylcyclohexane) hydrocarbons in the rat after repeated inhalation. Pharmacol Toxicol 1990;67:436-440.

11.          Korinth G, Geh S, Schaller KH, Drexler H. In vitro evaluation of the efficacy of skin barrier creams and protective gloves on percutaneous absorption of industrial solvents. Int Arch Occup Environ Health 2003;76:382-386.

12.          McDougal JN, Pollard DL, Weisman W, Garrett CM, Miller TE. Assessment of skin absorption and penetration of JP-8 jet fuel and its components. Toxicol Sci 2000;55:247-255.

13.          Eide I. A review of exposure conditions and possible health effects associated with aerosol and vapour from low-aromatic oil-based drilling fluids. Ann Occup Hyg 1990;34:149-157.

14.          Huo JZ, Aldous S, Campbell K, Davies N. Distribution and metabolism of 1,2,4-trimethylbenzene (pseudocumene) in the rat. Xenobiotica1989;19:161-170.

15.          Fukaya Y, Saito I, Matsumoto T, Takeuchi Y, Tokudome S. Determination of 3,4-dimethylhippuric acid as a biological monitoring index for trimethylbenzene exposure in transfer printing workers. Int Arch Occup Environ Health 1994;65:295-297.

16.          Jarnberg J, Johanson G, Lof A, Stahlbom B. Toxicokinetics of 1,2,4-trimethylbenzene in humans exposed to vapours of white spirit: comparison with exposure to 1,2,4-trimethylbenzene alone. Arch Toxicol 1998;72:483-491.

17.          Kostrewski P, Wiaderna-Brycht A. Kinetics of elimination of mesitylene and 3,5-dimethylbenzoic acid after experimental human exposure. Toxicol Lett 1995;77:259-264.

18.          Jarnberg J, Stahlbon B, Johanson G, Lof A. Urinary excretion of dimethylhippuric acids in humans after exposure to trimethylbenzenes. Int Arch Occup Environ Health 1997;69:491-497.

19.          Stahlbom B, Jarnberg J, Soderkvist P, Lindmark D. Determination of dimethylhippuric acid isomers in urine by high-performance liquid chromatography. Int Arch Occup Environ Health 1997;69:147-150.

20.          Ichiba M, Hama H, Yukitake S, et al. Urinary excretion of 3,4-dimethylhippuric acid in workers exposed to 1,2,4-trimethylbenzene. Int Arch Occup Environ Health 1992;64:325-327.

21.          Mikulski PI, Wiglusz R. The comparative metabolism of mesitylene, pseudocumene, and hemimellitene in rats. Toxicol Appl Pharmacol1975;31:21-31.

22.          Cerf J, Potvin M, Laham S. Acidic metabolites of pseudocumene in rabbit urine. Arch Toxicol 1980;45:93-100.

23.          Freundt KJ, Romer KG, Federsel RJ. Decrease of inhaled toluene, ethyl benzene, m-xylene, or mesitylene in rat blood after combined exposure to ethyl acetate. Bull Environ Contam Toxicol1989;42:495-498.

24.          Pyykko K. Effects of methylbenzenes on microsomal enzymes in rat liver, kidney and lung. Biochim Biophys Acta 1980;633:1-9.

25.          Janasik B, Jakubowski M, Jalowiecki P. Excretion of unchanged volatile organic compounds (toluene, ethylbenzene, xylene and mesitylene) in urine as result of experimental human volunteer exposure. Int Arch Occup Environ Health2008;81:443-449.

26.          Hissink, A. et al. (2007).  Model studies for evaluating the neurobehavioral effects of complex hydrocarbon solvents III.  PBPK modeling of white spirit constituents as a tool for integrating animal and human data.  Neurotoxicology 28:751-760.

27.          Muhammad, F. et al. (2005).  Effect of in vivo jet fuel exposure on subsequent in vitro dermal absorption of individual aromatic and aliphatic hydrocarbon fuel constituents.  Journal of Toxicological and Environmental Health Part A, 68:719-737.

Discussion on absorption rate:

There have not been any in vivo dermal absorption studies of C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids, but there have been in vitro studies of some constituents, particularly dodecane.  Due to the structural similarity of these molecules to other constituents of the C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids, it seems reasonable to assume that the solvents would have toxicokinetic properties similar to those of these constituents.  There is also dermal absorption studies available for the C9 aromatics.

C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids 

IN VIVO

 

Ten healthy adult volunteers (five males and five nonpregnant females) with no occupational exposure to jet fuel were recruited for participation. One of the volunteer’s forearms was placed palm up inside the exposure chamber, and two aluminum application wells (10 cm2 per well) were pressed against the skin to prevent JP-8 from spreading during the experiment. Neat JP-8 (1.0 mL) was applied to the volar forearm. The exposure chamber was sealed for the duration of the experiment (0.5 h). At the end of the exposure period, the two exposed skin sites were wiped with a gauze pad and tape-stripped as many as 10 times. Blood samples were drawn from the unexposed arm at baseline, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, and 3.5 h.

 

The permeability coefficients (cm/h) of the aliphatic hydrocarbons were determined to be: Decane 6.5E-06, Undecane 4.5E-07, and Dodecane 1.6E-06.

 

A simple mathematical model based on Fick’s laws of diffusion was used to predict the spatiotemporal variation of undecane and dodecane in the stratum corneum of human volunteers using the same data as above. The estimated values of the diffusion coefficients (Dsc, cm2/min×10E−8 +/- S.D.) were determined to be: undecane, 4.2 +/- 1.2 and Dodecane, 5.0 +/- 0.7.

 

IN VITRO

Several in vitro studies used porcine skin flaps to determine the absorption and disposition of several aliphatic compounds. There are some general conclusions of the absorption and disposition of C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids. All of the tested chemicals showed a lag time of about 1 h. The retention of aliphatic chemicals in stratum corneum is much higher than epidermis and dermis at all time points. Under infinite dose conditions, the chemicals diffused rapidly into stratum corneum and reached plateau levels within 1 h. The absorption of chemicals in stratum corneum at all time points were in the following order: tetradecane > dodecane > nonane. This shows a linear relationship between the carbon chain length and the absorption of the chemicals in the stratum corneum. The absorption pattern of chemicals in epidermis and dermis, in contrast to stratum corneum, demonstrated a parabolic relationship between the molecular weight of the hydrocarbon and their skin retention.

 

Dermal absorption values for several of the C9-C14 aliphatic, < 2% aromatic hydrocarbon fluids have been experimentally determined. The permeability coefficients (cm/h) for decane, undecane, and dodecane were determined to be 6.5*10E-6, 4.5*10E-07, and 1.6*10E-06, respectively. In a second experiment, the diffusion coefficient values (cm2/h) of for dodecane (DOD), tridecane (TRI), and tetradecane (TET) were determined to be (0.21 +/-0.02)*10E-6, (6.849 +/- 0.57)*10E-6, (0.209 +/- 0.04)*10E-6, respectively.

 

Binding to the stratum corneum can be determined by calculating the Log PC (octanol/water) values. There is an increase in binding of the aliphatic JP-8 components to with increasing Log PC value. Log PC values are 8.76 +/- 0.74, 13.15 +/- 1.05, 15.85 +/- 1.36 for dodecane (DOD), tridecane (TRI), and tetradecane (TET), respectively.

 

The flux, JSS (nmol/cm2 per h)*10E-2, values were determined to be 1.94 +/- 0.39, 13.80 +/- 0.82, and 1.40 +/- 0.20 for DOD, TRI, and TET, respectively. The permeability coefficients, Kp (cm/h)*10E-4, were 0.37 +/- 0.13, 18.46 +/- 1.50, 0.64 +/- 0.20 for DOD, TRI, and TET, respectively. The diffusion coefficient values, D (cm/h)*10E-6, were determined to be 0.21 +/- 0.02, 6.84 +/- 0.57, and 0.20 +/- 0.04 for DOD, TRI, TET, respectively. The lag time (hours) was determined to be 1.33 +/- 0.07, 0.89 +/- 0.17, and 1.62 +/- 0.34 hours for DOD, TRI, and TET, respectively. FTIR results suggest that all of the test chemicals significantly (P<0.05) extracted SC lipid and protein in comparison to control. TRI exhibited greater extraction of the SC lipid and protein as well as greater transport through the skin than other chemicals.

C9 aromatic fluids

ABSORPTION INHALATION EXPOSURE

 

Human Data

Exposure of human volunteers to trimethylbenzene vapour concentrations ranging from 5 -150 mg/m3 resulted in pulmonary retentions between 56-71% depending on the chemical species and the study (1, 2). Absorption into the blood stream of human volunteers exposed to a 25 ppm vapour of 1,3,5-trimethylbenzene for a period of 4 hours was rapid, and resulted in a mean steady-state blood level of 0.85 micromol/l after 1-2 hours of exposure(3). Similar results were observed in human volunteers exposed to 100 ppm (26). Likewise, rapid pulmonary absorption of 1,2,3-trimethylbenzene in human volunteers has also been demonstrated (4, 5).

In vitro human blood:gas partition coefficients for the trimethylbenzenes are high, ranging from 40.8 to 69.3, depending on the chemical species (6).  Thus the pulmonary absorption of trimethylbenzenes is ventilation-limited.  This is consistent with the apparent high rate of uptake of the trimethylbenzenes from the alveoli into the blood and the apparent slow rate of equilibration of 1,3,5-trimethylbenzene partial pressures in alveolar and inspired air in man (3).

 

Animal Data : The systemic absorption of inhaled trimethylbenzenes in rats is rapid with blood levels reaching a plateau after about 2 hours of exposure (7, 8).   The rate of uptake of inhaled 1,2,4-trimethylbenzene rats is 13.6 nmol×kg-1×min-1×ppm-1during nose-only exposure (9, 10). As in humans, 1,2,4-trimethylbenzene has a relatively high blood:gas partition coefficient and its uptake is ventilation-limited (10).  

 

Summary: The available human and animal data imply that: a high proportion of inhaled C9 aromatic substances are available for absorption; that rapid systemic absorption of C9 aromatics following inhalation exposure can be expected; and that pulmonary absorption of the C9 aromatic substances is ventilation limited.

 

DERMAL EXPOSURE

 

Human Data: Attempts at dermal absorption determinations in humans with trimethylbenzenes has been difficult due to their acute primary skin irritancy (3). Slow, low-level skin penetration of 1,2,4-trimethylbenzene through excised human skin in vitro, as measured using Franz static diffusion cells, can occur although steady state absorption conditions were not established following an 8 hour exposure period (11)

 

Animal Data: The mean in vitro rat dermal absorption flux of trimethylbenzenes present in a kerosene-based fuel (JP-8), was 1.25 micrograms/cm2/hour with a breakthrough time of 1 hour, as determined in Franz static diffusion cells.12 Similarly, in a study in which pigs were treated dermally with jet fuel for 1-4 days, and then skin removed and tested for dermal penetration under in vitro conditions, values of 0.49-1.01 micrograms/cm2/hour were reported for trimethylbenzene (27).

 

Summary: The available in vitro and animal data imply that C9 aromatics will be systemically absorbed following dermal exposure, albeit at low levels.

OVERVIEW OF PERCUTANEOUS ABSORPTION OF HYDROCARBON SOLVENTS

There are no studies of repeated dose toxicity of hydrocarbon solvents using the dermal route of administration. Accordingly, where it is necessary to calculate dermal DNELs, systemic data from studies utilizing other routes of administration, normally inhalation but also oral data, can be used in some situations.  In accordance with ECHA guidance, read across from oral or inhalation data to dermal should account for differences in absorption where these exist (R8, example B.6). In fact, hydrocarbon solvents are poorly absorbed in most situations, in part because some are volatile and do not remain in contact with the skin for long periods of time and also because, due to their hydrophobic natures, do not partition well into aqueous environments and are poorly absorbed into the blood. 

If these differences in relative absorption are introduced into the DNEL calculations to calculate external doses, the DNELs based on systemic effects are highly inflated. This seems potentially misleading as it implies that substances have different intrinsic hazards when encountered by different routes whereas in fact the differences are due ultimately to differences in absorbed dose. Accordingly, it is our opinion that it would be more transparent if the differences in absorption were taken into account in the exposure equations rather than in DNEL derivation. 

 

Shown below is a compilation of percutaneous absorption information for a number of hydrocarbon solvent constituents covering carbon numbers ranging from C5 to C14 as well as examples of both aliphatic and aromatic constituents. The low molecular weight aliphatic hydrocarbons (n-pentane, 2-methylpentane, n-hexane, n-heptane, and n-octane) were tested by Tsuruta (1982) using rat skin in an in vitro model system. As shown (Table 1), the highest percutaneous absorption value was 2 ug/cm2/hr for pentane. Lower values (< ~ 1 ug/cm2/hr) were reported for aliphatic hydrocarbons ranging from hexane to octane. Several authors have assessed the percutaneous absorption of higher molecular weight aliphatic constituents including Baynes et al. (2000), Singh and Singh (2003), Muhammad et al. (2005), and Kim et al., (2006). The first three of these authors used porcine skin models and reported that, except for one anomalous result with tridecane, the percutaneous absorption values for aliphatic constituents ranging from nonane to tetradecane were well below 1 ug/cm2/hr. Rat and human skin are considered to be more permeable than human skin (Kim et al., 2006), so these numbers can be considered conservative. 

 

Kim et al. (2006) reported results of percutaneous absorption studies with human skin under in vivo conditions. In this case, the assessment method was based on tape stripping. The authors reported percutaneous absorption values ranging from 1 – 2 ug/kg/day for decane, undecane and dodecane. These values are higher than those reported by other authors, most likely because this technique measures absorption into the skin but not through the skin as was done in the studies listed above. Accordingly, it seems likely that these numbers are conservative as well.

 

With respect to aromatic hydrocarbons, most of the reported percutaneous absorption values [Baynes et al. (2000); Singh and Singh (2003); Mohammad et al. (2005); and Kim et al. (2006)] are less than 2 ug/cm2/day. The only exceptions are the values for naphthalene from Mohammad et al. (2005) which range from 4.2-6.6 ug/cm2/hr. 

 

After considering all of the above, it seems reasonable to assume apparent that across the entire range of hydrocarbon solvent constituents, percutaneous absorption values are less than 2 ug/cm2/day. Accordingly, when systemic dermal DNELs are calculated using route to route extrapolations, the values will not be corrected for differences in absorption. Rather, 2 ug/cm2/hr will be used as a common percutaneous absorption rate for all hydrocarbon solvents for which dermal exposure estimates are provided. 

           

Table 1: Summarized information on percutaneous absorption of hydrocarbon solvent constituents (C5-C16). 

 

Constituent

Molecular Weight

nmol/min/cm2

nmol/hr/cm2

ug/cm2/hr

Reference

Aliphatic Constituents

 

 

 

 

 

Pentane

72

0.52

31.2

2.2

Tsuruta et al. 1982

 

 

 

 

 

 

2-methyl pentane

86

0.02

1.2

0.1

Tsuruta et al., 1982

 

 

 

 

 

 

n-hexane

86

0.02

0.6

0.5

Tsuruta et al., 1982

 

 

 

 

 

 

n-heptane

100

0.02

1.2

0.1

Tsuruta et al., 1982

 

 

 

 

 

 

n-octane

114

0.08 x 10-3

0.005

0.0005

Tsuruta et al., 1982

 

 

 

 

 

 

Nonane

128

 

 

0.03

Muhammad et al., 2005

Nonane

 

 

 

0.38

McDougal et al., 1999

 

 

 

 

 

 

Decane

142

 

 

2

Kim et al., 2006

Decane

 

 

 

1.65

McDougal et al., 1999

 

 

 

 

 

 

Undecane

156

 

 

0.06-0.07

Muhammad et al., 2005

Undecane

 

 

 

1.0

Kim et al., 2006

Undecane

 

 

 

1.22

McDougal et al., 1999

 

 

 

 

 

 

Dodecane

170

 

 

0.02-0.04

Muhammad et al., 2005

Dodecane

 

 

 

2

Kim et al., 2006

Dodecane

 

 

 

0.3

Singh and Singh, 2003

Dodecane

 

 

 

0.51

McDougal et al., 1999

Dodecane

 

 

 

0.1

Baynes et al. 2000

 

 

 

 

 

 

Tridecane

184

 

 

0.00-0.02

Muhammad et al., 2005

Tridecane

 

 

 

2.5

Singh and Singh, 2003

Tridecane

 

 

 

0.33

McDougal et al., 1999

Tetradecane

198

 

 

0.3

Singh and Singh, 2003

Hexadecane

 

 

7.02 x 10E-3

0.00004

Singh and Singh, 2002

 

 

 

 

 

 

Aromatic Constituents

 

 

 

 

 

Trimethyl benzene

120

 

 

0.49 - 1.01

Muhammad et al., 2005

Trimethyl benzene

 

 

 

1.25

McDougal et al., 1999

 

 

 

 

 

 

Naphthalene

128

 

 

6.6 - 4.2

Muhammad et al., 2005

Naphthalene

 

 

 

0.5

Kim et al., 2006

Naphthalene

 

 

 

1.4

Singh and Singh 2002

Naphthalene

 

 

 

1.8

Baynes et al. (2000)

Naphthalene

 

 

 

1.0

McDougal et al., 1999

 

 

 

 

 

 

1 methyl naphthalene

142

 

 

0.5

Kim et al., 2006

Methyl naphthalene

 

 

 

1.55

McDougal et al., 1999

 

 

 

 

 

 

2-methyl naphthalene

 

 

 

0.5

Kim et al., 2006

2-methyl naphthalene

 

 

 

1.1

Singh and Singh, 2002

 

 

 

 

 

 

 

 

 

 

 

 

Dimethyl naphthalene

156

 

 

0.62 – 0.67

Muhammad et al., 2005

Dimethyl naphthalene

 

 

 

0.59

McDougal et al. 1999

 

Table 2. Estimated percentages of various hydrocarbon solvent constituents absorbed

 

Based on the information provided below, an overall estimate of 1% for all hydrocarbon solvents seems reasonable. 

 

Category

Representative Substance

Estimate of Percent absorption

Proposal for category

Reference for percent value

 

 

 

 

 

1

Trimethyl benzene

0.2%

0.2%

Based on data in Muhammad et al. (2005)

2

Naphthalene

1.2%

1.2%

Riviere et al. 1999

3

Dodecane (75%)

0.63%

0.5%

Riviere et al., 1999

 

TMB (25%)

0.2%

 

Muhammad et al., 2005

 

 

 

 

 

4

Hexadecane (70%)

0.18%

0.5%

Riviere et al., 1999

 

Naphthalene (30%)

1.2%

 

Riviere et al., 1999

 

 

 

 

 

5

Pentane

 

 

 

 

 

 

 

6

Hexane

 

 

 

 

 

 

 

7

Heptane

0.14%

0.14%

Singh et al. 2003

 

 

 

 

 

8

Dodecane

0.63%

0.63%

Riviere et al. 1999

 

 

 

 

 

9

Hexadecane

0.18%

0.18%

Riviere et al., 1999

 

 

 

 

 

 

Kim, D., Andersen, M., and Nylander-French (2006). Dermal absorption and penetration of jet fuel components in humans. Toxicology Letters 165:11-21.

 

Muhammad, F., N. Monteiro-Riviere, R. Baynes, and J. Riviere (2005). Effect of in vivo jet fuel exposure on subsequent in vitro dermal absorption of individual aromatic and aliphatic hydrocarbon fuel constituents. Journal of Toxicology and Environmental Health Part A. 68:719-737.

 

Singh Somnath, Zhao Kaidi, Singh Jagdish. (2002). In vitro permeability and binding of hydrocarbons in pig ear and human abdominal skin. Drug and chemical toxicology, (2002 Feb) Vol. 25, No. 1, pp. 83-92.

 

Singh, S. and Singh, J. (2003). Percutaneous absorption, biophysical and macroscopic barrier properties of porcine skin exposed to major components of JP-8 jet fuel. Environmental Toxicology and Pharmacology 14:77-85.

 

 

Singh, S., Zhao, K., Singh, J. (2003). In vivo percutaneous absorption, skin barrier perturbation and irritation from JP-8 jet fuel components. Drug Chem. Toxicol 26:135-146.

 

McDougal, J., Pollard, D., Weisman, W., Garrett, C., and Miller, T. (2000). Assessment of skin absorption and penetration of JP-8 jet fuel and its components. Toxicological Sciences 25:247-255.

 

Muhammad, F., N. Monteiro-Riviere, R. Baynes, and J. Riviere (2005). Effect of in vivo jet fuel exposure on subsequent in vitro dermal absorption of individual aromatic and aliphatic hydrocarbon fuel constituents. Journal of Toxicology and Environmental Health Part A. 68:719-737.

 

Riviere, J., Brooks, J., Monteiro-Riviere, N., Budsaba, K., and Smith, C. (1999). Dermal absorption and distribution of topically dosed jet fuels jet A, JP-8 andJP-8(100). Toxicology and Applied Pharmacology 160:60-75.

 

Tsuruta, H. et al. (1982). Percutaneous absorption of organic solvents III. On the penetration rates of hydrophobic solvents through the excised rat skin. Industrial Health 20:335-345.