Description

Absorption, Distribution, and Clearance of 2,6-Di-tert-Butyl-4-nitrophenol (DBNP)

This document is a technical report summarizing research conducted by the Naval Health Research Center Detachment (Toxicology) (NHRC/TD). The research was supported by the Office of Naval Research (ONR).

Introduction and Background

The report concerns the chemical 2,6-Di-tert-Butyl-4-nitrophenol (DBNP). DBNP is a contaminant found on the interior surfaces of US submarines. It was first brought to attention in 1992 due to discoloration or yellowing of submarine interiors, predominantly on bulkheads but also appearing throughout the ship on surfaces, dishes, bedding, etc. In extreme cases, yellowing of the skin of some submariners was reported.

DBNP (CAS #728-40) is an intensely yellow crystalline material. It results from the nitration of 2,6-di-tert-butylphenol (DBP). DBP is used as an antioxidant additive in certain synthetic steam turbine lubricating oils (MILSPEC-L-17331H) and some synthetic hydraulic fluids. The nitration process occurs when oil mist vapor/aerosol from hydraulic lines (containing DBP) passes through the electrostatic vent fog precipitators used to remove contaminants from the submarine atmosphere. Due to existing stockpiles of DBP-containing oil, the potential for Navy personnel exposure will continue for several more years.

DBNP is capable of moving throughout the submarine via the ventilation system. Air concentrations of DBNP in various submarine locations ranged from less than 3 ppb to 13 ppb, with laboratory simulations reaching as high as 122 ppb. US sailors may be exposed to DBNP for 24 hours per day for periods up to 6 months.

Hypothesized Mechanism of Toxicity

Both DBP and DBNP may function physiologically as uncouplers of mitochondrial oxidative phosphorylation. This results in the inhibition of mitochondrial respiration and ATP production. By rendering the mitochondrial membrane permeable to protons, DBNP uncouples ATP synthesis, leading to increased respiratory rates and excess energy released as heat. This uncoupling may also alter the production of oxygen reactive species. The clinical effects seen in rats exposed to DBNP (prostration, extreme hyperthermia, muscle rigidity, death) are consistent with this hypothesis. DBNP was estimated to be a potent ATP uncoupler.

Routes of Exposure

DBNP is known to be highly lipophilic and nearly insoluble in water.

• Dermal and respiratory exposure are generally assumed to present minimal human risk due to these properties. Previous research in rats suggested very low toxicity for acute dermal exposure (up to 1000 mg/kg). The insolubility of DBNP is thought to preclude its absorption by the lung.
• Oral ingestion is considered a possible risk. DBNP deposited on foods, food-processing equipment, eating utensils, or in drinking water could be delivered to humans. Ingestion of foods high in lipid content (e.g., salad oils) might particularly facilitate delivery. Crystalline DBNP can be absorbed directly from the GI tract, and suspension in lipid carriers significantly increases its uptake into the blood. This route appears to provide the greatest human risk. The vehicle used to deliver DBNP is critically important to the observed toxicity.

Rat Study Details

The research summarized in the report involved a toxicokinetic evaluation of DBNP through oral gavage in male rats. The study aimed to gain information on the distribution and excretion of DBNP post an oral gavage.

• Animals: Male Sprague-Dawley rats were used, treated in accordance with specified guidelines.
• Dosing: Rats were weighed and dosed by oral gavage. A single oral dose of ring-labeled ^14C-DBNP was administered in a 99.2% canola oil:0.8% DMSO vehicle. Canola oil was chosen to represent a lipid carrier typical of ingested foods, and DMSO was added to maximize radiolabel presence in assayed tissues. Dosing groups received either 15 mg/kg or 40 mg/kg DBNP, with a control group receiving only the vehicle.
• Sample Collection: Blood was collected from the tail vein at various intervals for 10 days. Urine and feces were collected at 24-hour intervals for 10 days. Tissues (brain, heart, lungs, liver, kidney, testis, spleen, muscle, fat) were collected from euthanized rats at 24-hour intervals for 10 days.
• Methods: Samples were processed for scintillation counting to measure the ^14C radiolabel concentration.

Study Results

• Mortality: At the 40 mg/kg dose, 6 out of 15 rats died within 24 hours post-exposure. The LD50 for this treatment and vehicle in male Sprague-Dawley rats is assumed to be approximately 40 mg/kg. None of the 15 mg/kg dosed rats died. Necropsy of rats that died indicated edema/hemorrhage of the lungs or congestion of the chest cavity. Surviving rats at both doses exhibited lethargy, reduced startle response, and hyperthermia.
• Absorption: Orally gavaged ^14C-DBNP was detectable in the blood as early as 10 minutes post-exposure (40 mg/kg dose). Blood concentration increased approximately linearly for at least the first 10 hours, with little difference observed between the 15 mg/kg and 40 mg/kg doses during this initial period.
• Distribution: DBNP was differentially distributed to all tissue compartments sampled at levels higher than control between 24-96 hours post-exposure. By 24 hours, the highest concentrations of DBNP (or its metabolites) were found in the fat, kidney, lung, and liver. By the third day, the majority of the dose remained in the fat, kidney, or liver. Concentrations in the fat and liver were significantly greater than in other organs, measuring 200-1000% higher. Fat generally contained the greatest amount of radiolabel. The relatively small presence in the brain was unexpected, potentially suggesting difficulty crossing the blood-brain barrier.
• Clearance: Clearance occurred rapidly over the first 48 hours following a single dose. By 48 hours, radiolabel in fat and kidneys reduced by over 75%, while liver reduced by less than 50%. Generally, DBNP or its metabolites were cleared from all tissue compartments, including fat, by 96 hours post-exposure. However, detectable levels numerically higher than controls persisted in most organs for approximately 8 days.
• Excretion: DBNP is eliminated from the body primarily through the urine and feces as a glucuronide conjugate.

   *   With oral administration (in 80% DMSO vehicle), approximately 70% is absorbed from the GI tract into the blood, and 30% is excreted unchanged through the feces.
   *   Within the first 10 days following a single oral gavage, 82-90% is excreted in urine and feces (at 0.4 mg/kg dose in 80% DMSO).
   *   The majority of orally gavaged ^14C-DBNP is excreted in the feces. Fecal excretion accounted for 62% of the dose within 168 hours.
   *   Clearance in the urine accounted for up to 37% of the dose. Urinary excretion peaked around 96 hours post-exposure.
   *   Excretion via urine and bile requires conjugation by phase-II metabolism.

• Histopathology: The presence of DBNP in liver, kidneys, heart, lungs, striated muscle, and spleen is consistent with previously reported DBNP-induced histopathology in these organs. Reported effects include congestion/fatty accumulation in liver, epithelial degeneration in kidneys, waxy degeneration of the myocardium, edema/congestion of the lungs, and minimal degeneration in muscle.

Discussion and Human Risk Assessment

The study results appear consistent with previous findings for oral ingestion of DBNP and provide new data on absorption, distribution, and clearance. Orally ingested DBNP is rapidly absorbed, distributed to tissues (differentially based on lipid content and other factors), and generally eliminated in feces and urine within 72-96 hours. The vehicle used is a critically important factor in the toxicity observed. The canola oil/DMSO vehicle used in this study appeared to increase toxicity compared to other vehicles like CMC or 100% corn oil.

The persistence of DBNP in various tissues for as long as 5-6 days provides a mechanism by which accumulation with repeated exposure could occur. This is important for military risk assessment, as submarine personnel can orally ingest DBNP mixed with food high in lipid content daily for months. The substantial clearance from fat observed after a single dose might combine with subsequent doses to produce higher concentrations in other tissue compartments than predicted from single exposure data.

The observed severe adverse health effects and increased core temperature support the hypothesis that DBNP is a potent uncoupler of oxidative phosphorylation. However, it remains unknown whether humans are susceptible to significant health effects from DBNP ingestion at "real world" exposure levels. While rodent studies show toxicity from repeated high doses, it's considered highly unlikely humans would ingest such high quantities on a submarine. However, oral ingestion of smaller quantities suspended in food oils or dissolved in ethanol might provide a real-world human risk. Human risk may vary depending on the fat content of the food consumed, and ethanol could potentially substantially increase absorption.

To date, no acute medical symptoms or long-term illness has been reported among personnel exposed to DBNP. It is unknown whether repeated exposure over months or years to low doses is capable of inducing negative health effects. It cannot be assumed that the rodent model perfectly predicts human toxicity, or that DBNP presents a toxic risk to humans. Submariners represent a "healthy worker" population, and subtle effects from repeated low-dose exposure might not be detected for years.

Study Information

• Authors: K.R. Still, W.W. Jederberg, G.B. Briggs, A.E. Jung, S.L. Prues, G.D. Ritchie, and R.J. Godfrey.
• Performing Organization: Naval Health Research Center Detachment (Toxicology), Wright-Patterson AFB, OH.
• Support: Office of Naval Research (ONR).
• Report Date: June 2002. The research itself was conducted from February to April 2001.
• Distribution: Approved for public release; distribution is unlimited.
• Opinions: The opinions expressed are those of the authors and do not necessarily reflect the views of the Department of the Navy or the Naval Service.

==Abstract==This document is a technical report summarizing research conducted by the Naval Health Research Center Detachment (Toxicology) (NHRC/TD). The research was supported by the Office of Naval Research (ONR). The report concerns the chemical 2,6-Di-tert-Butyl-4-nitrophenol (DBNP), a contaminant found on the interior surfaces of US submarines. It was first brought to attention in 1992 due to discoloration or yellowing of submarine interiors, predominantly on bulkheads but also appearing throughout the ship on surfaces, dishes, bedding, etc. In extreme cases, yellowing of the skin of some submariners was reported. DBNP is capable of moving throughout the submarine via the ventilation system. Air concentrations of DBNP in various submarine locations ranged from less than 3 ppb to 13 ppb, with laboratory simulations reaching as high as 122 ppb. US sailors may be exposed to DBNP for 24 hours per day for periods up to 6 months.