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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March, 1985 - July, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
bioaccessibility
Qualifier:
no guideline followed
GLP compliance:
yes
Specific details on test material used for the study:
t-BP used in these toxicity studies was manufactured by Penwalt Corporation (Lucidol Division, Buffalo, NY); the chemical was identified by NMR, infrared, and ultraviolet spectroscopy. Cumulative data derived from iodometric titration, elemental analysis, HPLC, and thin layer chromatography indicated a purity of > 98.8%. The bulk chemical was stored at room temperature, protected from light. Quantitative reanalyses were performed within a month prior to the initiation and completion of the 13-week studies; no degradation of the material was evident.

For the stability and disposition studies, [14C]-t-butyl perbenzoate (Lot # 830107) was prepared by Pathfinder Laboratories, Inc. (St. Louis, MO). Labeled in the ring, the [14C]-t-BP had a specific activity of 10 mCi/mmol. Unlabeled t-butyl perbenzoate (Lot # 32120J) was procured from Aldrich Chemical Co. (Milwaukee, WI). The purities of the unlabeled and labeled material were determined using 2 HPLC systems with a Waters Associates liquid chromatograph (Waters Chromatography, Milford, MA) equipped with 2 model 6000A pumps, a model 660 solvent programmer, a model U6K injector, and a model 440 ultraviolet detector operated at 254 nm. The flow rate was 2 ml/min. The first HPLC system that was used to determine the purity incorporated a linear solvent gradient beginning with CH3CN:0.04M NH4OAc; pH 6.5 (50:50) and ending with CH3CN:0.04M NH4OAc; pH 6.5 (95:5) in 10 minutes; the system used a Whatman Partisil® 10/ODS-3 column (Whatman, Inc., Clifton, NJ). Unlabeled t-BP was pure by HPLC analysis; radiochemical purity of the [14C]-t-BP was 94 -97%. The second HPLC system employed a linear gradient using a Lichrosorb® diol column (E. Merck, Rahway, NJ) with a mobile phase of heptane for 5 minutes and then heptane to heptane/n-propanol (95:5) in 5 minutes. The [14C]-t-BP was 97% radiochemically pure; the unlabeled compound was essentially 100% pure.
Radiolabelling:
yes
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
Adult male F344/N rats used in the disposition studies were purchased from Charles River Breeders (Kingston, NY). The rats were examined for diseases and abnormalities upon arrival and quarantined for 2 weeks before being used in a study. Animals were fed Certified Purina Rat Chow and water, ad libitum, for the duration of the studies. Food and water were withheld 15 hours prior to dose administration. Animals were transferred to glass metabolism chambers the day prior to being used in an experiment.
Route of administration:
other: in vitro studies, dermal application and i.v. administration.
Details on exposure:
Stability Studies
Stability studies were performed with 14C-labeled t-BP in corn oil at concentrations of 3 and 30 mg/ml, and in various biological media at concentrations of 1.1, 0.11, or 0.011 mg/ml. Biological media studied included 40 mg/ml BSA in buffered saline; Sorensen's buffer, 0.067M (pH 7.4); rat serum; whole blood; 20% stomach contents in Sorensen's buffer (pH 4.1); HEPES buffer (pH 7.4), with and without 5 mM glutathione; and liver microsomes and soluble fractions, with and without 5 mM glutathione. [14C]-t-BP solutions in biological media were incubated at 37°C. Aliquots of 1 ml were withdrawn at 0, 5, 15, 30, and 60 minutes and extracted twice with ether. The pH of the sample was adjusted to 2, and the sample was extracted twice with ether. Radioactivity in the extracts was determined by scintillation spectrometry. The samples then were concentrated and reconstituted to 100-300 μl in ethanol and analyzed by HPLC.

t-BP/corn oil solutions were held at room temperature for for 0, 1, 2, or 24 hours. Aliquots of 10 μl were diluted with 60 μl of hexane and analyzed by HPLC. Initial conditions for the analysis were 100% hexane for 10 minutes followed by a 5-minute gradient of 100% hexane to 70:30 of hexane:hexane/ethanol (95:5). Analyses were performed using a Lichrosorb® diol column with a flow rate of 2 ml/min. Incubations (15 min) of 1.1 mg t-BP in 1 ml of liver microsomes, prepared from rats, were performed to analyze t-butanol content. GC/FID analysis was performed by direct injection of the incubation mixture onto an 80/100 Carbopak® column (Supelco, Bellefont, PA) at 120°C; nitrogen was used as the carrier gas, at a flow rate of 20 ml/min.

The stability of t-BP on isolated rat skin was assessed by direct application of t-BP solutions containing 0.1, 1, and 10 mg t-BP/20 μl ether, and 1.6, 2.0, and 2.3 x 106 DPM/20 μl ether, respectively. Skin was obtained from rats anesthetized with Ketamine®/Xylazine® by intraperitoneal injection. Their backs and sides were shaved; the animals were killed; and portions of the shaved skin were dissected into 2 cm2 sections. Skin sections were placed in Petri dishes containing moist paper towels kept at 37°C, and 20 μl of each t-BP solution described above was placed on a 1 cm2 area on each of 2 pieces of skin. The skin sections were kept at 37°C in a covered glass container with a small piece of moist paper towel. Skin samples receiving each of the 3 concentrations of t-BP were withdrawn and analyzed after 1 hour and after 24 hours. Each skin piece was rinsed with 5 ml of ether and 2 ml of ethanol, then sonicated in 20 ml of ether for 5 minutes. An aliquot of the extract was analyzed by HPLC using a Partisil® 10 ODS-3 column developed with a 20-minute linear gradient from 0.01M NH4OAc (pH 4) to CH3CN:0.01M NH4OAc, pH 4 (98:2).

Disposition Studies
Distribution and excretion studies were performed following intravenous and dermal dose administration. Intravenous doses were administered in a tail vein and consisted of a dose volume of 1 μl/gram body weight Sorensen's buffer containing 4% rat serum albumin (w/v) and t-BP to obtain a target concentration level of 4 mg/kg. Dermal doses were prepared from 14C-labeled and unlabeled material dissolved in ether to give target application doses of 0.38, 3.4, and 39 mg/kg t-BP. Dermal dosing solutions of 20 μl were applied to a 1 cm2 shaved area on the backs of anesthetized animals. The area was secured using a 4 x 4 cm2 square of adhesive backed foam with a 3 cm2 square hole cut from the center, placed around the dosed area, and secured with Superglue® (Loctite Corp., Cleveland, OH). A piece of hard-backed waxed paper was placed over the hole and secured with adhesive tape and Superglue®. Elastic adhesive bandage (Elastoplast®, Beirsdorf, Inc., South Norwalk, CT) then was placed over the entire area and glued around the edges to the skin.

Three animals receiving dermal applications at each dose level were placed in glass metabolism cages. Urine and feces were collected separately. Urine was collected at 2, 4, 6, 8, and 24
hours, and feces at 8 and 24 hours. Volatile organics and expired CO2 were collected by drawing air from the metabolism cage at 200 - 500 ml/min, through an ethanol trap at 0°C, and through a series of 2 traps each containing 400 ml of 1N NaOH. Blood was collected by cardiac puncture at the end of the experiment (esterase activity was inhibited by addition of 12 mM physostigmine). A portion of each blood sample was separated into plasma and packed RBCs by centrifugation. Breath trap solutions were stored at room temperature. Blood was stored in the dark at 4°C until analyzed. All remaining samples were stored in the dark at -20°C.

All animals were killed by an overdose of Ketamine®/Xylazine® adminstered intravenously. Samples of all major tissues plus possible target tissues were taken for analysis of t-BP-derived 14C. Plasma and urine analyses were performed to determine total radioactivity. Duplicate aliquots of plasma (0.1-0.2 ml) and urine (0.5 ml) were added to 10 ml of Scintiverse E® (Fisher Chemical Co., Pittsburgh, PA). Water or methanol was added as needed to obtain homogenous samples for scintillation counting. Feces and large tissues were homogenized in water. Entire small tissues and aliquots of the homogenates and blood were combusted in a Packard Model 306 oxidizer (Packard Instrument Co., Downers Grove, IL). Combusted samples were stored overnight in the dark before scintillation counting.
Details on absorption:
Skin:
The stability of t-BP on isolated skin at 37°C is shown in Table 2. After 1 hour, both the fraction of t-BP that could be removed from the skin by rinsing, and the proportion of this material remaining as parent compound, were greater at 10 mg/cm2 than at lower concentrations. It appeared that t-BP was quite stable on skin for 1 hour at a concentration of 10 mg/cm2, but degraded by approximately 34% and 48% at 1.0 and 0.1 mg/cm2, respectively. Absorption or binding of t-BP increased as the doses decreased and accounted for over 30% of the lower dose within 1 hour. At 24 hours, the amount of t-BP bound or absorbed to skin accounted for approximately 70% of all doses; the amount of parent compound detected in the rinse was minimal. The major decomposition product of t-BP detected in the rinse or extract of all skin samples in this study was benzoic acid.

In intact animals, the degree of absorption of a dermal dose of t-BP apparently was not affected by the size of the dose administered in the range studied (Table 3). These data indicate that approximately 13% to 14% of the administered radioactivity was absorbed from skin (see table 2 and 3).

An estimate of dermal absorption indicated that approximately 16% of each of the dermal doses was absorbed. The nature of the material absorbed (that is, parent or degradation products of t-BP) could not be determined due to the instability of t-BP on skin and in blood.
Details on distribution in tissues:
The distribution of t-BP-derived radioactivity following i.v. or dermal administration is shown in Table 5. As could be inferred from data presented in Tables 3 and 4, the levels of radioactivity remaining in tissues 24 hours after administration were low. Further, radioactivity retained in tissues 24 hours after dosing was relatively evenly distributed throughout the tissues; in most instances, tissue/blood ratios did not vary from unity by a factor of more than 2 or 3. Skin was an exception in the dermal studies, in that the concentration of t-BP-derived radioactivity in skin was usually at least 10 times higher than that in blood. This was not seen with i.v. administration and probably represents some cross contamination from the dose site in the dermal studies. Concentrations in the intestines were high, but these data were too variable to permit speculation as to the significance of this observation. Radioactivity remaining in the tissues at the dose site in the dermal studies was quite high. Retention of radioactivity in these tissues is consistent with the binding of t-BP and/or its degradation products to skin observed in the in vitro studies described in Table 2. It is interesting to note that retention at the dose site is proportional to the dose administered.
Details on excretion:
After dermal application in intact animals approximately 13% to 14% of the administered radioactivity was absorbed from skin and eliminated in urine within the first 24 hours after dosing. Elimination in feces and expired breath (data not shown) was minimal; combined, they never accounted for more than 1% of the radioactivity administered. When a dose of 3.7 mg/kg t-BP was administered i.v. to simulate 100% absorption, most of the dose was excreted in urine within 8 hours (Table 4). Excretion in feces was minimal, and less than 0.1% of the dose was eliminated in breath (data not shown).
Metabolites identified:
yes
Details on metabolites:
Benzoic acid and t-butanol made up 93% of the major degradation and/or metabolic products.
Bioaccessibility testing results:
The toxicity of parent substance, t-BP, is limited to the site of contact. This can be seen in the results from oral repeated dose studies where effects on the stomach were observed.t-BP would not be expected to gain access to the systemic circulation of humans. The degradation products of t-BP, which are t-butanol and benzoic acid, are likely to be absorbed into the systemic circulation.

in vitro stability studies

t-BP containing a 14C label in the benzoate portion of the molecule was used in all studies of the fate and stability of this compound. Through HPLC analysis, t-BP was determined to be stable for 24 hours in corn oil at room temperature, and 97% stable for 1 hour at room temperature in a solution of 4% rat serum albumin in Sorensen's buffer, pH 7.4. This stability was sufficient to permit preparation and administration of dose solutions for oral gavage in corn oil, and i.v. administration in buffered albumin. These preparations were used in the respective in vivo studies. t-BP was stable in HEPES buffer, pH 7.4, for up to an hour at 37°C; the addition of glutathione to the HEPES buffer, however, resulted in concentration and time dependent degradation. t-BP solutions of 0.011, 0.11, and 1.1 mg/ml in HEPES buffer, pH 7.4, containing 5 mM glutathione, were degraded by 22, 18, and 10% in 15 minutes, respectively.

In vitro studies established that t-BP was not stable in rat blood at 37°C. At all concentrations studied (0.011 to 16 mg/ml), more than 50% of the t-BP degraded within 15 minutes after addition to blood. t-BP appeared to be even less stable in experiments with human blood than in those with rat blood. Half-lives of 4 mg/ml in rat and human blood were estimated to be 10.4 and 4.0 minutes, respectively. Degradation or metabolism of t-BP in microsomal or soluble enzyme preparations from rat liver was extremely rapid; less than 1% of concentrations of 0.011,

0.11 or 1.1 mg/ml could be recovered as parent compound from incubations with either fraction after 15 minutes. Benzoic acid and t-butanol made up 93% of the major degradation and/or metabolic products. t-BP was stable in Sorensen's buffer, pH 4.1, for 1 hour, but degraded in a 20% suspension of stomach contents in this buffer in a concentration-dependent fashion. t-BP concentrations of 1.1, 0.11, and 0.011 mg/ml degraded by 0, 31, and 74%, respectively, in 1 hour at 37°C in a suspension of stomach contents.

Conclusions:
In vitro studies conducted by the National Toxicology Program showed thatconcentrations of 1.1, 0.11, and 0.011 mg/ml degraded by 0, 31, and 74%, respectively, in 1 hour at 37°C in a suspension of stomach contents. However,the substance is extremely rapidly degraded in rat and human blood. These experiments resulted in half lives of 4mg/L to be 10.4 and 4.0 minutes, respectively. When in incubated with microsomal or soluble enzyme preparations from rat liver less than 1% of the parent substance remained after 15 minutes. This shows that degradation is extremely rapid also in contact with liver enzymes. Also incubation with glutathione showed thatt-BP solutions of 0.011, 0.11, and 1.1 mg/ml in HEPES buffer, pH 7.4, containing 5 mM glutathione, were degraded by 22, 18, and 10% in 15 minutes, respectively. Overall these resultsindicating that any substance absorbed in the body will have a very short half-life.

Dermal studies determined that approximately 16% of dermal doses administered to rats was absorbed and rapidly eliminated without tissue accumulation.

Similarly, t-BP given intravenously was rapidly degraded and eliminated, primarily in urine, with no apparent accumulation in any tissue.

The toxicity of parent substance, t-BP, is limited to the site of contact. This can be seen in the results from oral repeated dose studies where effects on the stomach were observed.t-BP would not be expected to gain access to the systemic circulation of humans.

Benzoic acid and t-butanol made up 93% of the major degradation and/or metabolic products.

The degradation products of t-BP, which are t-butanol and benzoic acid, are likely to be absorbed into the systemic circulation, but they are relatively nontoxic. Both t-butanol and benzoic acid have been the subjects of other studies and they do not appear to produce other (chronic) toxicity.
Executive summary:

T-BP had half-lives of 4.0 and 10.4 minutes in human and rat blood, respectively. Enzymatic or chemical degradation in the presence of liver fractions was even more rapid, demonstrating that any t-BP absorbed into the body would be expected to have a very short half-life. Benzoic acid and t-butanol constituted at least 93% of the products of enzymatic and/or chemical degradation in in vitro systems. The stability of t-BP in a suspension of stomach contents was concentration dependent but was thought to be sufficient to permit some absorption of the parent molecule into stomach tissue. t-BP rapidly degrades in the presence of glutathione, one of numerous reducing agents found in biological systems, indicates that it should have a short half-life in intact animals.

t-BP was relatively stable on isolated skin; t-BP-derived radioactivity was absorbed into or bound to skin with continued contact (Table 2). Dermal absorption of t-BP-derived radioactivity was confirmed by the observation that when placed on the skin of living animals, approximately 16% of the radiolabel from a wide range of t-BP doses was absorbed and excreted in urine in 24 hours. Due to the rapid chemical and/or enzymatic degradation of t-BP in biological systems, it was not possible to determine if the radiolabel absorbed from skin represented parent compound or products of t-BP degradation. In any case, only traces of t-BP-derived radioactivity appeared to be retained in tissues following dermal or i.v. administration.

t-BP-derived material excreted in urine was not identified because preliminary studies showed a quantitative yield of t-butanol and benzoic acid on degradation and/or metabolism. Because the radiolabel was in the benzoic acid portion of the molecule, it was assumed that material excreted in urine represented metabolites of benzoic acid. Results of previous studies conducted in this laboratory indicated that both rats and mice metabolize more than 90 percent of the benzoic acid (derived from benzyl acetate) to hippuric acid, and excrete it in urine (Abdo et al., 1985). Further, in the previous study, metabolism and elimination of benzoic acid was observed to be linear with dose, with no evidence of saturation at doses up to an equivalent of approximately 380 mg/kg benzoic acid in the rat and over 700 mg / kg in the mouse.

Endpoint:
dermal absorption, other
Remarks:
in vitro and in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March, 1985 - July, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Key result
Absorption:
ca. 16 %
Conclusions:
Dermal studies determined that approximately 16% of dermal doses administered to rats was absorbed.
Endpoint:
basic toxicokinetics, other
Remarks:
OECD QSAR ToolBox 4.2
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Executive summary:

QSAR Toolbox Metabolism profile

 

The information on the substance was entered in OECD QSAR ToolBox 4.2

 

Tert-butyl perbenzoate (t-BP)

CAS 614-45-9

CC(C)(C)OOC(=O)c1ccccc1

 

-in vivo Rat metabolism simulator (9 metabolites).

tert-Butyl alcohol

CAS 75-65-0

CC(C)(C)O

 

Benzoic acid

CAS 65-85-0

OC(=O)c1ccccc1

 

Propane-1,2-diol

CAS57-55-6

CC(O)CO

 

2-hydroxy-2-methylpropanoic acid

CAS594-61-6

CC(C)(O)C(O)=O

 

2-hydroxy-2-methylpropanal

CAS20818-81-9

CC(C)(O)C=O

 

2-methylpropane-1,2-diol

CAS 558-43-0

CC(C)(O)CO

 

Acetone

CAS 67-64-1

CC(C)=O

 

Propan-2-ol

CAS 67-63-0

CC(C)O

 

2-hydroxypropanal

CAS 3913-65-3

CC(O)C=O

 

 

- Rat liver S9 metabolism simulator and Observed rat liver metabolism with quantitative data (2 metabolites).

tert-Butyl alcohol

CAS 75-65-0

CC(C)(C)O

 

Benzoic acid

CAS 65-85-0

OC(=O)c1ccccc1

 

Description of key information

There is reliable data from the National Toxicology Program for tert-butyl peroxybenzoate.

 

In vitro studies conducted by the National Toxicology Program showed thatconcentrations of 1.1, 0.11, and 0.011 mg/ml degraded by 0, 31, and 74%, respectively, in 1 hour at 37°C in a suspension of stomach contents. However,the substance is extremely rapidly degraded in rat and human blood. These experiments resulted in half lives of 4mg/L to be 10.4 and 4.0 minutes, respectively. When in incubated with microsomal or soluble enzyme preparations from rat liver less than 1% of the parent substance remained after 15 minutes. This shows that degradation is extremely rapid also in contact with liver enzymes. Also incubation with glutathione showed thatt-BP solutions of 0.011, 0.11, and 1.1 mg/ml in HEPES buffer, pH 7.4, containing 5 mM glutathione, were degraded by 22, 18, and 10% in 15 minutes, respectively. Overall these resultsindicating that any substance absorbed in the body will have a very short half-life.

 

Dermal studies determined that approximately 16% of dermal doses administered to rats was absorbed and rapidly eliminated without tissue accumulation.

 

Similarly, t-BP given intravenously was rapidly degraded and eliminated, primarily in urine, with no apparent accumulation in any tissue.

 

The toxicity of parent substance, t-BP, is limited to the site of contact. This can be seen in the results from oral repeated dose studies where effects on the stomach were observed.t-BP would not be expected to gain access to the systemic circulation of humans.

 

Benzoic acid and t-butanol made up 93% of the major degradation and/or metabolic products. As supporting data the OECD ToolBox (4.2) was also run and the results are in line with the in vivo findings. Next to Benzoic acid and t-butanol 7 additional metabolites are predicted. Just as the two major metabolites none of these metabolites are classified for long term systemic effects.

 

The degradation products of t-BP, which are t-butanol and benzoic acid, are likely to be absorbed into the systemic circulation, but they are relatively nontoxic. Both t-butanol and benzoic acid have been the subjects of other studies and they do not appear to produce other (chronic) toxicity.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
100
Absorption rate - dermal (%):
20
Absorption rate - inhalation (%):
100

Additional information