Dibenzoyl peroxide

Endpoint summary

Description of key information

Topically applied benzoyl peroxide penetrates unchanged through the stratum corneum or follicular openings of excised human skin and is converted metabolically to benzoic acid within the skin. A study in rhesus monkeys in vivo showed that this benzoic acid is systemically absorbed as benzoate and rapidly excreted in the urine in an unchanged form, without being conjugated to hippuric acid, as would be predicted to occur following oral administration.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

The generation of free radical intermediates and in vitro and in vivo skin penetration of benzoyl peroxide (BPO) was extensively reviewed by Binder et al. (1995) and summarized as below:

Free radical intermediates

There are several potential pathways for the degradation of BPO in chemically defined and biological systems. The thermal decomposition of BPO, which is used for initiating free radical polymerizations in the plastics industry, occurs by the homolytic pathway shown in Fig. 1A (Swain, et al., 1958; Janzen, et al., 1972). However, BPO is relatively stable at physiological temperatures. Early studies demonstrated that BPO is converted to benzoic acid in skin (Nacht, et al., 1981; Morsches and Holzmann, 1982).

Work from the laboratory of T. W. Kensler has provided evidence that in mouse keratinocytes, BPO is degraded according to the one electron pathway shown in Fig. 1, with copper (Cu¹⁺) serving as the electron donor (Kensler, et al., 1988; Swauger, 1991; Swauger, et al., 1991). The evidence supporting this conclusion is summarized below.

When [¹⁴C-carbonyl]-BPO was incubated with primary keratinocytes from neonatal SENCAR mice, or cell extracts prepared from keratinocytes, the major stable metabolite was benzoic acid (Kensler, et al., 1988; Swauger, 1991). In reaction mixtures containing extracts of primary keratinocytes, 7.5% of the radioactivity was evolved as [14C]-CO₂(Swauger, 1991). This evolution of CO₂was inhibited by the metal chelator O-phenanthroline, implicating the involvement of metals in the reaction. Incubation of BPO with copper (Cu¹⁺) was also found to cause breakdown of the diacyl peroxide to benzoic acid. However, CO²evolution was not detected in incubations with copper (Swauger, 1991; Swauger, et al., 1991). Spin trapping and electron paramagnetic resonance (EPR) spectroscopy were used to characterize free radical intermediates formed during BPO metabolism. When the spin trap N-t-butyl-phenylnitrone (PBN) was incubated with keratinocyte extracts and BPO, or copper and BPO, benzoyloxyl-PBN radical adducts were detected (Swauger, 1991). In earlier work from the same laboratory, EPR spectra characteristic of carbon-centered radical adducts were detected when PBN or the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) were incubated with primary keratinocytes (Kensler, et al., 1988). These radical adducts were attributed to the reaction of phenyl radicals with the spin traps. Overall, these results support the conclusion that in cultured mouse keratinocytes benzoyloxyl radicals are formed from BPO. These yield benzoic acid through hydrogen abstraction, while a relatively minor pathway involves the decarboxylation of the benzoyloxyl radical yielding the phenyl radical and CO₂(Fig 1B).

In extracts of primary keratinocytes the decomposition of BPO to benzoic acid was only partially inhibited by prior heat inactivation or incubation at 4° C as compared to 37° C, suggesting that the initial scission of the peroxide bond is not an enzymatic process, but rather depends on metals (Swauger, 1991). The metal involved probably is copper since, as noted above, Cu¹⁺causes the formation of benzoyloxyl radicals from BPO. Also, copper activates BPO to a species, presumably the benzoyloxyl radical that can induce DNA single strand breaks, whereas, other transition metals including molybdenum, ferric and ferrous iron, and zinc do not (Swauger, et al., 1991). In contrast to scission of the peroxide bond, the decarboxylation of the benzoyloxyl radical is temperature-dependent and blocked by heat inactivation of keratinocyte extracts, suggesting that this reaction is a specific, enzyme-mediated process (Kensler, et al., 1988; Swauger, 1991). In further support of this conclusion, phenyl radical adducts were not detected when BPO was reacted with copper in the absence of cell extracts (Swauger, 1991; Swauger, et al., 1991).

While copper has been implicated in the degradation of BPO by mouse keratinocyte extracts, BPO interacts with other metals. The induction of lipid peroxidation by BPO in rabbit dental pulp microsomes was dependent on the presence of transition metals (Terakado, et al., 1984). Copper (Cu²⁺) was most effective (Cu¹⁺was not examined), but ferrous and ferric iron were moderately effective, and zinc, nickel, and magnesium were slightly effective in stimulating lipid peroxidation by BPO in this system.

Rat liver microsomes have also been found to metabolize BPO to the same products as mouse keratinocytes (Greenley and Davies, 1993). In incubations with BPO, DMPO, and microsomes, benzoyloxyl, phenyl and hydroxyl radical adducts were detected, as well as an additional carbon-centered radical adduct This latter spin adduct apparently was due to abstraction of hydrogen from dimethyl sulfoxide, which was included to increase the solubility of BPO. Radical formation in this system was dependent on reducing equivalents from NADPH or NADH and was probably mediated by cytochrome P-450, based on inhibitor studies with other peroxides. The basis for formation of hydroxyl radicals in this system was not established. One possible explanation suggested by the authors was oxidation of the spin trap by cytochrome P-450 to a radical-cation, followed by hydration. These investigators also studied the photodegradation of BPO by UV light emitted by an unfiltered mercury/xenon arc lamp, to aid in analysis of EPR spectra generated in the experiments with microsomes. Again, benzoyloxyl, phenyl and hydroxyl radical adducts were detected. Under these conditions the hydroxyl radical adduct was presumed to be due to photo-ionization and then hydration of the spin trap.

In contrast to the results obtained with rat liver microsomes, BPO did not yield detectable spin adducts when incubated with intact rat liver mitochondria in the presence of DMPO or PBN (Kennedy, et al., 1989a, b). BPO inhibits mitochondrial respiration in the micromolar concentration range, which leads to production of hydroperoxyl and hydroxyl radical adducts in preparations of respiring submitochondrial particles (SMP) (Kennedy, et al., 1989b). This effect is consistent with inhibition of respiratory electron transport, and was not observed with nonrespiring SMP, and no BPO-derived radicals were detected. In intact mitochondria the radical species apparently are effectively scavenged, and with SMP preparations production of radical adducts could be completely blocked by addition of catalase and superoxide dismutase. BPO was rapidly degraded when incubated with mitochondria, and this degradation was inhibited about 75% by heat inactivation of the organelles. No lipid soluble metabolites of BPO were detected in extracts of BPO­ treated mitochondria, and the only water soluble product detected was benzoic acid. The metabolism of BPO by mitochondria appears to proceed through a two electron pathway (Fig. 1A), which is not free radical-mediated and leads only to the production of benzoic acid (Kennedy, et al., 1989a, b).

The potential of BPO to form free radical intermediates has also been studied with freshly isolated or cultured human keratinocytes by Iannone, et al., 1993. These investigators used EPR and the spin traps 3,5-dibromo-4-nitrosobenzene sulfonic acid and a-(4-pyridyl-1-oxide) -N-t-butylnitrone in their analyses. Although radical adducts were detected when human keratinocytes were incubated with cumene hydroperoxide or t-butyl hydroperoxide, none were detected in incubations with BPO. This finding may be important to understanding potential differences in the responses of human and mouse skin to BPO. Unfortunately, the experiment. al conditions used in these studies with human epidermal keratinocytes were not identical to those used in earlier studies with murine cells, so direct comparisons are somewhat uncertain. It would be valuable to have comparative studies performed with cells from both species in parallel under the same conditions.

Timmins and Davies (1993) used novel techniques to examine the potential of organic peroxides to form free radical intermediates in intact mouse skin. Test substances were applied to excised skin placed in a custom-made EPR spectrometer sample cell, then EPR spectra were recorded. Free radical generation by the peroxides was observed by two methods: 1) detection of the EPR spectrum of the ascorbyl radical formed by interaction of peroxide-derived radicals with endogenous ascorbate, or 2) spin trapping by pretreatment of the skin with DMPO prior to the peroxide treatment. Using these approaches, ascorbyl radicals and DMPO-radical adducts were detected with cumene hydroperoxide, t-butyl hydroperoxide and t-butyl peroxybenzoate. However, BPO and lauroyl peroxide did not produce measurable quantities of ascorbyl radicals or DMPO-radical adducts. The authors attributed the lack of detectable radical production by the two diacyl peroxides to relatively poor penetration ·through the stratum corneum compared to that for the lower molecular weight hydroperoxides tested. Removal of the stratum corneum by tape-stripping was found to markedly increase the concentration of ascorbyl radicals induced by t-butyl hydroperoxide, demonstrating that the ascorbyl radical is not generated in the stratum corneum, and that this layer is a barrier to the penetration of the peroxide. Although it is possible that BPO treatment did cause free radical formation at levels below the limits of detection, the results of Timmins and Davies (1993) suggest that treatment of intact mouse skin with BPO does not necessarily result in radical generation. The fact that the normal barrier function of the stratum corneum can limit the effects of higher molecular weight peroxides is relevant to the extrapolation of the mouse skin effects of BPO to humans.

Timmins and Davies (1993) used Lac a mice for their EPR studies with intact skin, whereas Kensler, et al. (1988) and Swauger (1991) used SENCAR mice for their analyses of radical production in keratinocytes and keratinocyte extracts. It is not known whether the Lac a mouse responds to the tumor promoting activity of BPO. Since different strains and stocks of mice vary substantially in their sensitivity to tumor promotion by BPO, it would be of value to compare the production of free radicals by BPO in intact skin from various strains or stocks of known responsiveness.

Skin penetration and metabolism in vitro

Nacht, et al. (1981) and Yeung, et al. (1983) examined the penetration and disposition of BPO with surgical specimens of human skin. Each skin sample was placed in a two chambered diffusion cell, and acted as a diaphragm separating the chambers. The lower chamber was filled with physiological saline. [¹⁴C]-labeled BPO was applied to the skin surface at concentrations ranging from 2.5 - 10% in a lotion or aqueous emulsion vehicle. Over 8 hours about 2 - 3% of the applied radioactivity was recovered in the receptor chamber, and all of this was in the form of benzoic acid, while 2.6 - 4% was retained within the skin. Approximately half of the radioactivity extracted from skin was unchanged BPO, and the remainder was benzoic acid. Under the conditions of these experiments, most of the applied BPO remained unchanged on the surface of the skin. These data, and similar results reported by Morsches and Holzmann (1982), demonstrated that BPO is converted to benzoic acid in human skin.

Skin penetration and metabolism in vivo

Wepierre, et al. (1986) examined the penetration and metabolism of BPO in hairless Sprague-Dawley rats. A gel containing 10% [14C]-BPO was topically applied, under conditions which prevented grooming, then treated animals were euthanized at 3, 8 and 24 hours after treatment. After removal of the remaining test substance, the stratum corneum was collected by tape stripping and the skin was frozen and sectioned with a cryostat parallel to the skin surface. The total amount of BPO absorbed into the skin ranged from 12 - 18% of the applied dose, and most of this was found in the upper stratum corneum as BPO. While radioactivity decreased with depth in the skin, the relative amount of benzoic acid increased from the stratum corneum to the deep dermis. A stable concentration gradient of radioactivity from the stratum corneum to the deep dermis was observed at the three sampling limes. In the stratum corneum, epidermis and dermis, benzoic acid constituted about 23, 59 and 74% of the radioactivity, respectively, with the remainder being BPO.

Sahut., et al. (1985) reported that topically applied BPO is rapidly eliminated in New Zealand rabbits. These investigators applied 10% BPO in three different lotion or gel vehicles daily to rabbits for 33 days, and measured plasma benzoic acid concentrations at 0,5,12, 19, 26, and 33 days. Plasma levels of benzoic acid peaked within 1/2 hour after dosing and then fell off sharply. The concentrations of benzoic acid in plasma 24 hours after dosing were low, but greater than endogenous levels. However, in rabbits treated repetitively with BPO over 33 days there was no evidence of long-term accumulation of benzoic acid.

Nacht, et al. (1981) and Yeung, et al. (1983) examined the percutaneous penetration and metabolism of BPO in vivo in rhesus monkeys. An acetone solution of carrier-free [¹⁴C]-BPO was applied to the forearms of rhesus monkeys restrained in metabolism chairs (Nacht, et al., 1981). After 24 hours any remaining test substance was removed by washing and the monkeys were transferred to metabolism cages. Approximately 45% of the applied radioactivity was recovered in urine over 6 days, and 95% of the urinary radioactivity was in the form of unconjugated benzoic acid (Nacht, et al., 1981). No hippuric acid was detected in urine, indicating that the renal excretion was sufficiently fast that BPO-derived benzoic acid did not reach the liver, where it would have been conjugated with glycine to form hippurate. Three minor, polar, urinary metabolites were detected by thin layer chromatography after topical application, but not after intramuscular injection of the peroxide. These were not identified. By seven days after dosing, radioactivity was no longer detectable in urine. When [¹⁴C]-BPO was topically applied to rhesus monkeys in a lotion vehicle at concentrations ranging from 2.5 to 10%, there was a concentration­ dependent excretion of radioactivity in urine (Yeung, et al. 1983). Benzoic acid constituted 99.5% of the urinary radioactivity. Four minor urinary metabolites were detected, but not identified. Two of these were more polar and two were Jess polar than benzoic acid, but no hippurate was detected. If some fraction of topically applied BPO reached the systemic circulation, it would be expected to be metabolized by the liver to benzoic acid (Greenley and Davies, 1993; Kennedy, et al., 1989a. b), and then conjugated to form hippurate. The lack of detection of hippuric acid in the urine of monkeys, provides evidence that topically applied BPO is not systemically distributed as the unmetabolized parent compound. Consistent with this conclusion Morsches and Holzmann (1982) were able to detect benzoic acid, but not BPO, in the serum of leg ulcer patients treated with an oil/water emulsion containing 20% BPO. However, the analytical methods used were insensitive, with a limit of detection of 1 µg/ml BPO.

Seubert, et al. (1984) investigated the penetration of BPO into the stratum corneum of human volunteers from a commercial acne medication containing 10% BPO. The stratum corneum was sampled at various time points at different sites by repetitive tape stripping, and BPO was shown to be at least partially converted to benzoic acid in this epidermal layer. Under the conditions of this study, in which excess BPO was washed off after 2 minutes, the stratum corneum did not serve as a reservoir of the drug.

In summary, the major metabolite of BPO in skin is benzoic acid, which is rapidly eliminated in urine after entering the bloodstream. As indicated in Fig. 1, the formation of benzoic acid from BPO does not necessarily require free radical intermediates. The available data provide evidence that topically applied BPO does not enter the systemic circulation as the parent compound. BPO is unlikely to induce any systemic toxicity as a result of its metabolism to benzoic acid. Benzoate is a normal dietary constituent of low toxicity, and sodium benzoate has been found to be noncarcinogenic in a chronic drinking water study (Toth, 1984).

Discussion on the oral absorption rate

There are no data on the oral absorption of BPO. However, more than 50% of BPO is hydrolysed at each of pH 4, 7 and 9 (at 50±0.5ºC) after 2.4 hours in abiotic conditions and benzoic acid, the expected main degradate, was present in the hydrolysed solutions at each test pH (Sydney, 2010). Therefore, an hydrolysis of BPO to benzoic acid is expected to occur in the stomach after an oral administration. Should any residual BPO be absorbed, it would be destroyed by liver peroxidases. In man and rat, almost 100% of ingested benzoic acid can be accounted for within 24 hours as urinary hippuric acid (Bridges et al., 1970). Given the rapidity of the hydrolysis reaction to benzoic acid, and the readily absorption of benzoic acid, the oral bioavailability of BPO can be considered to be close to 0 as BPO and 100% as benzoic acic.

Discussion on the dermal absorption rate

Species comparison of in vitro skin penetration of benzoyl peroxide shown that the substance is well more absorbed by SKH1 hairless mouse than by F 344 rats, B6C3F1 and human cadaver skin (Nash, 1996). Furthermore, from the study of Nacht et al. (1981), the dermal absorption of benzoyl peroxide can reasonably be estimated lower than 50 % in human (in vitro: percutaneous penetration of benzoyl peroxide through excised human skin was less than 5 % in this study/ in vivo: in monkeys, when benzoyl peroxide dissolved in acetone was applied at low dose to the ventral forearms, about 45 % of the applied radioactivity was recovered in urines). However, this value did not reflect the systemic bioavailability of BPO, which is closed to zero, as BPO is converted to benzoic acid in the skin before to reach the blood stream.