26-Week Dermal Oncogenicity Study Evaluating Pure trans -Capsaicin in Tg.AC Hemizygous Mice (FBV/N)

Abstract

The objective of this study was to assess the oncogenic potential of trans-capsaicin when administered weekly via topical application to the dorsal skin of Tg.AC mice for 26 weeks. Male and female Tg.AC mice (25 mice/sex/group) received dose formulations containing trans-capsaicin dissolved in diethylene glycol monoethyl ether (DGME). The positive control was tetradecanoylphorbol-13-acetate (TPA) dissolved in DGME. Appropriate controls, including a topical lidocaine local anesthetic pretreatment (4% w/w), were maintained. All groups were dosed once weekly, except for the TPA group, which was dosed twice per week. Analysis of the macroscopic observations after the final sacrifice revealed no noteworthy treatment-related findings, with the exception of dermal masses that were randomly dispersed throughout all treatment groups for both males and females. The frequency of dermal masses in the capsaicin-treated groups (at a dose level of up to 102 mg/kg and an application rate of 25.6 mg/cm²/kg/week) was not elevated in comparison to either concurrent vehicle or untreated controls. In contrast, a notable increase in the frequency of dermal masses was observed in the TPA-treated mice compared to both the concurrent vehicle and untreated controls. Dermal application of capsaicin resulted in no increased incidence of preneoplastic or neoplastic skin lesions. In contrast, over half the male and female mice exposed to TPA had multiple skin papillomas; the majority of the TPA-treated animals either died early or was humanely euthanized due to tumor load. Spontaneously occurring neoplasms were not appreciably increased in capsaicin-treated animals. Capsaicin-related non-neoplastic microscopic findings were seen sporadically in both genders and included acanthosis, hyperkeratosis/parakeratosis (primarily females), epidermal crusts, subepidermal fibrosis, epidermal ulcerations/erosions, and chronic-active inflammation. There was no evidence of a dose response in either the incidence or severity of these findings. The lidocaine- (at a dose level of 162 mg/kg and at an application rate of 40.5 mg/cm²/kg/week) and DGME-treated (at a dose level of 4.0 g/kg and at an application rate of 1 g/cm²/kg/week) control groups also did not display any evidence of increase in dermal masses. Based on these results, trans-capsaicin, lidocaine, and DGME should be considered nononcogenic in the Tg.AC mouse dermal model.

The trans-geometric isomer of capsaicin, or trans-8-methyl-N-vanillyl-6-nonenamide (Figure 1), is a highly selective agonist for the transient receptor potential vanilloid receptor 1 (TRPV1; also known as VR1 according to older nomenclature) (Cortright and Szallasi 2004). TRPV1 is a ligand-gated, nonselective, cation channel preferentially expressed in small-diameter, primary afferent neurons (C-fibers and Aδ-fibers), especially no-ciceptive sensory nerves. TRPV1 responds to noxious stimuli, including capsaicin, heat, and extracellular acidification, and integrates simultaneous exposures to these stimuli (Cortright and Szallasi 2004). Based on the highly selective agonistic property of capsaicin towards TRPV1 receptors, drug products containing pure synthetic trans-capsaicin are under evaluation as a treatment for chronic neuropathic pain conditions after topical administration (Bley 2004).

Capsaicin is also the most abundant pungent molecule in chili peppers and thus represents an important ingredient in spicy foods. Although there are two geometric isomers of capsaicin, only trans-capsaicin occurs naturally (Cordell and Araujo 1993), and thus the term ‘capsaicin’ is used generically to refer to the trans-geometric isomer. The capsaicin content of chili peppers ranges from 0.1% to 1% w/w (Govindarajan and Sathyanarayana 1991). In addition to its role as a food additive throughout the world, there is also substantial human experience with capsaicin in the form of nonprescription (in the United States) or prescription (in the European Union) topical analgesics, and self-defense products (e.g., pepper spray). Separate from its role as a modulator of the sensory nervous system, capsaicin has a history of use for the improvement of gastrointestinal ailments and lowering of blood pressure, and herbal extracts are available as food supplements (as an example, see http://www.naturesway.com/NaturesWay/productdetail.aspx?productid = 11500).

Published information on the potential genotoxicity of capsaicin is inconsistent (Table 1). Both positive and negative effects have been found in classical genetic toxicology assays (Surh and Lee 1995; Azizan and Blevins 1995). Results from eight sets of bacterial point mutation assays (including Ames assays using various strains of Salmonella typhimurium) with capsaicin of varying origins were published between 1981 and 1995; various forms of S9 activation were performed in seven of the eight assays. Four of these assay sets resulted in positive responses and four in negative responses. Point mutation tests in Chinese hamster V79 cells were conducted twice, resulting in one positive response and one negative response. Data from one micronucleus and sister-chromatid exchange study in human lymphocytes were interpreted to show that capsaicin is genotoxic (Marques et al. 2002). Capsaicin was also reported to induce DNA strand breaks in human SHSY-5Y neuroblastoma cells (Richeux et al. 1999). However, two recent publications based on studies using only pure capsaicin and conducted according to strict Good Laboratory Practice (GLP) standards show that pure capsaicin is nongeno-toxic (Chanda et al. 2004; Proudlock, Thompson, and Longstaff 2004).

In contrast to numerous in vitro studies, there are only three reports investigating the carcinogenic potential of capsaicin extracts in animal models. The in vivo micronucleus test was conducted once in mice and considered positive (Nagabhushan and Bhide 1985). Capsaicin extracted from chili peppers induced duodenal adenocarcinomas in mice (Toth, Rogan, and Walker 1984) and acted as a stomach and liver tumor promoter in BALB/c mice (Agarwal et al. 1986). However, the capsaicin materials tested in these studies were natural extracts and may not exhibit the same toxicological or safety profile as pure trans-capsaicin.

The objective of the present study was to assess the oncogenic potential of pure trans-capsaicin when administered weekly via topical application to the dorsal skin of the Tg.AC transgenic mice for 26 weeks. The hemizygous Tg.AC mouse model has been used historically for dermally applied nongenotoxic compounds to detect carcinogenicity potential more rapidly than conventional rodent carcinogenicity bioassays (Thompson, Rosenzweig, and Sistare 1998; Thompson et al. 2003; Torrey et al. 2005). Additionally, diethylene glycol monoethyl ether (DGME) was used in the present study as a vehicle to dissolve and administer the positive control tetradecanoylphorbol-13-acetate (TPA) and the test article; DGME has not been reported previously as a vehicle in any in vivo oncogenicity study. To the best of our knowledge, lidocaine cream (4% w/w), a commonly used topical anesthetic, has also never been tested for dermal oncogenic response.

MATERIALS AND METHODS

The studies reported here were conducted according to the principles of Good Laboratory Practice (GLP) standards.

Capsaicin

The trans-capsaicin (CAS no. 404-86-4) used in the studies described in this paper was manufactured under current Good Manufacturing Practice (cGMP) conditions and had ≥99% purity. The only quantifiable impurity was the cis-stereoisomer (Figure 1) of capsaicin (CAS no. 25775-90-0), also known as zu-capsaicin or civamide. For the purposes of this article and consistent with general usage, the trans-capsaicin used in all of the studies will be referred to simply as capsaicin.

Animal Treatments

All procedures conducted on animals in this study were in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare. TGAC-T (hemizygous), FVB/NTac-Tg(v-Ha-ras)TG.ACled mice for this study were purchased from Taconic Farms (Germantown, New York, USA). Animals were allowed to acclimatize for at least 1 week. Mice used in the study at initiation of treatment were approximately 8 to 10 weeks old and weighed between 17 and 32 g. Animals were individually housed in suspended, stainless-steel cages. Mice were fed Certified Rodent Diet no. 8728C (Harlan Teklad) ad libitum and had access to water ad libitum. Samples of the water and diet were routinely analyzed for specified microorganisms and environmental contaminants. Environmental controls for the animal room were set to maintain 18°C to 26°C, a relative humidity of 30% to 70%, a minimum of 10 air changes/h, and a 12-h light/12-h dark cycle. Animals were assigned to the study using a computerized procedure designed to achieve body weight balance with respect to groups. Prior to group assignment, animals were excluded from the selection pool/sex to produce minimal variation. After group assignment, the mean body weight for each group/sex was statistically different at the 5.0% probability level, as indicated by analysis of variance F probability. After the acclimation period, mice were divided in seven groups as shown in Table 2. Following randomization, each study animal was assigned a unique number by means of an implantable microchip identification device in the lower lumbar region.

Dose levels for capsaicin were selected based on data from a 1-month range-finding study in FVB/N mice (data not shown). Briefly, in that study, FVB/N mice received capsaicin at 0.64, 1.28, 2.56, and 5.12 mg/animal/week for 4 weeks. Dermal irritation in the form of erythema, desquamation or scaling, and fissuring and escharing were noted in some of the mice at the 5.12 mg/animal/week dose group only. Based on that observation, 2.56 mg/animal/week was selected as the highest dose level for the 26-week study. Additionally, in the pilot study, plasma sample analysis revealed that the mice were exposed systemically to capsaicin after topical application and the exposure generally increased with increasing dose levels. After topical application, capsaicin slowly appeared in the plasma, with T _max (time to reach maximum plasma concentration) values ranging from 4 to 12 h. Measurable values were still detectable in several animals at 24 h post dose, especially at the 2.56 and 5.12 mg/animal/week dose levels. The C _max (maximum plasma concentration) for capsaicin in males was 9.17, 28.4, 43.0, and 51.5 ng/ml and in females was 24.2, 24.7, 71.4, and 84.8 ng/ml in the 0.64, 1.28, 2.56, and 5.12 mg/animal/week dose groups, respectively. The increases in the AUC_0−t for males and females were essentially proportional to the increases in dose level. The dermal route of administration was selected, as it is the intended route of administration in humans for most capsaicin-based medical products.

On the day prior to the first dose of the main study, and at least weekly thereafter (as needed), an area of dorsal skin larger but including the application site was clipped free of hair to allow uniform application of doses and clear observation of the application site. An area, approximately 2 cm by 2 cm of dorsal skin at the intrascapular region, was the site of application. Backs of group 7 (untreated) animals were also clipped.

Groups 1 though 4 were treated once weekly for 26 weeks at a fixed dose volume of 0.1 ml/animal/week. Treatment continued through the day before necropsy. The test (capsaicin) or vehicle-control (DGME; Sigma-Aldrich, St. Louis, MO) article was applied uniformly using a microliter pipette directly to the back of each animal. As capsaicin is known to produce localized pungency, a local anesthetic, lidocaine 4% cream (L· M·X4^®, Ferndale Laboratories, Ferndale, MI, USA) was applied (approximately 0.1 ml/animal, just enough to cover slightly beyond the application area) to the treatment site approximately 30 to 45 min before the application of the test or vehicle-control article. After 30 to 45 min, any residual lidocaine cream was wiped off with dry gauze before the test or vehicle control article application. Approximately 3 h after application of the test or vehicle-control article, the application area was cleaned with a specially designed cleansing gel (containing approximately 90% polyethylene glycol), which is intended to thoroughly remove capsaicin from skin) (approximately 0.2 m/animal on gauze). Group 5 mice (serving as the local anesthetic control) were administered lidocaine only, once a week dermally for 26 weeks at approximately 0.1 ml/animal/week, just enough to cover slightly beyond the 4-cm² treatment area. After 30 to 45 min, any residual lidocaine was wiped off with dry gauze. Group 6 mice received the positive-control article (TPA; Sigma-Aldrich) dermally with a calibrated pipette directly and uniformly to the application site twice per week for 26 weeks at a fixed dose volume of 0.2 ml per application. Approximately 3 h after application of the positive-control article, the application area was cleaned with the cleansing gel (approximately 0.2 ml/animal on gauze). Group 7 mice served as untreated control.

Body weights were taken once prior to treatment, prior to dosing on the first day of dosing, and weekly thereafter. Food consumption was measured weekly. Dermal or local irritation observations were recorded daily for all animals and groups during week 1 prior to dosing, at least weekly thereafter, and on the day of scheduled sacrifice; the application site was scored/graded using a modified Draize technique.

A necropsy was performed on animals that died or were sacrificed at an unscheduled interval. All positive-control animals having 15 or more dermal masses (clumped multiple dermal masses) or 15 or more individual dermal masses/animal were humanely euthanized. After 26 weeks of treatment, all surviving animals were weighed, anesthetized by sodium pentobarbital injection, and exsanguinated. A necropsy was performed on each animal. Tissues (when present) from each animal were preserved in 10% neutral-buffered formalin or Modified Davidson’s fixative. For all animals, macroscopic lesions, a representative skin section (clear of dermal masses) from the treated and untreated areas and skin (treated and untreated) containing dermal masses, if present, were examined microscopically.

Dose Concentration Verification

Capsaicin and TPA in DGME were analyzed using a reverse-phase, high-performance liquid chromatography (HPLC) on weeks 1, 4, 13, and 26.

Statistical Evaluation

One-way analysis of variance (ANOVA) (Winer 1971) was used to analyze body weight and food consumption data. If the ANOVA was significant (p ≤ .05), Dunnett’s t test (Dunnett 1955, 1964) was used for control versus treated group comparisons.

RESULTS

Concentration verification analysis revealed that the capsaicin concentrations of the prepared formulations ranged from 93.9% to 108.3% and the TPA concentrations ranged from 88.4% to 106.6% of target (data not shown).

The numbers of males and females surviving to terminal sacrifice were lowest for those receiving TPA, with 19 of 25 males and 17 of 25 females either dead or moribund prior to terminal sacrifice (Table 3). Among mice dosed with DGME or capsaicin, early deaths and moribund sacrifices ranged from 1 of 25 (DGME males) to 9 of 25 (0.64 mg capsaicin/animal/week, females). Among males and females dosed with TPA, early death/moribundity was mostly due to large numbers of skin papillomas. Hematopoietic system alterations (erythrocytic leukemia and lymphosarcoma) and odontogenic (tooth) neoplasms were also commonly associated with some early death/moribundity. These conditions have been described in an article by Mahler et al. (1998) as spontaneous neoplasms in Tg.AC mice. Therefore, exposure to the test article did not appear to be a causative factor for early death/moribund sacrifice.

Once-a-week topical application of capsaicin to male and female Tg.AC mice for 26 weeks did not result in an increased incidence of preneoplastic or neoplastic (papillomas) skin lesions (see Table 4). Moreover, neither the lidocaine topical anesthetic nor DGME caused an increased incidence of preneoplastic or neoplastic skin lesions. In contrast, over half the male and female mice exposed to TPA (twice a week) had multiple skin papillomas; the majority of the positive-control animals died early or were sacrificed in a moribund condition. Spontaneously occurring neoplasms were not appreciably increased in capsaicin-treated animals.

Capsaicin-related non-neoplastic microscopic findings of the treated skin were seen sporadically in both sexes treated with 0.64, 1.28, or 2.56 mg/animal/week and included acanthosis (thickening of the epidermis), hyperkeratosis/parakeratosis (primarily females), epidermal crusts, subepidermal fibrosis, epidermal ulcerations/erosions, and chronic-active inflammation (Table 4). There was no evidence of a dose response in incidence or severity of these findings. Acanthosis (12/25), crust (serocellular) formation (11/25), and subepidermal fibrosis (9/25) were more frequent in the high-dose males, and ulcer/erosion and chronic-active inflammation were more frequent in the mid- and high-dose males (3/25, 4/25; 1/25, 3/25, respectively). In the females, hyperkeratosis/parakeratosis (14/25) was most frequent in the low-dose animals, acanthosis had a higher incidence in the low- and mid-dose groups (10/25, 12/25, respectively), and epidermal crust formation (6/25) was more frequent in high-dose animals. A low incidence of numerous morphologically similar lesions was also present in the treated skin collected from control animals, suggesting that the control article and/or treatment procedure, such as weekly shaving of the treatment site, contributed to lesion incidence and severity.

Clinical signs of toxicity were noted in groups 1 to 6 and no noteworthy signs of toxicity were observed in the untreated group 7 animals; these signs included rough haircoat and random masses in all treated mice (groups 1 to 6). No remarkable dermal atonia, desquamation, edema, erythema, eschar, or fissuring were observed (data not shown).

No dose-related decreases in mean body weight or overall (weeks 1 to 26) mean body weight change were noted for any of the capsaicin-treated group 2, 3, or 4, compared to vehicle control (data not shown). The food consumption data revealed no remarkable changes and differences among the different groups (data not shown).

Macroscopic findings were similar for both unscheduled-and terminal-sacrifice animals, with the most frequently observed gross lesions consisting of masses in the nonglandular (forestomach) and glandular region of the stomach, skin (other than treatment site), oral cavity, salivary gland, and treated skin (Table 5). Squamous papilloma was the most frequent microscopic diagnosis for the masses in the treated skin (Table 4) and skin other than the treatment site (often involving the anterior one half of the body) (Table 6). Squamous papilloma was also the most frequent microscopic diagnosis for the masses in the forestomach (Table 6). Oral cavity masses consisted primarily of odontogenic tumors, such as ameloblastoma and odontoma (Table 6), and the salivary gland masses consisted of one adenoma and one carcinoma (data not shown). None of these masses were considered unusual for this strain of mice or related to administration of the test article. Non-neoplastic macroscopic findings, such as abrasion or crust formation in skin, were randomly dispersed throughout treatment groups 1 to 7 for both males and females, with greater incidences observed in the mid- and high- capsaicin, and positive-control groups (Table 5). Other gross observation occasionally noted in males consisted of distended urinary bladder. Differentiation between the influence of the test article, control articles, and/or treatment procedure on non-neoplastic gross alterations could not be determined microscopically.

All remaining macroscopic and microscopic pathology findings, including those involving the spleen, liver, lung, head, and/or skin (other than treated skin), were either consistent with changes commonly associated with the Tg.AC mouse (Mahler et al. 1998) or considered to be incidental and unrelated to test article administration (data not shown).

DISCUSSION

The present study constitutes the first in vivo evaluation of the oncogenic potential of pure capsaicin, the primary pungent ingredient of chili peppers, in a transgenic rodent model. The Tg.AC mice used in our study have been shown to have a high sensitivity for the detection of skin papillomas caused by nongenotoxic carcinogens (Torrey et al. 2005). Moreover, this model is used increasingly to evaluate the carcinogenic potential of nongenotoxic compounds and drug substances in a shorter time than possible using conventional carcinogenicity bioassays. The concentration of capsaicin applied to the treated skin areas was quite high, as the lowest capsaicin dose level in the present study (0.64 mg/0.1 ml) corresponds to a concentration of 20.9 mM. Accordingly, the absence of significantly increased dermal masses or preneoplastic and neoplastic lesions in skin exposed to very high concentrations of capsaicin entails that capsaicin should not be considered oncogenic under the current exposure and application conditions.

Capsaicin has previously been studied in animals for possible mutagencity, carcinogenicity, and target organ toxicities. However, almost all of these studies used pepper plant extracts, which are likely to display varying degrees of capsaicinoid content and possibly diverse impurity profiles. A typical capsaicin extract is a mixture of trans-capsaicin (cis-capsaicin does not occur naturally) and other capsaicinoids (including norhydrocapsaicin, dihydrocapsaicin, homocapsaicin, homodihydrocapsaicin). The actual percent of capsaicin and other capsaicinoids will vary depending on the peppers used and method of extraction. In fact, the United States Pharmacopoeia defines capsaicin as a product that contains >55% capsaicin and the combination of capsaicin and dihydrocapsaicin to be >75%; total capsaicinoid content may be as little as 90% ( United States Pharmacopoeia 2005). Additionally, pepper extracts are expected to contain chemical entities other than vanilloid compounds; these impurities may be the contributing factor in some of the toxicities observed. For instance, we have reported (Chanda et al. 2004) that pure capsaicin displays a different genotoxicity profile than that of extracts described in some previous literature.

In contrast to the high dose levels used in toxicology studies, human exposure to dietary capsaicinoids (a mixture of capsaicin, dihydrocapsaicin, norhydrocapsaicin, homocapsaicin, and ho-modihydrocapsaicin) in the United States and in European countries is about 1.5 mg/day, which translates into, at most, 0.025 mg/kg/day. In Mexico and in the Asian countries like Korea, Thailand, and India, the daily intake of capsaicinoids can be as high as 150 mg/day, which translates into about 2.5 mg/kg/day (Govindarajan and Sathyanarayana 1991). After reviewing existing literature and data, a recent report from the European Commission’s Scientific Committee on Food (2002) concluded that the available evidence does not allow establishment of a safe maximum exposure level for capsaicinoids in food.

Some epidemiological studies have suggested a correlation of stomach cancer incidences with geographic areas known to consume a chili pepper-rich diet (e.g., Mexico) (Archer and Jones 2002; Lopez-Carrillo, Avila, and Dubrow 1994; Lopez-Carrillo et al. 2003). However, the inference of causal connections between capsaicin and cancer based on these epidemiological studies is problematic, as factors like smoking and exposure to environmental pollutants that are found commonly in developing countries are difficult to exclude.

There is also substantial published literature on the anticarcinogenic effects of capsaicin, as capsaicin has been shown to inhibit growth of or induce apoptosis in a wide variety of tumor and cancer cell lines (Surh et al. 1995; Surh and Lee 1995; Surh 2002). Recent studies confirm and extend these observations to cell lines derived from human colon cancer (Kim et al. 2004), human C6 glioma (Qiao et al. 2005), human hepatoma (Kim, Kang, and Lee 2005), human breast epithelial cells (Kang et al. 2003), human leukemia (Ito et al. 2004), and murine bladder tumor (Lee et al. 2004). Corresponding activity has been observed in vivo against both human leukemic and prostrate tumor PC-3 xenographs in mice, with few or no toxic effects on the mice reported (Ito et al. 2004; Sanchez et al. 2006; Mori et al. 2006).

Mechanisms postulated to account for capsaicins’ anticancer activities include inhibition of nuclear factor kappa B (NF κ B) activation (Agarwal et al. 2004; Demirbilek et al. 2004), increase of reactive oxygen species production (Ito et al. 2004), inhibition of mitochondrial respiration (Hail and Lotan 2002), inhibition of a tumor-specific surface hydroquinone (NADH) oxidase with protein disulfidethiol interchange activity (Chueh et al. 2004), affecting ras-downstream signaling molecules (Kang et al. 2003), and the inhibition of angiogenesis both in vitro and in vivo, due to reductions of vascular endothelial growth factor (VEGF)-induced proliferation, DNA synthesis, chemotactic motility, capillary-like tube formation, and p38 mitogenactivated protein kinase activation (Min et al. 2004).

DGME was used in the present study to dissolve and administer TPA and capsaicin; to the best of our knowledge, DGME has not been reported previously as a vehicle for TPA in any in vivo rodent oncogenicity study. Acetone, methanol, and ethanol are the most commonly used vehicles for positive controls in these studies as they are known to produce robust tumor responses when used to deliver TPA topically either two or three times per week (Stoll et al. 2001). Our data suggest that DGME is both nononcogenic and has the ability to provide significant dermal drug delivery to mice. The high solubility of many compounds in DGME, coupled with its excellent skin penetration properties (Panchagnula and Ritschel 1991), makes it an attractive vehicle for widespread use in toxicological investigations.

The present study also confirmed the widely held impression that lidocaine is not mutagenic, as the lidocaine control group exhibited no increase in dermal masses or preneoplastic and neoplastic lesions relative to the untreated control group. Though skin penetration or systemic exposure of lidocaine in mice were not measured, the cream used in the study (L·M·X4, previously called ELA-Max) is a commonly used topical local anesthetic and has shown effectiveness in humans (Lehr et al. 2005; Friedman et al. 2001), implying an ability to deliver lidocaine into human skin. As mouse skin is more permeable to almost all drug substances than human skin (Ghosh et al. 2000), it is expected that lidocaine will also enter into mouse skin at effective concentrations. As L·M·X4 has also produced systemic exposure in humans (Friedman et al. 2001), it is predicted to produce significant systemic exposure in mice as well. The widely held impression that lidocaine is not genotoxic is based on data from the Ames, chromosome aberration (Hagiwara et al. 2006) and in vivo mouse micronucleus tests (www.fda.gov/cder/foi/label/2003/21496_duocaine_lbl.pdf). Interestingly, there are no publications regarding an assessment of lidocaine’s potential for carcinogenicity in vivo. In fact, lidocaine-containing products contain the explicit warning that long-term studies in animals have not been performed to evaluate the carcinogenic potential of lidocaine. However, metabolites of lidocaine have been shown to be carcinogenic in laboratory animals: a 2-year oral toxicity study of 2,6-xylidine has shown increased adenomas and carcinomas of the nasal cavity (Kornreich and Montgomery 1990).

In conclusion, based on the data presented here, capsaicin (at a dose level of up to 102 mg/kg and an application rate of 25.6 mg/cm²/kg/week), lidocaine (at a dose level of 162 mg/kg and at an application rate of 40.5 mg/cm²/kg/week) and DGME (at a dose level of 4.0 g/kg and at an application rate of 1 g/cm²/kg/week) should be considered nononcogenic in the Tg.AC mouse dermal model. Taken together with the two rigorously conducted genotoxicity panels using pure capsaicin (Chanda et al. 2004; Proudlock, Thompson, and Longstaff 2004), this naturally occurring molecule—which is present in many food, self-defense, and medical products—displays a very low carcinogenic potential.