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Topical Application of Representative Multifunctional Acrylates Produced Proliferative and Inflammatory Lesions in F344/N Rats and B6C3F1 Mice, and Squamous Cell Neoplasms in Tg.AC Mice
1 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA Correspondence: Address correspondence to: Rajendra S. Chhabra, National Institute of Environmental Health Sciences, P.O. Box 12233, Mail Drop EC-35, Research Triangle Park, NC 27709, USA; e-mail:chhabrar{at}niehs.nih.gov
Widespread human exposure to multifunctional acrylates is of concern, due to their inherent reactivity and irritating properties. Trimethylolpropane triacrylate (TMPTA) and pentaerythritol triacrylate (PETA) are industrially important representatives of multifunctional acrylates. The current studies characterized the toxicity of 3-month topical administration of technical grade TMPTA and PETA in F344/N rats and B6C3F1 mice, and evaluated the carcinogenic potential of TMPTA and PETA in hemizygous Tg.AC (v-Ha-ras) transgenic mice. Administration of 0.75, 1.5, 3, 6, and 12 mg/kg TMPTA and PETA for 3 months resulted in hyperplastic, degenerative, and necrotic lesions, accompanied by chronic inflammation of the skin, with severities generally increasing with dose. Lesions were slightly more severe in rats, when compared with mice, and illustrate the irritant potential of TMPTA and PETA. A similar dosage regimen was used for the 6-month study with Tg.AC mice. Topical application of TMPTA and PETA to Tg.AC mice showed dose-dependent increases in squamous cell papillomas at the site of application, with decreases in the latency of their appearance in mice receiving 3 mg/kg or greater. Papillomas, the reporter phenotype in Tg.AC mice, were accompanied by a few squamous cell carcinomas, along with hyperplastic and inflammatory lesions. Although chronic inflammation might have contributed to the development of the skin lesions, the dose-related nature of the induction of the skin papillomas in Tg.AC mice by TMPTA and PETA may reflect a potential for carcinogenicity.
Key Words: Dermal • multifunctional acrylates • transgenic mouse • toxicity • carcinogenicity • squamous cell papillomas • inflammation
Trimethylolpropane triacrylate (TMPTA) and pentaerythritol triacrylate (PETA) are representatives of the multifunctional alkyl acrylates class, used industrially in the production of UV-curable inks, coatings, and paints. Widespread human exposure is anticipated, mainly through the skin, as a result of contact with products containing acrylates, or during their manufacturing, processing, handling, and application. Occupational exposure to acrylate-containing compounds usually involves industrial mixtures of acrylates, rather than individual chemicals, which have been reported to cause contact or allergic dermatitis and conjunctivitis (Bjorkner et al., 1980; Nethercott, 1978; Nethercott et al., 1983). TMPTA and PETA have been previously shown to be sensitizers in the guinea pig maximization test, although contrasting results have been reported with other methods (Andrews and Clary, 1986). Slight to moderate irritation occurred in rabbits 72 hours following a single exposure to TMPTA and PETA, while applications for 2 weeks caused skin necrosis, but no systemic effects (Andrews and Clary, 1986). Studies with C3H/HeJ mice that received a topical dose of 3 mg of a 15% solution of PETA in acetone, 3, times a week, for their life span, produced no skin or visceral neoplasms (DePass et al., 1985). Similarly, no skin tumors were observed in C3H/HeJ mice when 100 mg/kg of TMPTA in mineral oil was applied topically, twice a week, for up to 80 weeks, although acanthosis and fibrosis of the skin were frequently observed (Andrews and Clary, 1986). Lymphomas involving spleen or lymph nodes in 6 out of 50 exposed animals were reported following PETA applications for 80 weeks in the latter study. A subsequent unpublished pathology review determined that these lesions were not neoplasms (Van Miller et al., 2003), however, no additional details are available. The current studies examined the toxicity of topical technical grade TMPTA and PETA to Fischer 344/N rats, B6C3F1 mice, and hemizygous Tg.AC (v-Ha-ras) mice. F344/N rats and B6C3F1 mice are used routinely in studies conducted by the National Toxicology Program (NTP), and were used to characterize the subchronic toxicity of TMPTA and PETA. The genetically manipulated Tg.AC mouse, created by Leder et al. (1990), carries a transcriptionally silent transgene consisting of a v-Ha-ras oncogene fused to a zeta-globin promoter and an SV-40 polyadenylation sequence. The transgene confers these animals the characteristic of genetically initiated skin, as it is transcriptionally silent unless activated by UV light (Trempus et al., 1998), full-thickness wounding (Cannon et al., 1997), or topical chemical exposure (Spalding et al., 1993, 1999). Skin papillomas develop quickly, following transgene activation, in response to the topical application of both tumor promoters and genotoxic and nongenotoxic carcinogens (Spalding et al., 1999; Tennant et al., 2001). Thus, Tg.AC mice were included in these studies for testing the carcinogenic potential of TMPTA and PETA. These studies were part of a broader NTP effort to evaluate alternative in vivo models for rapid short-term identification of chemical carcinogens.
Test Chemicals Technical grade trimethylolpropane triacrylate (TMPTA) was purchased from Aldrich Chemical Co. (Milwaukee, WI). The purity of TMPTA was approximately 80%, and impurities identified by HPLC/MS included 4 structurally related acrylates or adducts, namely trimethylolpropane diacrylate, trimethylolpropane triacrylate acrylic acid adduct, trimethylolpropane triacrylate-trimethylolpropane monoacrylate adduct, and trimethylolpropane triacrylate-trimethylolpropane diacrylate adduct. Technical grade pentaerythritol triacrylate (PETA) was purchased from Sartomer Co. (Exton, PA). HPLC analyses of PETA showed an approximate purity of 45%, and impurity components tentatively identified by HPLC/MS consisted of structurally related adducts, dimers and acrylates, as well as TMPTA and related esters and adducts. Technical grade TMPTA and PETA were used in these studies for being the mixtures of relevance with regard to human exposure. In addition, due to their reactivity, these chemicals are not available in their pure form. The 12-O-Tetradecanoylphorbol-13-acetate (TPA) was obtained from Sigma Chemical Co. (St. Louis, MO; purity 99%) and acetone was procured from Spectrum Chemical Manufacturing Co. (Gardena, CA; purity 99%). Stability of dose formulations in acetone was verified periodically by gas chromatography.
Animals
Study Design
Six-Month Studies Volumetric pipettes equipped with disposable tips were used to deliver, and as needed, to spread the dose over the application site. The dose was applied to a standard area from the mid-back to the interscapular area, with the longitudinal axis oriented along that of the animal. An area slightly larger than the application site was clipped at least 24 hours prior to initial dose administration and weekly thereafter. Control animals, receiving acetone alone, were clipped at the same frequency as dosed animals.
Clinical Observations and Histopathology Diagnostic criteria and features used to diagnose squamous cell tumors of the skin (papillomas and carcinomas) have been previously described by Elwell et al. (1990) and Peckham and Heider (1999). In brief, squamous cell papillomas were benign, generally exophytic neoplasms consisting of proliferative squamous epithelium arrranged in prominent folds or ridges with a fibrovascular stromal core. The neoplastic epithelium was well differentiated with no evidence of invasion of the basement membrane. Squamous cell carcinomas were generally more anaplastic with evidence of invasion of the basement membrane.
Statistical Methods
Three-Month Studies with F344/N Rats Topical exposure to TMPTA and PETA for 3 months had no effect on the survival or body weights, with the exception of a 9% decrease in the final body weight of male rats dosed with 12 mg/kg of PETA. Irritation at the site of application of TMPTA and PETA were noted in rats exposed to 12 mg/kg. Microscopic changes at the site of application included epidermal hyperplasia, hyperkeratosis, epidermal degeneration and necrosis, and chronic active inflammation, with incidence and severity generally increasing with dose (Table 1); control animals displayed no significant changes. Epidermal hyperplasia was of minimal-to-mild severity and characterized by focally extensive to diffuse increased thickness of the epidermis, from the normal 1 to 3 cell layers thick in controls, to 4 to 6 layers in exposed rats. Hyperplasia was accompanied by hyperkeratosis and an increased thickness of the superficial keratin layer. Severity of epidermal degeneration was minimal to mild in TMPTA- and generally minimal in PETA-treated animals. Degeneration was a focal change and consisted of epidermal vacuolization, presumably due to intra- or intercellular fluid accumulation. Epidermal necrosis was significantly increased in 50% of the female rats treated with the high dose of TMPTA, and 50% of male rats treated with 6 mg/kg PETA, albeit without a clear dose-response (Table 1).
Necrosis was focal, minimal to mild, and consisted of partial to full thickness coagulative change of the epidermis and was likely a sequela of degeneration. Suppurative inflammation consisted of epidermal infiltration by neutrophils, often accompanied by degeneration or necrosis of the epidermis. Chronic active inflammation, seen as a mixed inflammatory cell infiltrate, was minimal to mild, and present in most dose groups of both TMPTA and PETA exposures. Sebaceous glands were slightly enlarged and prominent (hyperplasia) at the site of application.
Three-Month Studies with B6C3F1 Mice
Six-Month Studies with Hemizygous Tg.Ac Mice Survival, Body Weight and Clinical Findings Exposure to TMPTA and PETA had no effect on the survival of Tg.AC mice. Mean body weights of Tg.AC mice treated with TMPTA or PETA were within 6% of controls throughout the study. Clinical findings included in-life observations of papillomas at the site of application in males and females exposed to PETA at 3 mg/kg or greater, and papillomas in males and females exposed to TMPTA at 6 and 12 mg/kg (Table 3). Papillomas appeared earlier in the study in animals treated with 6 and 12 mg/kg when compared with 3 mg/kg.
Histopathology—Skin Topical application of TMPTA and PETA resulted in squamous cell neoplasms observed histologically at the site of application. Incidences of the skin neoplasms were significantly increased in males and females treated with 6 and 12 mg/kg TMPTA, with multiple papillomas, often contiguous, in all effected Tg.AC mice treated with 12 mg/kg and males treated with 6 mg/kg (Table 4). Squamous cell papillomas were present at the site of application in most males and females treated with 3 mg/kg or greater PETA (Table 4). The morphology of the squamous cell papillomas was typical, an exophytic growth of well-differentiated squamous epithelium covering arborizing fronds of connective tissue. One female in each group treated with 1.5, 6, and 12 mg/kg TMPTA developed squamous cell carcinomas at the site of application, which appeared to arise within papillomas (Figure 4); while Tg.AC mice exposed to PETA developed carcinomas in males treated with 3 mg/kg and males and females administered 12 mg/kg (Table 4). Carcinomas had extensions of atypical squamous cells into underlying dermis and subcutis, and evidence of localized invasion.
Nonneoplastic lesions of the skin occurred at the site of application of TMPTA and PETA, and included epidermal hyperplasia, hyperkeratosis, and chronic active inflammation (Table 4). Significantly increased incidences of epidermal hyperplasia occurred in Tg.AC mice administered TMPTA at 3 mg/kg or greater, characterized by increased thickness of the epidermis, and was generally diffuse in areas between papillomas. Occasional focal nodular hyperplasia was considered a precursor lesion to squamous papilloma. Significantly increased incidences of epidermal hyperplasia were seen in males administered PETA at 3 mg/kg or greater and females administered 1.5 mg/kg or greater. Hyperplasia was accompanied by hyperkeratosis, an increased thickness of the keratin layer, with significant increases in groups treated with 3 mg/kg or greater TMPTA or PETA. Chronic active inflammation was diagnosed in the dermis, generally in Tg.AC mice treated with 6 and 12 mg/kg TMPTA and 3 mg/kg PETA, in nonneoplastic areas. Inflammation associated with neoplasms also occurred, but were diagnosed separately.
Histopathology Other Organs The incidences of hematopoietic cell proliferation were significantly increased in various tissues of some TMPTA and PETA-treated groups (Table 5). Hematopoietic cell proliferation consisted of increases in erythroid and granulocytic precursors in the splenic red pulp, liver sinusoids, or nodal parenchyma. Exposure to 6 or 12 mg/kg TMPTA and PETA resulted in a change present in 1 or more organs; characterized by variable morphology. This lesion was not present in control animals, and in its more severe state, it was diagnosed as myelodysplasia (Table 5). Milder lesions were diagnosed as cellular infiltration, and occurred in several animals exposed to TMPTA and PETA (Table 5). Lesions were characterized predominantly by myeloid infiltration/proliferation that tended to be perivascular in the liver and lungs. Infiltrating cells were predominantly granulocytes (eosinophils and neutrophils), with lesser numbers of mononuclear cells. While the liver was the most commonly affected organ, the change was also commonly observed in the mediastinal, axillary, and mesenteric lymph nodes, but also involved the epididymis and spleen.
Chronic active inflammation of the liver was observed in male mice administered PETA, with significantly increased incidences in groups treated with 0.75 (11/15; p  0.05), 3 (13/15; p 0.01), and 6 (13/15; p 0.01) mg/kg (5/15, 11/15, 10/15, 13/15, 13/15, and 5/15). In addition, female mice administered PETA showed thymocyte necrosis in 5 out of 15 animals of the 12 mg/kg group (control 0/15; p 0.05).
The primary target of TMPTA and PETA toxicity, following topical application for 3 months to F344/N rats and B6C3F1 mice, was the skin. Overall, both for TMPTA and PETA, the incidences of most epidermal lesions were higher, and at times more severe, at lower doses in male F344/N rats and B6C3F1 mice, compared to those of females. The major exception was the increased incidence of epidermal necrosis in female F344/N rats exposed to 12 mg/kg TMPTA; this lesion was absent in male rats. Necrotic lesions were not observed in female F344/N rats and B6C3F1 mice exposed to PETA, and there were no marked sex-related differences for either TMPTA or PETA-related effects on the dermis. The skin lesions of TMPTA and PETA following repeated exposures was consistent with previous reports, which showed skin necrosis in rabbits, following 2-week topical applications, without any systemic effects (Andrews and Clary, 1986). According to principles for dose selection utilized by the NTP, minimal proliferative lesions at the site of chemical application do not preclude the use of a dose as the MTD (maximum tolerated dose or minimum toxic dose) in a 2-year study (Bucher, 2000). Doses that produce marked inflammation, necrosis, and ulceration of the epidermis, on the other hand, are generally avoided. Ordinarily, the presence of mild necrosis and mild suppurative inflammation at the 12 mg/kg dose in the current studies would be enough histopathological evidence to preclude the selection of this top dose for a 2-year study. However, these lesions were less severe and/or occurred in fewer animals or not at all in the lower dose groups, consisted of focal lesions of minimal-to-mild severity, and were being selected for a different mouse strain and for a shorter-term 6-month study. Thus, 12 mg/kg was selected as the top dose group, and additional lower dose levels were included to provide a margin of safety in the event of excessive toxicity at the high dose. Accordingly, necrotic lesions were not observed in the 6-month study with Tg.AC mice. The Tg.AC mouse, containing features of genetically initiated skin, presented an appropriate model for testing the carcinogenic potential of acrylates. This study was part of an NTP effort to prospectively evaluate the strengths and limitations of alternative in vivo methods. The usefulness of the Tg.AC model in facilitating the identification of carcinogens had been previously established, in a review coordinated by the International Life Sciences Institute (ILSI) (Eastin et al., 2001). The incidence of spontaneous skin tumors in Tg.AC mice is low overall, and the model responds to both genotoxic and nongenotoxic carcinogens (Tennant et al., 2001). The current study with hemizygous Tg.AC mice showed dose-response increases in the number of animals that developed papillomas at the site of application following treatment with TMPTA and PETA. There were significant decreases in the latency of papilloma appearance, with time of initial papilloma appearance in the highest dose being comparable to the TPA positive control. Microscopically, the occurrence of carcinomas at the base of papillomas suggested that they might have developed from the papillomas. The dose-related nature of papillomas, the skin reporter phenotype in Tg.AC mice, with neoplasms appearing as early as 10 weeks into the study, suggests that TMPTA and PETA may have carcinogenic potential, or at the very least, promoting activity. The appearance of papillomas in the skin of Tg.AC mice was generally accompanied by dose-related increases in epidermal hyperplasia, hyperkeratosis and dermal chronic active inflammation, mostly of minimal-to-mild severity. It is likely that the nonneoplastic lesions were primary, rather than secondary to neoplasm development, as they occurred in association with tumors, but also in areas where neoplasms were not present. Additionally, nonneoplastic lesions similar to those seen in the Tg.AC mice were also observed in 3-month studies with B6C3F1 mice, however, the necrosis and degeneration observed in the B6C3F1 mouse were not seen in the Tg.AC.
Although previously demonstrated for select tumor-promoting agents such as phorbol esters and peroxides, the relationship of sustained cellular hyperplasia and the development of skin tumors is very complex (Slaga et al., 1995). Recent studies have suggested that chronic inflammation can promote cancer development through several pathways, namely, the activation of the NF Previous long-term studies with TMPTA in C3H/HeJ mice, showed no remarkable skin irritation, even though skin acanthosis and fibrosis were frequently observed, following topical application of 100 mg/kg, twice a week, for 80 weeks (Andrews and Clary, 1986); a weekly dose approximately 3-fold higher than the high dose used in the current study (12 mg/kg, 5 days per week). A study with PETA with doses of 120 mg/kg, 3 times a week (approximately 6-fold higher than the high dose in the current study), showed no incidence of tumors in the skin or other organs of C3H/HeJ mice, following topical application for the life span of the animals (DePass et al., 1985). It is unclear how these previous studies compare to the current study, due to differences in study design and animal strain. Systemic effects in the current study included increases in hematopoietic cell proliferation in various tissues, including the liver, spleen, and lymph nodes. Hematopoietic cell proliferation might have occurred as a result of inflammation at the site of application and/or release of hematopoietic cytokines and growth factors from proliferative epidermal cells. It is also possible that systemic effects resulted from a direct effect resulting from oral exposure, as a result of grooming. However, because the hematopoietic cell proliferation was though to be associated with the myeloid rather than the erythroid component, it is more likely that the systemic response was related to the inflammatory response in the skin. Exposure to TMPTA and PETA also resulted in a systemic change that involved one to several organs, and had characteristics that variably resembled hematopoietic, inflammatory, and neoplastic processes. Because of the variable morphology and uncertain biological behavior, assigning an appropriate diagnostic term was problematic. In its most severe form, the lesion may have resulted from infiltration and/or proliferation, and was diagnosed as myelodysplasia (Mahler et al., 1998). In milder cases, component cells appeared more infiltrative and the change was diagnosed as cellular infiltration. Myelodysplasia has been previously reported in another Tg.AC study with rotenone, which showed extensive hyperplasia and in-flammation (Eastin et al., 1998). Because ras mutations are frequently found in myeloproliferative disorders (Liu, 1990), and the Tg.AC mouse has the activated v-Ha-ras oncogene, it is possible that myelodysplasia may occur in response to inflammatory stimuli. Additional studies are needed to clarify these assumptions. Additional systemic effects in the current Tg.AC study included the development of squamous cell papillomas of the forestomach of female mice treated with the high dose of TMPTA. Forestomach papilloma is a relatively common spontaneous finding in Tg.AC mice that occurs at rates of 10 to 25% in hemizygous mice (Mahler et al., 1998; Eastin et al., 2001). The rates in the current study (60%), although above these incidences, did not follow a clear dose-response, and were significant only at the high dose in female mice treated with TMPTA, but not PETA-treated animals. Earlier studies with acrylates in homozygous Tg.AC mice demonstrated the carcinogenic potential of tripropyleneglycol diacrylate (TRPGDA), but not ethyl acrylate (EA) (Nylander-French and French, 1998). These results did not support previous chronic dermal toxicity studies with TRPGDA, in which C3H/HeJ mice treated with comparable doses for 80 weeks showed no increases in skin or visceral tumors (Andrews and Clary, 1986); but were in accordance with other studies where no skin tumors developed in C3H/HeJ mice treated topically with EA for their lifespan (DePass et al., 1984). Interestingly, ethyl acrylate administered by gavage produced irritation of the mucosa and squamous cell papillomas and/or carcinomas in the forestomach of F344/N rats and B6C3F1mice (NTP, 1986), but produced no tumors in F344 rats and B6C3F1 mice exposed via inhalation for 27 months (Miller et al., 1985). The disparity from the positive oral study with EA, compared to negative results obtained with other routes of administration, was thought to be related to the sustained hyperplasia at the forestomach, the site of application, which in turn, might be dependent on chemical dose and concentration (Nylander-French and French, 1998). Dermal toxicity, thus, would be dependent on the effect of chemical structure on dermal absorption and chemical-skin interaction. The disagreement between the TRPGDA studies needs to be further investigated. A recent study by Van Miller et al. (2003) attempted to address the equivocal findings in carcinogenicity studies with acrylates. A previous review of chronic dermal toxicity studies of triethyleneglycol diacrylate (TREGDA) and tetraethyleneglycol diacrylate (TTEGDA) reported severe skin damage and a low incidence of skin tumors when chemicals were applied at a dose of 2.5 mg, twice a week, for 80 weeks to C3H/HeJ mice (Andrews and Clary, 1986). Van Miller et al. (2003) showed no development of skin tumors when TREGDA doses approximately 10-fold lower were applied, despite skin irritation and basal epithelial cell proliferation (Van Miller et al., 2003). According to the authors, the severe skin damage was most likely the cause of tumorigenesis in the early acrylate studies, and should be controlled in carcinogenicity evaluations of this class of chemicals. The current studies with TMPTA and PETA illustrate the irritant potential of multifunctional acrylates in F344/N rats and B6C3F1 mice. In addition, they demonstrate that topical application of TMPTA and PETA induced squamous cell papillomas, the reporter phenotype that reflects the activation of transgene expression in Tg.AC mice, which indicates a potential for carcinogenicity, or at the very least, promoting activity. Although chronic inflammation might have contributed to the development of the skin lesions, these studies suggest that TMPTA and PETA may yield positive results in a long-term carcinogenicity bioassay.
The authors are grateful to Drs. Skip Eastin, Gail Pearse, and Judson Spalding for reviewing this manuscript. The authors also acknowledge the contribution of all NTP personnel involved in the execution and interpretation of these studies.
Toxicologic Pathology, Vol. 33, No. 6,
631-640 (2005)
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Abstract
Introduction
B pathway via the pro-inflammatory cytokine TNF, or upregulation of cytokine IL-6 or IL-8 (