Raymond M. David

PPARα Agonist-Induced Rodent Tumors: Modes of Action and Human Relevance

Widely varied chemicals—including certain herbicides, plasticizers, drugs, and natural products—induce peroxisome proliferation in rodent liver and other tissues. This phenomenon is characterized by increases in the volume density and fatty acid oxidation of these organelles, which contain hydrogen peroxide and fatty acid oxidation systems important in lipid metabolism. Research showing that some peroxisome proliferating chemicals are nongenotoxic animal carcinogens stimulated interest in developing mode of action (MOA) information to understand and explain the human relevance of animal tumors associated with these chemicals. Studies have demonstrated that a nuclear hormone receptor implicated in energy homeostasis, designated peroxisome proliferator-activated receptor alpha (PPARα), is an obligatory factor in peroxisome proliferation in rodent hepatocytes. This report provides an in-depth analysis of the state of the science on several topics critical to evaluating the relationship between the MOA for PPARα agonists and the human relevance of related animal tumors. Topics include a review of existing tumor bioassay data, data from animal and human sources relating to the MOA for PPARα agonists in several different tissues, and case studies on the potential human relevance of the animal MOA data. The summary of existing bioassay data discloses substantial species differences in response to peroxisome proliferators in vivo, with rodents more responsive than primates. Among the rat and mouse strains tested, both males and females develop tumors in response to exposure to a wide range of chemicals including DEHP and other phthalates, chlorinated paraffins, chlorinated solvents such as trichloroethylene and perchloroethylene, and certain pesticides and hypolipidemic pharmaceuticals. MOA data from three different rodent tissues—rat and mouse liver, rat pancreas, and rat testis—lead to several different postulated MOAs, some beginning with PPARα activation as a causal first step. For example, studies in rodent liver identified seven “key events,” including three “causal events”—activation of PPARα, perturbation of cell proliferation and apoptosis, and selective clonal expansion—and a series of associative events involving peroxisome proliferation, hepatocyte oxidative stress, and Kupffer-cell-mediated events. Similar in-depth analysis for rat Leydig-cell tumors (LCTs) posits one MOA that begins with PPARα activation in the liver, but two possible pathways, one secondary to liver induction and the other direct inhibition of testicular testosterone biosynthesis. For this tumor, both proposed pathways involve changes in the metabolism and quantity of related hormones and hormone precursors. Key events in the postulated MOA for the third tumor type, pancreatic acinar-cell tumors (PACTs) in rats, also begin with PPARα activation in the liver, followed by changes in bile synthesis and composition. Using the new human relevance framework (HRF) (see companion article), case studies involving PPARα-related tumors in each of these three tissues produced a range of outcomes, depending partly on the quality and quantity of MOA data available from laboratory animals and related information from human data sources.

Proposed Mode of Action for In Utero Effects of Some Phthalate Esters on the Developing Male Reproductive Tract

Phthalate esters containing a straight-chain backbone of 4–6 carbons have demonstrated testicular toxicity and infertility in adult and pre-adolescent rats, mice, hamsters, and ferrets. In recent years, these same phthalates have been shown to interfere with the normal development of the male reproductive tract in rodents and rabbits. The review presented here summarizes studies that provide evidence of a mode of action for these effects. The data indicate that C4–C6 phthalate esters inhibit processes in the Leydig cell, such as the synthesis of testosterone (T) and production of insulin-like factor 3 (insl3), both of which are required for normal development of male genitalia. A proposed secondary effect of reduced androgen production is on Sertoli cells, resulting in failure to proliferate and interference with cell-cell communication (gap-junction intracellular communication) leading to the development of large multinucleate gonocytes. The possibility that phthalates act directly on the Sertoli cells to interfere with intracellular communication is not excluded. The strength, consistency, and plausibility of the proposed mode of action and alternate modes of action are discussed.

Reversibility of the chronic effects of di(2-ethylhexyl)phthalate.

Fischer-344 rats treated with 12,500 ppm (728 and 879 mg/kg/d for male and females, respectively) and B6C3F1 mice treated with 6,000 ppm (1,227 and 1,408 mg/kg/d, respectively) di(2-ethylhexyl)phthalate (DEHP) in the diet for 78 weeks were allowed to recover for an additional 26 weeks on control diet. Blood was analyzed at weeks 78 and 104 from 10 animals per sex per group; animals were sacrificed at weeks 79 and 105 for histopathologic examination. The results are compared with data from animals continuously exposed to these dietary levels for 104 weeks (10, 11). Body weights and food consumption were measured monthly. BUN, albumin, and globulin that were significantly different for rats exposed to DEHP throughout 104 weeks, were comparable to controls for the recovery group. Reversibility of chronic effects on erythrocyte count, hemoglobin, and hematocrit values was apparent only for female rats. Chronic exposure demonstrated effects on liver, kidney, and testes weights. All organ weight effects except for testes for the Recovery group of rats, and all organ weight effects for mice, were reversible. Pigmentation of Kupffer cells and renal tubules present in chronically treated rats were not observed for the Recovery group. Lesions in the testes and pituitary gland were not reversible in rats. This may be a reflection of the senescence of the hypothalamic-gona d axis in rats. Cessation of exposure for mice resulted in amelioration of effects in the kidneys, liver, and testes. The extent of reversibility suggests that many chronic effects may be associated with a metabolic phenomenon such as peroxisome proliferation, which also reverted to control levels after 26 weeks of recovery.

Lack of sensitization for trimellitate, phthalate, terephthalate and isobutyrate plasticizers in a human repeated insult patch test

Two hundred and three human volunteers were tested for evidence of sensitization to several plasticizers following 3 weeks of dermal application three times a week. Tris(2-ethylhexyl)mellitate (TOTM; 1%, v/v), 2,2,4-trimethyl-1,3-pentanediol-diisobutyrate (TXIB; 1%, v/v), di(2-ethylhexyl)terephthalate (DEHT; 0.5%, v/v) and diethylphthalate (DEP; 2%, v/v) were applied to the skin of volunteers under semi-occlusive patch for 3 consecutive weeks and the reactions to a challenge application noted following a 2-week rest period. Slight erythema was observed in four individuals exposed to TOTM, two of which resolved within 96 h and one that occurred only after 96 h. Slight erythema was noted in three subjects exposed to TXIB, one of which resolved by 96 h and one that occurred only after 96 h. Two subjects had slight erythema to DEHT, one that resolved by 96 h and one that occurred only after 96 h. One reaction occurred with DEP at 96 h after challenge. Of the positive responses, one subject reacted to all test substances. No subject had a response grade of 1.0 or greater. Because of the low response, the overall conclusion is that none of the plasticizers demonstrated evidence of sensitization or irritation.

Evaluation of subchronic toxicity of n-butyl acetate vapor

The subchronic toxicity of n-butyl acetate (nBA), a common industrial solvent, was tested in rats in a 13-week inhalation study. Male and female Sprague-Dawley (SD) rats were exposed to concentrations of 0, 500, 1500 or 3000 ppm nBA for 6 h per day, 5 days per week for 13 consecutive weeks. Transient signs of sedation were observed only during exposure to the 1500 and 3000 ppm concentrations. Body weights for the 1500 and 3000 ppm groups were significantly reduced. Feed consumption values for the 1500 and 3000 ppm groups were significantly lower than the control group. Weights of the liver, kidneys and spleen were significantly lower for the 3000 ppm male group; testes and adrenal gland weights for the 1500 and 3000 ppm groups and the lung weight for the 3000 ppm male group were significantly higher than for the control group. Signs of irritation of the glandular stomach and necrosis in the non-glandular stomach were observed in 3000 ppm female rats. Degeneration of the olfactory epithelium along the dorsal medial meatus and ethmoturbinates of the nasal passages of some 1500 and all 3000 ppm rats was also seen. The severity was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. No effects were observed in the lungs of any group. The no-observed-effect level (NOEL) for this study is considered to be 500 ppm. The data presented here are relevant to the toxicity risk assessment of n-butanol due to the rapid hydrolysis of nBA in vivo.

Lack of nephrotoxicity and renal cell proliferation following subchronic dermal application of a hydroquinone cream.

Hydroquinone (HQ) is used in over-the-counter formulations of skin-lightening creams sold in the United States and European Union. HQ was introduced into these formulations to provide a safe and effective alternative to mercury and other less effective ingredients. Recent studies involving subchronic oral exposure of male F344 rats to HQ have shown nephrotoxicity and renal tubule cell proliferation (English et al., 1994), while chronic exposures of male F344 rats were reported to cause renal cell adenomas (NTP, 1989). Previous subchronic dermal toxicity studies (CTFA, 1986; NTP, 1989) with HQ failed to detect nephrotoxicity; however, these studies were not specifically designed to assess renal structure and function. More sensitive endpoints were used in the present subchronic study to address concerns over potential toxicity from repeated dermal exposure to HQ. Male and female F344 rats were given topical applications with 0, 2.0, 3.5, or 5.0% HQ in an oil-in-water emulsion cream for 13 wk (5 days/wk). Body weights, feed consumption and water consumption were monitored, and animals were observed for clinical signs of toxicity and dermal irritation. Blood taken at termination was analysed for haematological and clinical chemistry effects. Erythema, which abated when exposure stopped, was the only dermatological effect seen at the HQ-cream application sites. Cell proliferation in the kidneys was evaluated after 3, 6 and 13 wk of treatment using bromodeoxyuridine (BrdU) labelling, but no changes indicative of sustained cell proliferation were seen. The renal histopathological lesions noted after oral exposure to HQ were not present after dermal exposure. Thus, topical exposure to HQ does not result in the renal toxicity observed in previous studies with F344 rats given HQ orally.

2-Ethylhexanoic Acid: Subchronic Oral Toxicity Studies in the Rat and Mouse

Groups of 10 male and 10 female Fischer 344 rats and B6C3F1 mice were fed diets containing either 0.0, 0.1, 0.5 or 1.5% 2-ethylhexanoic acid (EHA) for 13 wk. Additional groups of 10 male and 10 female rats or mice. were fed either 0.0 or 1.5% EHA for 13 wk followed by a 4-wk recovery (non-treatment) period. Based on food consumption and body weight, the EHA diets provided doses of 61, 303 or 917 mg/kg/day for male rats and 71, 360 or 1068 mg/kg/day for female rats. The EHA diets provided doses of 180, 885 or 2728 mg/kg/day for male mice and 205, 1038 or 3139 mg/kg/day for female mice. No mortality or significant clinical signs of toxicity were observed during the study. Body weights and food consumption of both rats and mice fed 1.5% EHA were lower beginning after the first week of treatment, consistent with a reduction in food consumption. Other groups were unaffected by treatment. After 13 wk, lower triglyceride levels occurred in male mice fed 1.5% EHA and female mice fed 0.5 or 1.5% EHA, but not in other groups. Cholesterol levels were higher in all male rat test groups and in female rats and male and female mice fed either 0.5 or 1.5% EHA, although this effect was reversible following a 28-day recovery period. The principal effects of EHA involved the liver or metabolic processes associated with the liver. The 0.5 and 1.5% diets in both rats and mice were associated with increased relative liver weight and histological changes in hepatocytes, specifically hepatocyte hypertrophy and reduced cytoplasmic vacuolization. Observed histopathological and clinical pathological changes were reversible following recovery. These results indicate that EHA does not produce persistent. overt toxicity in rats or mice following subchronic dietary exposure at concentrations up to 1.5% in feed. The no-observed-adverse-effect level (NOAEL) for male rats was 61 mg/kg/day and the no-observed-effect level (NOEL) for female rats was 71 mg/kg/day, while 180 and 205 mg/kg/day represent NOELs for male and female mice, respectively.

Histopathologic changes in the kidneys of male F344 rats from a 2-year inhalation carcinogenicity study of tetrahydrofuran: A pathology working group review and re-evaluation

Risk evaluation and hazard classification for tetrahydrofuran (THF) is based partly on the incidences of renal tumors in male F344/N rats reported in a 2-year carcinogenicity study by the National Toxicology Program (NTP). A Pathology Working Group (PWG) was commissioned to conduct an independent review of the kidney slides from this bioassay (along with two subchronic studies) to assess renal changes in light of recent scientific work on pathogenesis of pre-neoplastic and neoplastic lesions in rat kidney. PWG pathologists confirmed the NTP assessment that adenomas were non-statistically increased in animals exposed to the highest level of THF. However, when pre-neoplastic and neoplastic lesions were combined, there was no difference between control and THF-exposed groups. Also, the majority of these proliferative lesions were in rats with severe chronic progressive nephropathy (CPN). Accordingly, the PWG concluded that renal lesions in the control and THF-exposed groups resulted primarily from regenerative processes associated with advanced CPN. Based on an alpha(2u)-globulin/hyaline droplet response observed in a 4-week study with THF, the PWG could not exclude the possibility of both advanced CPN and low-grade alpha2u-g nephropathy contributing to the renal proliferative lesions developing chronically in high-dose males. Neither condition has a pathologic counterpart in humans.

Subchronic inhalation toxicity study of a water-dispersible polyester in rats

AQ55 is a high molecular weight, water-dispersible, amorphous polyester used in applications where the exclusion of solvents and conventional surfactants is desirable, such as water-based adhesives, coatings, emulsions, paint primers, cosmetics and detergents. Potential health effects were evaluated in rats exposed by inhalation for about 13 wk to mean concentrations of 0, 2.4, 19.6 or 199 mg/m3 AQ55 polymer. No mortality occurred and body weights were unaffected. Mean relative liver weights in all treated male groups were slightly higher than control weights, but were not judged to be treatment related. Absolute liver weights and all other organ weights except lung weights were normal. Haematology, clinical chemistries and gross pathology were unremarkable. Exposure-related changes in the 199 mg/m3 groups included increased mean absolute and relative lung weights, accumulations of macrophages and acute inflammatory cells in alveolar and bronchial lumina, and increased numbers of macrophages in sinusoids of peribronchial lymph nodes. Minor accumulation of macrophages in alveolar lumina was the only exposure-related change in the 19.6 mg/m3 group. No exposure-related effects were seen in the 2.4 mg/m3 group. AQ55 produced no systemic toxicity, and aerosols of AQ55 do not appear to be toxic to pulmonary tissues following subchronic inhalation exposure.

The Synergistic Effect of Feed Restriction and Endotoxemia on Motor Activity

The United States Environmental Protection Agency and the Organization for Economic Cooperation and Development have recommended incorporating measurement of motor activity into routine toxicity studies to provide a screen for potential neurotoxic effects. However, there is little information on how to interpretate motor activity levels when an animals clinical state is altered by systemic toxicity. Because systemic toxicity often includes nonspecific effects, such as reduced feed consumption, this physiologic condition was mimicked by limiting the feed of healthy rats to 10–15 g/rat/day and by limiting appetite through induction of endotoxemia. Injection of animals with endotoxin lowered motor activity by 50%. Combining feed restriction with endotoxin treatment resulted in reduced motor activity 2 days after injection, when signs of endotoxemia were not apparent. Animals treated with endotoxin but fed ad libitum had motor activity levels comparable to the control group 2 days after injection. The results suggest that motor activity levels can be altered by reduced feed consumption in the presence of subclinical systemic toxicity. Significant feed restriction in the absence of systemic toxicity appears unlikely to result in lowered motor activity levels. Key Words: Neurotoxicity-Motor activity-Endotoxin-Systemic toxicity-Feed restriction.