Chronic toxicity/oncogenicity study of styrene in CD-1 mice by inhalation exposure for 104 weeks.

Groups of 70 male and 70 female Charles River CD-1 mice were exposed whole body to styrene vapor at 0, 20, 40, 80 or 160 ppm 6 h per day 5 days per week for 98 weeks (females) or 104 weeks (males). The mice were observed daily; body weights, food and water consumption were measured periodically, a battery of hematological and clinical pathology examinations were conducted at weeks 13, 26, 52, 78 and 98 (females)/104 (males). Ten mice of each gender per group were pre-selected for necropsy after 52 and 78 weeks of exposure and the survivors of the remaining 50 of each gender per group were necropsied after 98 or 104 weeks. An extensive set of organs from the control and high-exposure mice were examined histopathologically, whereas target organs, gross lesions and all masses were examined in all other groups. Styrene had no effect on survival in males. Two high-dose females died (acute liver toxicity) during the first 2 weeks; the remaining exposed females had a slightly higher survival than control mice. Levels of styrene and styrene oxide (SO) in the blood at the end of a 6 h exposure during week 74 were proportional to exposure concentration, except that at 20 ppm the SO level was below the limit of detection. There were no changes of toxicological significance in hematology, clinical chemistry, urinalysis or organ weights. Mice exposed to 80 or 160 ppm gained slightly less weight than the controls. Styrene-related non-neoplastic histopathological changes were found only in the nasal passages and lungs. In the nasal passages of males and females at all exposure concentrations, the changes included respiratory metaplasia of the olfactory epithelium with changes in the underlying Bowman's gland; the severity increased with styrene concentration and duration of exposure. Loss of olfactory nerve fibers was seen in mice exposed to 40, 80 or 160 ppm. In the lungs, there was decreased eosinophilia of Clara cells in the terminal bronchioles and bronchiolar epithelial hyperplasia extending into alveolar ducts. Increased tumor incidence occurred only in the lung. The incidence of bronchioloalveolar adenomas was significantly increased in males exposed to 40, 80 or 160 ppm and in females exposed to 20, 40 and 160 ppm. The increase was seen only after 24 months. In females exposed to 160 ppm, the incidence of bronchiolo-alveolar carcinomas after 24 months was significantly greater than in the controls. No difference in lung tumors between control and styrene-exposed mice was seen in the intensity or degree of immunostaining, the location of tumors relative to bronchioles or histological type (papillary, solid or mixed). It appears that styrene induces an increase in the number of lung tumors seen spontaneously in CD-1 mice.

Chronic toxicity/oncogenicity study of styrene in CD rats by inhalation exposure for 104 weeks.

Groups of 70 male and 70 female Charles River CD (Sprague-Dawley-derived) rats were exposed whole body to styrene vapor at 0, 50, 200, 500, or 1000 ppm 6 h/day 5 days/week for 104 weeks. The rats were observed daily, body weights and food and water consumption were measured periodically, and a battery of hematologic and clinical pathology examinations was conducted at weeks 13, 26, 52, 78, and 104. Nine or 10 rats per sex per group were necropsied after 52 weeks of exposure and the remaining survivors were necropsied after 104 weeks. Control and high-exposure rats received a complete histopathologic examination, while target organs, gross lesions, and all masses were examined in the lower exposure groups. Styrene had no effect on survival in males, but females exposed to 500 or 1000 ppm had a dose-related increase in survival. Levels of styrene in the blood at the end of a 6-h exposure during week 95 were proportional to exposure concentration. Levels of styrene oxide in the blood of rats exposed to 200 ppm or greater styrene were proportional to styrene exposure concentration. There were no changes of toxicologic significance in hematology, clinical chemistry, urinalysis, or organ weights. Males exposed to 500 or 1000 ppm gained less weight than the controls during the first year and maintained the difference during the second year. Females exposed to 200, 500, or 1000 ppm gained less weight during the first year; those exposed to 500 or 1000 ppm continued to gain less during months 13-18. Styrene-related non-neoplastic histopathologic changes were confined to the olfactory epithelium of the nasal mucosa. There was no evidence that styrene exposure caused treatment-related increases of any tumor type in males or females or in the number of tumor-bearing rats in the exposed groups compared to controls. In females, there were treatment-related decreases in pituitary adenomas and mammary adenocarcinomas. Based on an overall evaluation of eight oncogenicity studies, there is clear evidence that styrene does not induce cancer in rats.

Ecotoxicity hazard assessment of styrene.

The ecotoxicity of styrene was evaluated in acute toxicity studies of fathead minnows (Pimephales promelas), daphnids (Daphnia magna), amphipods (Hyalella azteca), and freshwater green algae (Selenastrum capricornutum), and a subacute toxicity study of earthworms (Eisenia fostida). Stable exposure levels were maintained in the studies with fathead minnows, daphnids, and amphipods using sealed, flowthrough, serial dilution systems and test vessels. The algae were evaluated in a sealed, static system. The earthworms were exposed in artificial soil which was renewed after 7 days. Styrene concentrations in water and soil were analyzed by gas chromatography with flame ionization detection following extraction into hexane. Test results are based on measured concentrations. Styrene was moderately toxic to fathead minnows, daphnids, and amphipods: fathead minnow: LC50 (96 hr), 10 mg/liter, and NOEC, 4.0 mg/liter; daphnids: EC50 (48 hr), 4.7 mg/liter, and NOEC, 1.9 mg/liter; amphipods: LC50 (96 hr), 9.5 mg/liter, and NOEC, 4.1 mg/liter. Styrene was highly toxic to green algae: EC50 (96 hr), 0.72 mg/liter, and NOEC, 0.063 mg/liter; these effects were found to be algistatic rather than algicidal. Styrene was slightly toxic to earthworms: LC50 (14 days), 120 mg/kg, and NOEC, 44 mg/kg. There was no indication of a concern for chronic toxicity based on these studies. Styrene's potential impact on aquatic and soil environments is significantly mitigated by its volatility and biodegradability.

Subchronic inhalation studies of styrene in CD rats and CD-1 mice.

Groups of 10 male and 10 female Charles River (CRL) CD (Sprague-Dawley-derived) rats were exposed to styrene vapor at 0, 200, 500, 1000, or 1500 ppm 6 hr per day 5 days per week for 13 weeks. Styrene had no effect on survival, hematology, or clinical chemistry. Males at 1500 ppm weighed 10% less after 13 weeks and males and females at 1000 and 1500 ppm consumed more water than controls. Histopathologic changes were confined to the olfactory epithelium of the nasal mucosa. Groups of 20 male and 20 female CRL CD-1 and B6C3F1 mice were exposed to styrene vapor at 0, 15, 60, 250, or 500 ppm 6 hr per day 5 days per week for 2 weeks. Mortality was observed in both CD-1 and B6C3F1 mice exposed to 250 or 500 ppm; more female mice, but not males, died from exposure to 250 ppm than from 500 ppm. Groups of 10 male and 10 female CRL CD-1 mice were exposed to styrene vapors at 0, 50, 100, 150, or 200 ppm 6 hr per day 5 days per week for 13 weeks. Two females exposed to 200 ppm died during the first week. Liver toxicity was evident in the decedents and in some female survivors at 200 ppm. Changes were observed in the lungs of mice exposed to 100, 150, or 200 ppm and in the nasal passages of all treatment groups, those exposed to 50 ppm being less affected. Satellite groups of 15 male rats and 30 male mice were exposed as described above for 2, 5, or 13 weeks for measurement of cell proliferation (BrdU labeling). No increase in cell proliferation was found in liver of rats or mice or in cells of the bronchiolar or alveolar region of the lung of rats. No increase in labeling index of type II pneumocytes was seen in mouse lungs, while at 150 and 200 ppm, an increased labeling index of Clara cells was seen after 2 weeks and in occasional mice after 5 weeks. Large variations in the labeling index among animals emphasize the need for large group sizes. For nasal tract effects, a NOAEL was not found in CD-1 mice, but in CD rats, the NOAEL was 200 ppm. For other effects, the NOAEL was 500 ppm in rats and 50 ppm in mice.

Contact sensitivity to captafol in BALB/c mice.

The fungicide captafol has been reported as causing irritant and allergic contact dermatitis in humans and in guinea pigs. This study investigated the ability of purified captafol to cause contact sensitization in BALB/c mice. Female mice were pretreated with an intraperitoneal injection of cyclophosphamide or saline. Applications of captafol (18.7 or 37.4 mg/ml), dinitrofluorobenzene (DNFB; 5 mg/ml) or solvent (4:1 acetone:ethanol mixture) were administered to the shaved abdomen on 2 consecutive days, or on days 1, 2, 8, 15, 22 and 29. Following challenge with captafol (37.4 mg/ml) or DNFB (2 mg/ml) on the right ear 6 days after the last induction, ear thickness ratios (right ear/left ear) were significantly larger after challenge in captafol-induced and DNFB-induced mice compared to control mice. A slightly larger response was observed with the smaller induction dose level of captafol and with multiple inductions over the course of a month. The overall maximum response to captafol was not increased by pretreatment with cyclophosphamide. Histologically, ears from captafol-induced and DNFB-induced mice showed edema and cellular infiltration. This study demonstrated the ability of captafol to produce contact hypersensitivity in the BALB/c mouse.

Evaluation of calcium magnesium acetate and road salt for contact hypersensitivity potential and dermal irritancy in humans.

Calcium magnesium acetate (CMA) and road salt are both de-icing agents to which workers may be dermally exposed. A commercial formulation of CMA (Chevron Ice-B-Gon Deicer) and road salt were tested in a human repeat insult patch test to evaluate the contact hypersensitivity potential of these materials and to evaluate irritation following single or multiple applications. 72 of the initial 82 panelists completed the study. CMA and road salt (each at 10% and 30% w/w in distilled water; 0.3 ml) were administered under occlusive patches on the forearm for 14 h 3 x per week for 3 weeks. The panelists were challenged 2 weeks later; 2 panelists who had mild reactions were subsequently rechallenged 6 weeks later. Neither CMA nor road salt produced contact hypersensitivity in any panelists. Following the first application, moderate acute irritation was observed only at 1 skin site exposed to 30% road salt. Repeated exposure to CMA or road salt produced mild to moderate irritation. The highest incidence of moderate irritation was observed with 30% road salt. Thus, neither material is expected to cause significant dermal effects in exposed workers. CMA is expected to cause dermal irritation equivalent to or less than that caused by road salt.

Allergic hypersensitivity to the insecticide malathion in BALB/c mice.

The widely used insecticide malathion (diethylmercaptosuccinate, S ester with O,O-dimethylphosphorodithioate) has been reported to cause allergic responses in some exposed people and in guinea pigs. In this study, IgE antibody-mediated and cell-mediated hypersensitivity to malathion was evaluated in female BALB/c mice. To elicit malathion-specific antibodies of the IgE class, a conjugate of the anhydride of the diacid metabolite of malathion (MMA) with keyhole limpet hemocyanin was administered ip with aluminum hydroxide as adjuvant. Sera collected following three sequential sensitizations were tested for specific IgE with the passive cutaneous anaphylaxis (PCA) test in Sprague-Dawley rats. MMA coupled to bovine serum albumin was used as the challenge antigen. Specific IgE was produced following the second and third sensitization in the mice receiving 10 and 100 micrograms of conjugate and following the third sensitization in the mice receiving 1 microgram of conjugate. A paper radioallergosorbent test (PRAST) was as sensitive as and showed a good correlation with the PCA assay for samples analyzed by both procedures. Malathion applied epicutaneously on 2 days or over 4 weeks failed to elicit delayed-type hypersensitivity as determined by change in ear thickness, 5-[125I]iodo-2'-deoxyuridine incorporation in the ear, and histology of the ear following ear challenge, with or without pretreatment of the mice with cyclophosphamide. MMA-specific IgE antibodies were not detected by the PCA test in the serum of mice treated epicutaneously for 4 weeks.

Allergic hypersensitivity to the herbicide 2,4-D in BALB/c mice.

Following several reports of human allergic hypersensitivity, the ability of 2,4-D (2,4-dichlorophenoxyacetic acid) to elicit 2,4-D-specific IgE antibodies and delayed-type hypersensitivity in BALB/c mice was studied. 2,4-D-specific IgE antibodies were detected in mouse sera following the second intraperitoneal immunization with 1, 10, or 100 micrograms 2,4-D-keyhold limpet hemocyanin conjugate with aluminum hydroxide adjuvant. Specific IgE was determined with the rat passive cutaneous anaphylaxis test using a conjugate of 2,4-D with bovine serum albumin for challenge. The highest antibody titers and a measurable response in all mice were seen in the group that received 1 microgram of 2,4-D conjugate. Dinitrophenyl-specific IgE was measured at all intervals examined in mice immunized with a dinitrophenyl-keyhole limpet hemocyanin conjugate. 2,4-D applied epicutaneously on 2 d or over 4 wk failed to elicit delayed-type hypersensitivity as measured by change in ear thickness, incorporation of 5-[125I]iodo-2'-deoxyuridine, or histology following challenge on the ear. No 2,4-D-specific IgE antibodies were detected in serum during the 4-wk sensitization period. Similar treatment with a known sensitizer, dinitrofluorobenzene, produced delayed hypersensitivity. Following 4 wk of dosing, low titers of dinitrophenyl-specific IgE antibodies were elicited.