Merrel Robinson

Carcinogenicity of chloroform in drinking water to male Osborne-Mendel rats and female B6C3F1 mice

The carcinogenic activity of chloroform administered at 0, 200, 400, 900, and 1800 mg/liter in drinking water was studied in male Osborne-Mendel rats and female B6C3F1 mice. A second control group was included in the study and was restricted to the water consumption of the high-dose group. Animals were maintained on study for 104 weeks. Group sizes were adjusted at low doses such that a detectable tumor response would result at the lowest dose if there was a linear relationship with dose, and the higher doses produced responses similar to previous carcinogenesis bioassays of chloroform. The primary finding was that chloroform increased the yield of renal tubular adenomas and adenocarcinomas in male rats in a dose-related manner. For the high-dose group, which corresponded to a time-weighted average dose of 160 mg/kg per day for 104 weeks, there was a 14% incidence of renal tubular adenomas and adenocarcinomas, vs 1% in the control group. This compares to a 24% incidence observed when 180 mg/kg per day of chloroform was administered for 78 weeks in earlier studies. In contrast, chloroform in the drinking water of mice failed to increase the incidence of hepatocellular carcinomas in female B6C3F1 mice. The highest dose group received a time-weighted average dose of 263 mg/kg for 104 weeks, resulting in a 5% combined incidence of hepatocellular adenoma and carcinoma relative to a 6% incidence in the control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogenic activity of acrylamide in the skin and lung of Swiss-ICR mice

Doses of acrylamide ranging from 12.5 to 50 mg/kg were administered orally to female ICR-Swiss mice over 3 days for each of 2 weeks (total doses of 75, 150 and 300 mg/kg). Two weeks later some of the animals were started on a promotion schedule involving the application of 2.5 micrograms TPA/mouse 3 times weekly. Development of tumors was observed weekly in the skin, and in the lungs at 1 year. Acrylamide was found to initiate squamous cell adenoma and carcinomas in the skin and increased the yield of adenomas and carcinomas in the lung. Skin tumor development was dependent upon 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion whereas lung tumor induction was not. These data extend previous observations of carcinogenic activity of acrylamide in the skin of SENCAR mice and lungs of strain A/J mice to a third strain of mouse, the ICR-Swiss.

Evaluation of mutagenic and carcinogenic properties of brominated and chlorinated acetonitriles: by-products of chlorination

The present study was undertaken to determine if chlorinated and brominated acetonitriles formed during the chlorination of drinking water possess mutagenic and/or carcinogenic properties. Chloroacetonitrile (CAN), dichloroacetonitrile (DCAN), trichloroacetonitrile (TCAN), bromochloroacetonitrile (BCAN), and dibromoacetonitrile (DBAN) were tested for their ability (1) to produce point mutations in the Salmonella/microsome assay, (2) to induce sister chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells in vitro, (3) to produce micronuclei in polychromatic erythrocytes in CD-1 mice, and (4) to act as tumor initiators in the skin of Sencar mice. DCAN and BCAN were found to be direct-acting mutagens in Salmonella. All five haloacetonitriles induced SCE in CHO cells in vitro. This activity paralleled the extent of chlorine substitution and was further enhanced in the dihaloacetonitrile series when bromine was substituted for chlorine. None of the haloacetonitriles showed evidence of activity in the mouse micronucleus assay. DBAN, BCAN, and CAN initiated tumors in the mouse skin with topical applications followed by a 20-week promotion schedule of 12-O tetradecanoylphorbol-13-acetate applications (p less than 0.02). These data indicate that the haloacetonitriles do display mutagenic and carcinogenic properties in some test systems and the hazard associated with their occurrence in drinking water and production within the gastrointestinal tract require further evaluation.

Fourteen-and Ninety-Day Oral Toxicity Studies of Methyl Tertiary-Butyl Ether in Sprague-Dawley Rats

Male and female Sprague-Dawley rats were gavaged with methyl tertiary-butyl ether (MtBE) for 14 or 90 days to evaluate subacute and subchronic toxicity. Five daily dose levels ranged from 0 to 1428 mg/kg body weight for the 14 day study and 0 to 1200 mg/kg body weight for the 90-day exposure. Controls received the corn oil vehicle. At or above dose levels of 1200 mg/kg, MtBE-induced anesthesia lasted about 2 h, followed by uneventful recovery. Diarrhea was common in all treatment groups, but no deaths were attributed to MtBE toxicity. In the subacute study, lung weights were reduced in high-dose females. Trends in the 14-day exposure also included increased cholesterol in both females and males and decreased blood-urea nitrogen (BUN) and creatinine in females. In the 90-day study, females exhibited elevated cholesterol and decreased BUN, while creatinine was decreased in high-dose males. Microscopic findings in most organs were unremarkable, except for high-dose males where renal changes were compatible with alpha 2-globulin nephropathy and were considered to have little toxicologic significance for humans. Both studies indicated that dose levels below those which induce anesthesia (1200 mg/kg) do not result in significant pathophysiologic changes.

Subacute and Subchronic Toxicity of Ethylene Glycol Administered in Drinking Water to Sprague-Dawley Rats

Subacute (10-day) and subchronic (90-day) toxicity studies of ethylene glycol (EG) were conducted in male and female Sprague-Dawley rats to provide the U.S. Environmental Protection Agencys (EPA) Office of Drinking Water with toxicity data for final preparation of a Health Advisory for the chemical. Ethylene glycol was administered in drinking water at concentrations of 0.5, 1.0, 2.0, and 4.0% for both sexes in the 10-day study. Based on a projected consumption rate of 100 ml/kg/day, the respective doses on a mg/kg/day basis would be 554, 1108, 2216, and 4432. These dose levels were also used in the 90-day study for females, but dose levels for the males in the 90-day study were 0.25, 0.5, 1.0, and 2.0% (227, 554, 1108, and 2216 mg/kg/day). At time of sacrifice necropsies were performed and tissues were prepared for histological evaluation. Blood samples were taken for hematology and clinical chemistry determinations. Body weights were measured weekly. Water and food consumption were determined three times weekly. No mortality occurred in the 10-day study. In the 90-day study 8/10 females and 2/10 males in the high dose group died prior to sacrifice. Body weights were suppressed in a dose response fashion for males and females. Hemoglobin, hematocrit, erythrocytes, and leukocytes were all significantly decreased in female rats receiving 4% EG for 10 days. The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.

Carcinogenic activity associated with halogenated acetones and acroleins in the mouse skin assay

Several chlorinated acetones have been identified in drinking water and these, as well as a number of chlorinated acroleins, are produced by chlorination of humic acid solutions. Many of these chlorinated compounds and the brominated acrolein analog were positive in the Ames Assay in the laboratory. To determine if carcinogenic activity was associated with these chemicals the following acetone derivatives: monochloro (MCA); 1,1-dichloro (1,1-DCA), 1,3-dichloro (1,3-DCA), 1,1,1-trichloro (1,1,1-TCA), 1,1,3-trichloro (1,1,3-TCA), and substituted acroleins: 2-chloro (CAC), 3,3-dichloro (DCAC), 2,3,3-trichloro (TCAC) and 2-bromo (BAC), were applied topically to SENCAR mice (25, 30, or 40/group) at the following dose levels: 50 mg/kg (MCA and 1,1,3-TCA); 50, 75 and 100 mg/kg (1,3-DCA); 100, 200 and 400 mg/kg (CAC, DCAC, and TCAC); 200 and 300 mg/kg (BAC); and 400, 600, and 800 mg/kg (1,1-DCA, and 1,1,1-TCA). Doses were applied six times over a 2-week period in 0.2 ml ethanol per application. 1,3-DCA was also tested with single doses of 37.5, 75, 150 and 300 mg/kg in 0.2 ml ethanol. Control animals received 0.2 ml ethanol per application as a single dose or multiple doses to match corresponding studies. Two weeks after the final dose, 1.0 microgram TPA in 0.2 ml acetone was applied three times weekly for 20 weeks. After 24 weeks the percentage of animals with tumors for dose groups above were: MCA (8); 1,1,3-TCA (10); 1,3-DCA, multiple doses (48, 45, 32); CAC (30, 28, 38); DCAC (3, 0, 0); TCAC (10, 5, 0); BAC (54, 43); 1,1-DCA (0, 5, 0); 1,1,1-TCA (10, 5, 0); 1,3-DCA, single doses (47, 47, 63, 20); controls (12--Table 3, 9--Table 4 average). These data show that 1,3-DCA, CAC and BAC, when applied topically, initiate tumors in the mouse skin. These chemicals administered orally in a 2% emulphor solution, at doses described in Table 3, did not initiate tumors in the mouse skin.

Effect of gavage vehicle on hepatotoxicity of carbon tetrachloride in CD-1 mice: corn oil versus Tween-60 aqueous emulsion.

This investigation was conducted to evaluate the effect of gavage vehicles on altering the severity of the subchronic hepatotoxicity of carbon tetrachloride (CCl4). Male and female CD-1 mice were gavaged with 0, 1.2, 12, and 120 mg/kg CCl4 in either a corn oil or 1% Tween-60 vehicle once daily for 5 consecutive days per week for 90 days. The study revealed that the hepatotoxicity was greater in the mid- and high-dose groups of mice that had received CCl4 administered in corn oil. Increases in serum enzyme activities were detected in the mid-dose groups of mice that were gavaged with CCl4 in corn oil. The serum enzyme activities were significantly higher in the high-dose groups of animals in which CCl4 was administered in corn oil. Histopathological findings indicated that hepatocellular changes following the administration of CCl4 at the mid- and high-dose levels were more frequent and more severe when CCl4 was given in corn oil than when it was administered in Tween-60. The experimental findings indicate that the no-observed-adverse-effect level from CCl4 exposure was lowered by an order of magnitude (from 12 to 1.2 mg/kg) and that the hepatotoxicity of CCl4 was enhanced in the high-dose treatment groups when corn oil was employed as the gavage vehicle.

Carcinogenic effects in A/J mice of particulate of a coal-tar paint used in potable water systems

Coal tar paints are among the products used as inside coatings for water pipes and storage tanks to retard corrosion in potable water supply systems. Four different formulations of these paints were tested in earlier work by this laboratory in the Ames mutagenesis and the mouse skin carcinogenesis bioassays. The paint most active in these assays were then tested in a particulate form in the lung adenoma assay with A/J mice. The paint was applied to clean glass plates, cured, collected and homogenized in 2% Emulphor. Doses of this coal tar suspension were administered by gavage at 1.0, 10.0 and 55.0 mg in 0.2 ml per mouse 3X weekly for 8 weeks. The total doses of coal tar paint were 24, 240, and 1320 mg/mouse. Benzo[a]pyrene (BaP), administered in a parallel schedule to a total dose of 6 mg/mouse, served as positive control. A negative control group received an equivalent volume of 2% Emulphor. Animals were killed at 9 months of age (8 months after first dose) and lung adenomas counted. A dose-related response, in the average number of lung tumors per mouse, was observed with the coal tar particulate. There were also squamous cell tumors of the forestomach in 42% of the mice receiving 55.0 mg coal tar paint per application.

Gross and microscopic lesions in the female SENCAR mouse skin and lung in tumor initiation and promotion studies.

The skin and lung tissues from SENCAR mice used as part of the Environmental Protection Agencys (EPAs) Carcinogenesis Testing Matrix were examined. This study included SENCAR mice used in three different short-term bioassay protocols in which the skin papilloma assay was used to identify initiators, promoters, and complete carcinogens. Also included were the pathology findings from SENCAR mice used in the combined bioassay in which the skin assay and the lung adenoma assay were conducted simultaneously. The gross and microscopic features of treatment-associated and spontaneous lesions of the skin and lung of the SENCAR mouse used in these studies are defined and the lesions most commonly observed are described. Generally, gross observations and microscopic findings in both the skin and lung tissues were poorly correlated. Although there are several definite criteria on which gross interpretations of the various skin and lung lesions can be made, with the exception of pedunculated squamous cell papillomas and the classic squamous cell carcinomas, the various lesion types had a wide variety of clinical presentations that severely compromised the accuracy of gross diagnosis. Further, in the case of benign skin neoplasms, malignant transformation of these tumors most often occurred at the base of the lesion and was initially hidden from gross observation. As a result, approximately 50% of the neoplasms interpreted clinically as benign tumors (papillomas and keratoacanthomas) were actually malignant neoplasms. Moreover, many lesions determined grossly to be nontumorous were in fact found to be neoplastic when examined microscopically. The SENCAR mouse was found to be more responsive in the lung adenoma assay than other strains examined with exception of the Strain A. Although accurate interpretation of the lung lesions in the SENCAR was compromised by nonneoplastic treatment-associated and/or spontaneous lesions, the feasibility of using the SENCAR skin and lung as target tissues in two-stage combined carcinogenesis studies merits further consideration. ImagesFIGURE 1.FIGURE 2.FIGURE 3.FIGURE 4.FIGURE 5.FIGURE 6.FIGURE 7.FIGURE 8.FIGURE 9.FIGURE 10.FIGURE 11.FIGURE 12.FIGURE 13.FIGURE 14.FIGURE 15.FIGURE 16.FIGURE 17.

Ten- and Ninety-Day Toxicity Studies of 1,2–Dichlorobenzene Administered by Oral Gavage to Sprague-Dawley Rats

Ten- and ninety-day toxicity studies of 1,2-dichlorobenzene (DCB) were conducted in male and female Sprague-Dawley rats to meet the needs of the U.S. Environmental Protection Agency for toxicity data on this chemical for use in their determination of possible health risks related to human exposure. 1,2-Dichlorobenzene was administered at doses of 37.5, 75, 150, and 300 mg/kg/day (10-day), and 25, 100, and 400 mg/kg/day (90-day) in corn oil by oral gavage; control animals received corn oil. At time of sacrifice, gross necropsies were performed and selected tissues were weighed and prepared for histological evaluation. Blood was taken for hematology and clinical chemistries. In the 10-day study, exposure of 300 mg DCB/kg body weight to male rats resulted in a statistically significant decrease in final body weight, organ weights (heart, kidneys, spleen, testes, and thymus), and relative organ weights (spleen and thymus). A significant increase in absolute and relative liver weights was also noted in this dose group. Males also displayed significant increases in water consumption (300 mg/kg group), ALT (300 mg/kg) and leukocyte count (150 and 300 mg/kg). A significant increase in the incidence of hepatocellular necrosis was seen in the 300 mg/kg group of males compared to controls. In the 90-day study, male rats exposed to 400 mg DCB/kg displayed a statistically significant decrease in body weight, organ weight (spleen), and relative organ weight (spleen). The absolute weights of kidney and liver and the relative weights for heart, kidney, liver, lung, brain, and testes were increased significantly for this dose group. The absolute and relative weights of both the kidney and liver were significantly increased in the female 400 mg/kg dose group. The only clinical chemistry parameters statistically different than control were increased ALT (100 and 400 mg/kg groups), BUN and total bilirubin in the male 400 mg/kg group and total bilirubin in the 400 mg/kg female group. Histopathological evaluation showed hepatocellular lesions associated with DCB treatment which included centrolobular degeneration and hypertrophy, and single cell necrosis in male and females receiving 400 mg DCB/kg. The NOAEL observed in this study is 25 mg/kg/day.