Judy A. Stober

The Carcinogenicity of Dichloroacetic Acid in the Male B6C3F1 Mouse

Groups of male B6C3F1 mice (N = 50) were provided drinking water containing 2 g/liter sodium chloride (control) and 0.05, 0.5, and 5 g/liter dichloroacetic acid (DCA). Treatment of 30 animals in each group was carried out to 60 or 75 weeks. In a separate experiment, mice exposed to 3.5 g/liter DCA and the corresponding acetic acid control group were killed at 60 weeks. Groups of 5 mice were killed at 4, 15, 30, and 45 weeks. Time-weighted mean daily doses of 7.6, 77, 410, and 486 mg/kg/day were calculated for 0.05, 0.5, 3.5, and 5 g/liter DCA treatments. Animals exposed to 3.5 and 5 g/liter DCA had final body weights that were 87 and 83%, respectively, of the control value. Relative liver weights of 136, 230, and 351% of the control value were measured for 0.5, 3.5, and 5 g/liter, respectively. At 60 weeks mice receiving 5.0 g/liter DCA had a 90% prevalence of liver neoplasia with a mean multiplicity of 4.50 tumors/animal. Exposure to 3.5 g/liter DCA for 60 weeks resulted in a 100% tumor prevalence with an average of 4.0 tumors/animal. The prevalence of liver neoplasia and tumor multiplicity at 60 and 75 weeks in the 0.05 g/liter DCA (24.1%; 0.31 tumors/animal) and in the 0.5 g/liter group (11.1%; 0.11 tumors/animal) did not differ significantly from the control value (7.1% and 0.07 tumors/animal). No liver tumors were found in the group treated with acetic acid. Hyperplastic nodules were seen in the 3.5 (58%; 0.92/animal) and 5 g/liter DCA groups (83%; 1.27/animal). There was a significant positive dose-related trend in the age-adjusted prevalence of liver tumors. These data confirm the hepatocarcinogenicity of DCA administered in the drinking water to male B6C3F1 mice for 60 weeks. The results together with those in an earlier report from this laboratory suggest, for the conditions under which these assays were conducted, a threshold concentration of at least 0.5 g/liter followed by a steep rise to a maximum tumor incidence at 2 g/liter DCA.

Hepatocarcinogenicity of chloral hydrate, 2-chloroacetaldehyde, and dichloroacetic acid in the male B6C3F1 mouse☆

The chlorinated acetaldehydes, chloral hydrate (CH) and 2-chloroacetaldehyde (CAA), have been identified as chlorination by-products in finished drinking water supplies. Although both chemicals are genotoxic, their potential for carcinogenicity had not been adequately explored. The studies reported here are chronic bioassays conducted with male B6C3F1 mice exposed to levels of 1 g/liter CH and 0.1 g/liter CAA via the drinking water for 104 weeks. Distilled water (H2O) served as the untreated control and dichloroacetic acid (DCA; 0.5 g/liter), another chlorine disinfection by-product, was included. The mean daily ingested doses were approximately 166 mg/kg/day for CH, 17 mg/kg/day for CAA, and 93 mg/kg/day for DCA. Evaluations included mortality, body weight, organ weights, gross pathology, and histopathology. The primary target organ was the liver as the organ weights and pathological changes in the other organs (spleen, kidneys, and testes) were comparable between the treated groups and the H2O control group. Liver weights were increased for all three test chemicals at the terminal euthanasia with the greatest increase seen in the CH and DCA groups. Hepatocellular necrosis was induced by all three test chemicals, and it was also most prevalent and severe in the CH and DCA groups. A significant increase in the prevalence of liver tumors was seen for all three chemicals. The strongest response was with DCA, in which 63% of the 104-week survivors had hepatocellular carcinomas (carcinomas) and 42% possessed hepatocellular adenomas (adenomas) and the combined prevalence for carcinomas plus adenoma was 75%. The corresponding prevalence rate for carcinomas, adenomas, and combined tumors were 46, 29, and 71%; 31, 8, and 38%; and 10, 5, and 15% for CH, CAA, and H2O, respectively. In addition to the tumors we evaluated the prevalence of a possible preneoplastic lesion, the hepatocellular hyperplastic nodule (nodules), a lesion which occurred in all three treated groups but not in the H2O group.

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.

Developmental toxicity of halogenated acetonitriles: Drinking water by-products of chlorine disinfection

The developmental toxicity of acetonitrile and 5 halogenated derivatives was examined with an in vivo teratology screen adapted for use in the Long-Evans rat. The screen was extended to an evaluation of growth till postnatal Days 41-42, and weight of several organs at sacrifice. Acetonitrile was without developmental effects even at doses toxic to the dam. Of the halogenated compounds, treatment with trichloroacetonitrile (TCAN) and dichloroacetonitrile (DCAN) resulted in reduced fertility and increased early implantation failure. There was no effect on litter size in females bearing live litters, but pup birth weight was reduced in all litters exposed to halogenated compounds. Perinatal survival of the pups was adversely impacted by DCAN and TCAN. Postnatal growth till Day 4 was reduced by DCAN and bromochloroacetonitrile (BCAN) while growth till Day 42 was consistently affected only by TCAN. Some general observations were made on the usefulness of the criteria used in the screen, and TCAN, the most toxic of the halogenated compounds, was selected for further in-depth evaluation.

Developmental Toxicity of Dichloroacetonitrile: A By-Product of Drinking Water Disinfection

Dichloroacetonitrile (DCAN), a by-product of drinking water disinfection formed by reaction of chlorine with background organic materials, was evaluated for its developmental effects in pregnant Long-Evans rats. Animals were dosed by oral intubation on Gestation Days 6-18 (plug = 0) with 0, 5, 15, 25, or 45 mg/kg/day. Tricaprylin was used as a vehicle. The highest dose tested (45 mg/kg) was lethal in 9% of the dams and caused resorption of the entire litter in 60% of the survivors. Embryolethality averaged 6% per litter at the low dose and 80% at the high dose and was statistically significant at 25 and 45 mg/kg/day. The incidence of soft tissue malformations was dose related and was statistically significant at doses toxic to the dam (45 mg/kg). These anomalies were principally in the cardiovascular (interventricular septal defect, levocardia, and abnormalities of the major vessels) and urogenital (hydronephrosis, rudimentary bladder and kidney, fused ureters, pelvic hernia, cryptorchidism) systems. The frequency of skeletal malformations (fused and cervical ribs) was also dose related and significantly increased at 45 mg/kg. The no-observed-adverse-effect dose for toxicity in pregnant Long-Evans rats was established by statistical analysis to be 15 mg/kg/day.

Effect of wastewater spray irrigation on rotavirus infection rates in an exposed population

The Lubbock Infection Surveillance Study was conducted between June 1980 and October 1983 to detect potential increases in enteric infection and disease incidence in a community surrounding a wastewater spray irrigation site. This report concerns the incidence of rotavirus infections in study participates during that period. Rotavirus infection was defined as a > 2-fold increase in rotavirus serum antibody between blood collections which occurred approximately every 6 months. Antibody was detected by an enzyme-linked immunosorbent assay (ELISA). Of the 368 participants who provided ⩾ 2 blood specimens, 67 seroconversions to rotavirus were detected, an average annual rate of 6.8 infections/100 subjects. One subject was infected twice. Seroconversions were observed in 32.7% (3298) of children ⩽ 16 years of age and in 12.7% (33260) of adults (⩾ 17 years old) with a single infection. Thus, significantly more (P < 0.0001) rotavirus infections occurred in children. Baseline rotavirus serum antibody titers were found to be significantly lower (P = 0.047) in subjects who seroconverted. However, many children and adults with high titers were also infected. More seroconversions were observed between June and December (January) than between December (January) and June. Wastewater spray irrigation had no detectable effect on the incidence of rotavirus infection.

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.

Sources of variation in the computer-assisted motion analysis of rat epididymal sperm

Random and nonrandom factors associated with sample preparation and the automated analysis (CellSoft) of rat cauda epididymal sperm motion were studied. Random factors included inherent system variation at both the individual cell level and at the multiple cell level. Repeated analyses of identical tracks across grey level revealed a statistical interaction between grey settings and curvilinear velocity. However, in multiple track analyses, grey level was seen to be a factor only at higher settings. Nonrandom factors included time after sample preparation, dilution medium, and sample preparation procedures. Using a nicked preparation of the entire cauda epididymis from Long-Evans rats, the effects of time were studied on sperm suspended in 1) phosphate-buffered saline + 10 mg BSA/mL, 2) TEST yolk buffer, and 3) Medium 199. In PBS/BSA, the percent motile sperm estimate decreased (50% to 30%) over an hour, while the curvilinear velocity increased (127 to 142 microns/sec). Both sperm motion parameters were maintained in the TEST yolk buffer and in the Medium 199, although at lower values for the latter. Evaluation of the relative contribution of several factors, nested within sample, to the overall variance of three separate motion endpoints revealed that there was a large variation from field to field, negligible variation between overall CellSoft analyses of 200 cells or more, low variation at the preparation aliquot level, and moderate variation at the animal level. In planning experiments to test for effects on sperm motion endpoints, consideration of the relative contribution of the individual study factors to the overall variance of the parameter estimates will result in more sensitive experimental designs.

Comparison of two routes of chemical administration on the lung adenoma response in strain AJ mice

This study was undertaken to determine the ability of a series of 19 compounds representing different chemical classes of carcinogens to induce lung tumors in strain A/J mice after either ip or po administration. Aflatoxin B1, dibutylnitrosamine, 1,2-dimethylhydrazine, and methylnitrosourea induced a significant increase in the lung tumor response in both sexes after ip and po administration. Azaserine was active in both sexes only after ip administration. Benzene, 1,2-dibromoethane, and epichlorohydrin, following ip administration, produced significant increases in the tumor response in at least one sex. Aflatoxin B1, azaserine, benzene, 1,2-dibromoethane, dibutylnitrosamine, and epichlorohydrin were more active when given ip than after po administration. In contrast, dimethylhydrazine and methylnitrosourea were more active (in females only) when given po. 2-Acetylaminofluorene, azobenzene, chloroform, 1,4-dioxane, FANFT (N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide), lead subacetate, methylmethanesulfonate, beta-naphthylamine, beta-propiolactone, safrole, and 2,4,6-tri-chlorophenol did not induce lung tumors in strain A/J mice. These data confirm previous observations on the importance of the route of chemical administration on the lung tumor response in strain A mice, and on the inability of the lung tumor bioassay to detect certain liver and bladder carcinogens and unstable alkylating agents.