Greg R. Olson

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.

Differential Effects of Silver Nanoparticles and Silver Ions on Tissue Accumulation, Distribution, and Toxicity in the Sprague Dawley Rat Following Daily Oral Gavage Administration for 13 Weeks

There are concerns within the regulatory and research communities regarding the health impact associated with consumer exposure to silver nanoparticles (AgNPs). This study evaluated particulate and ionic forms of silver and particle size for differences in silver accumulation, distribution, morphology, and toxicity when administered daily by oral gavage to Sprague Dawley rats for 13 weeks. Test materials and dose formulations were characterized by transmission electron microscopy (TEM), dynamic light scattering, and inductively coupled mass spectrometry (ICP-MS). Seven-week-old rats (10 rats per sex per group) were randomly assigned to treatments: AgNP (10, 75, and 110 nm) at 9, 18, and 36 mg/kg body weight (bw); silver acetate (AgOAc) at 100, 200, and 400 mg/kg bw; and controls (2 mM sodium citrate (CIT) or water). At termination, complete necropsies were conducted, histopathology, hematology, serum chemistry, micronuclei, and reproductive system analyses were performed, and silver accumulations and distributions were determined. Rats exposed to AgNP did not show significant changes in body weights or intakes of feed and water relative to controls, and blood, reproductive system, and genetic tests were similar to controls. Differences in the distributional pattern and morphology of silver deposits were observed by TEM: AgNP appeared predominantly within cells, while AgOAc had an affinity for extracellular membranes. Significant dose-dependent and AgNP size-dependent accumulations were detected in tissues by ICP-MS. In addition, sex differences in silver accumulations were noted for a number of tissues and organs, with accumulations being significantly higher in female rats, especially in the kidney, liver, jejunum, and colon.

The Effects of Subchronic Chlorate Exposure in Sprague-Dawley Rats

Male and female Sprague-Dawley rats were exposed to drinking water containing 3.0, 12.0 or 48.0 mM sodium chlorate. The mean drinking water consumption varied between exposure groups from 100-200 ml/kg/day. Female exposure groups consistently drank more water (23-42%) than male exposure groups thereby receiving more chlorate/kg/day at every exposure level. There were no compound related deaths; however, both males and females in the high exposure groups had significant weight loss during the 90-day exposure period. Also, in these same groups females had mild but significant decreases in the following relative organ weights; adrenals, thymus and spleen, while the relative brain weight was increased. In males, the heart, kidneys and liver were mildly decreased while the brain and testes were mildly increased. Red blood cell counts and percent hematocrit were decreased in both sexes in the high dose group. Pituitary gland (pars distalis) vacuolization and thyroid gland colloid depletion were prominent in both sexes in mid and/or high dose animals. A NOAEL of 0.36 mM chlorate/kg b.w./day in males and 0.50 mM chlorate/kg b.w./day in females were established.

Fourteen‐day Toxicity Study of 1,3,5‐Trinitrobenzene in Fischer 344 Rats

Toxic effects of 1,3,5‐trinitrobenzene (TNB) in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg kg−1 diet) for 14 days. Food intake by female rats in 400, 800 and 1200 mg TNB diet groups was reduced and resulted in a significant decrease in absolute body weights (BW). Food and water consumption by male rats in high‐dose groups (800 and 1200 mg TNB kg−1 diet) was also reduced and resulted in a significant decrease in body weight. The calculated average TNB intake (from 1200 mg TNB kg−1 diet) was 92 mg kg−1 BW day−1 for male rats and 80 mg kg−1 BW day−1 for females. A decrease in testicular weight in males and an increase in spleen weight of both sexes in high‐dose groups was noted. In addition, histopathological examinationsrevealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hematopoiesis), brain (hemorrhage, malacia and gliosis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in Heinz bodies and increased methemoglobin concentration as compared to controls in both sexes. A lowest observed adverse effect level of 4.41 mg TNB kg−1 BW day−1 was established based on the findings of this study.

Ten and Ninety-Day Toxicity Studies of chloropicrin in Sprague-Dawley Rats

The toxicity of chloropicrin (CP) was assessed following its administration to rats via oral gavage for either 10 or 90 consecutive days at dose levels of 10, 20, 40, and 80 mg/kg and 2, 8 and 32 mg/kg, respectively. Control rats received corn oil at a dose of 1.0 ml/kg. Toxicological observations included organ and body weight measurements, necropsy and histopathology observations, urinalysis, clinical chemistry and hematology determinations. The most remarkable toxicological finding in both studies was the corrosive property of CP on forestomach tissue. Inflammation, necrosis, acantholysis, hyperkeratosis and epithelial hyperplasia of the forestomach were seen in all dose groups of the 10-day study. Similar changes were detected in only the high dose group in the 90-day study. Decreased red blood cell parameters were noted in the highest dose groups in both studies, possibly due to blood loss via the damaged stomach lining. CP may have been aspirated into the lungs of animals in the high dose group in the ninety day study resulting in pulmonary complications leading to the deaths of 60% of the males and 80% of the females starting at week five. The 8 mg/kg dose group in the ninety day study was considered to be the no observed adverse effect level.

Ten and Ninety-Day Toxicity Studies of 1,2-Dichloroethane in Sprague-Dawley Rats

Male and female Sprague-Dawley rats received 1,2-dichloroethane in corn oil by gavage for 10 or 90 consecutive days. The doses for the 10-day study were 10, 30, 100, and 300 mg/kg; the 90-day study doses were 37.5, 75, and 150 mg/kg. There were ten animals per sex per dose group. In the 10-day study, all female animals died in the high dose group and only 2 of 10 males survived. Final body weights and weight gain along with hematology and clinical chemistry findings were not different from controls. The only relative organ weight which was significantly different was the liver in males exposed to 100 mg/kg. The main histopathological lesion exhibited was multifocal to diffuse inflammation of the mucosal and submucosal layers of the forestomach in the 100 mg/kg dose group. This change was minimal in both males and females. In the 90-day study there were no treatment-related effects pertaining to clinical observations. Body weight gain and total food consumption were significantly decreased in high dose males. There were slight but significant differences in hemoglobin, hematocrit, red blood cell count, platelets, albumin, and alkaline phosphatase values in the 75 and/or 150 mg/kg groups in one or both sexes. In males, relative brain, kidney, and liver weights were significantly increased at 75 and 150 mg/kg. There were also differences in spleen, adrenal, and testes weights (absolute and/or relative). In females, absolute and/or relative kidney and liver weights were significantly increased at 150 mg/kg (liver) and at 75 and 150 mg/kg (kidney). There were no apparent treatment-related effects pertaining to mortality, ophthalmology, gross pathology, or histopathology.

Toxicity of Tetryl (N-Methyl-N,2,4,6–Tetranitroaniline) in F344 Rats

The toxicity of tetryl (N-methyl-N,2,4,6–tetranitroaniline) in male and female F344 rats was evaluated after adminstration in the diet for 14 or 90 days. The 14–day study diet concentrations used were 0, 500, 1250, 2000, 2500, and 5000 ppm; the 90–day study diet concentrations were 0, 200, 1000, and 3000 ppm tetryl in the diet. The calculated average daily tetryl intake was 32.1, 82.5, 130.3, 178.9, and 374.4 mg/kg body weight (BW) for females and 31.8, 80.0, 121.0, 170.5, and 349.7 mg/kg BW for males in the 14–day study. For the 90–day studies, the daily tetryl intake was 14.2, 68.8, and 199.0 mg/kg BW for females and 13.0, 62.4, and 179.6 mg/kg BW for males. In the 14–day study, there was a significant decrease in body weights (males), whereas relative(organ/body weight) liver and spleen (females), and kidney (males) weights were significantly increased in the 5000–ppm dose group. Hematological effects observed were decreased hemoglobin and hematocrit and an increased number of reticulocyts in females (2000 to 5000 ppm). Methemoglobin levels in males (2000 to 5000 ppm) and females (5000 ppm) and total blood protein and albumin levels in all groups of males and females (except 500 ppm) were significantly increased. Histopathological changes were observed in kidneys (deposition of cytoplasmic droplets) of all dose groups of male rats. In the sub–chronic (90–day) study, feed intake was reduced in all dose groups, but a significant decrease in terminal body weights was observed in females (1000 and 3000 ppm) and males (3000 ppm). An increase in the relative liver, kidney (1000–3000 ppm), and spleen (3000 ppm) weights were noted in both sexes. The hemoglobin content and red blood cell count were decreased whereas the reticulocyte count was elevated (3000 ppm) in both sexes at 45 and 90 days. Methemoglobin levels were increased in both sexes (1000 and 3000 ppm). Histopathologicalchanges were noted in the spleen (pigment deposition and erythroid cell hyperplasia) of both sexes (3000 ppm) and kidneys (tubular degeneration and cytoplasmic droplets containing alpha-2-micro globulin) of male rats (1000 to 3000 ppm). A no observed adverse effect level (NOAEL) for both sexes was 13 mg/kg BW/day was determined.

Subchronic Toxicity of 1,3,5-Trinitrobenzene in Fischer 344 Rats

The subchronic toxicity of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 rats was evaluated by feeding a powdered certified laboratory diet containing 0, 66.7, 400 and 800 mg TNBl kg diet for 90 days. The calculated average TNB intake was 4.29, 24.70, and 49.28 mg/kg body weight (BW)day for females and 3.91, 22.73, and 44.16 mg/ kg BWI day for males. Food intake in the 400 and 800 mg/kg diet dose groups of both sexes was decreased throughout the study and resulted in a significant decrease in absolute body weights. A significant decrease in relative testicular weights and a significant increase in the relative liver weight were observed in male rats receiving 400 or 800 mg TNB/kg diet. A significant increase in the relative spleen weights of both sexes receiving 400 or 800 mg TNB diet was noted. The relative liver weight was also increased only in female rats maintained on the 800 mg TNB diet. Histopathological examinations revealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets) in all male dose groups and testes (seminiferous tubular degeneration) in rats receiving 400 and 800 mg TNB diet groups. The spleen was also affected (extramedullary hematopoiesis) in both sexes in the 400 and 800 mg dose groups. Hematological studies at both 45 (data not given) and 90 days in the 400 and 800 mg dose groups indicated decreased values for red blood cell counts and hemoglobin content, while reticulocytes and methemoglobin levels were increased. Clinical chemistry parameters were unaffected. Based on kidney toxicity and hematological effects, a Low Observed Adverse Effect Level (LOAEL) of 3.91 mg/kg BWI day was suggested for subchronic toxicity studies on TNB.

Toxicity Studies of Epichlorohydrin in Sprague-Dawley Rats

Adult male and female Sprague-Dawley rats received epichlorohydrin via gavage in distilled water for 10 consecutive days at dose levels of 3, 7, 19, and 46 mg/kg-day, and for 90 days at dose levels of 1, 5, and 25 mg/kg-day. Epichlorohydrin did not adversely effect mortality, but toxicity, at the higher doses, was evident by: 1) losses in body weight gain and organ weights, 2) reductions in food and water consumption, and 3) in the hematological and microscopic examinations in both study periods. Significant decreases in erythrocyte count, hemoglobin, and hematocrit levels were found in the high dose level in males after 10 and 90 days. Dose-related increases in kidney and liver weights were observed in both sexes at 25 mg/kg-day in the 90-day study and in various organs for both 19 and 46 mg/kg-day in the 10-day study. Histopathological examination identified the forestomach as the primary target organ for both sexes and in both studies with significant dose-related increases in mucosal hyperplasia (acanthosis) and hyperkeratosis. Based on the data presented, a lowest observable adverse effect level (LOAEL) for oral exposure of Sprague-Dawley rats to epichlorohydrin is 3 mg/kg-day for 10 days and 1 mg/kg-day is suggested as the no observed adverse effect level (NOAEL) for a 90 day oral exposure. These conclusions were the same whether the lesions were analyzed for each sex individually or whether the data in each study was pooled.