13-wk Study Research Articles

Assessment of the carcinogenicity of stevioside in F344 rats

The carcinogenic potential of stevioside, a compound that is used as a sweetener for food and drink, was examined in F344 rats of both sexes. Stevioside was added to powdered diet at concentrations of 0 (control), 2.5 and 5%. The doses were selected on the basis of results from a 13-wk subchronic toxicity study and administered to groups of 50 male and 50 female rats ad lib. for 104 wk. All surviving rats were killed at wk 108. Body weight gains were slightly depressed in line with the dose of stevioside, in both sexes, and a significant decrease in the final survival rate was observed for the 5% treated males. Histopathologically, however, there was no significantly altered development of neoplastic or non-neoplastic lesions attributable to the stevioside treatment in any organ or tissue, except for a decreased incidence of mammary adenomas in females and a reduced severity of chronic nephropathy in males. It is concluded that stevioside is not carcinogenic in F344 rats under the experimental conditions described.

New Nonpurified Diet (NTP-2000) for Rodents in the National Toxicology Program's Toxicology and Carcinogenesis Studies

The NIH-07 open-formula nonpurified diet was the selected diet for rodents in the National Toxicology Program's toxicology and carcinogenesis studies from 1980 to 1994. Protein and mineral concentrations of the NIH-07 diet may have increased some diet- and age-associated lesions such as nephropathy. A number of experimental nonpurified diets with lower protein and higher fat and fiber (∼15% protein, 7–8.5% fat, and 9–14% crude fiber) than the NIH-07 diet were formulated and evaluated in Fischer 344 (F344) rats. Decreasing protein content of the diet decreased protein consumption by ∼30% and decreased severity of nephropathy without affecting growth. Increased fat intake seemed to have decreased the incidence or severity of leukemia, a lethal neoplasm of F344 rats. Increasing fiber content without decreasing the caloric density lowered body weight gain and slowed growth of mammary tumors. Higher fat and/or fiber intake decreased the incidences of adrenal pheochromocytomas and medullary hyperplasia in male rats. Nonpurified diets with lower protein and higher fat and fiber concentrations than the NIH-07 diet decreased or delayed diet- and age-associated lesions and increased survivals in 2-y studies. On the basis of these results, a new cereal-based nonpurified diet, designated as NTP-2000, was formulated with ∼14.5% protein, ∼8.2% fat, ∼9.3% fiber and a calcium:phosphorus molar ratio of ∼1.3. The NTP-2000 diet was compared with the NIH-07 diet in a 13-wk study in F344 rats. The NTP-2000 diet was adequate for growth, did not affect the hematological parameters and did not cause substantial changes in blood chemistry, serum enzyme or serum electrolyte values. The NTP-2000 diet decreased liver and kidney weights, prevented nephrocalcinosis and decreased the severity of diet- and possibly age-associated lesions.

Carcinogenicity study of β-cyclodextrin in F344 rats

The carcinogenicity of β-cyclodextrin, a cyclic, water-soluble carbohydrate comprising seven glucose units, was examined in Fischer 344 (F344) rats. Groups of 50 males and 50 females were given the compound in their diet at concentrations of 0 (control), 2.5 or 5% for 104 wk. Surviving rats were then given a basal diet for a further 5 wk and killed at 109 wk. The dose levels were selected from the results of a 13-wk subchronic toxicity study. Dose-dependent inhibitory effects of β-cyclodextrin on growth were observed in both sexes of the treated groups. The survival rates, mean survival times and range, however, demonstrated no significant differences between the control and treated groups. A variety of tumours developed in all groups, including the control group, but all the neoplastic lesions were histologically similar to those known to occur spontaneously in this strain of rat, and no statistically significant increase in the incidence of any tumour was found for either sex of the treated groups. Thus, it is concluded that under the present experimental conditions, the high dose, about 340–400 times higher than the current daily human intake from ingestion as a food additive and from pharmaceutical use, does not have any carcinogenic potential in F344 rats.

Non-carcinogenicity of 2,2′-[(4-Aminophenyl)imino]bisethanol sulfate in a long-term feeding study in fischer 344 rats

2,2′-[(4-aminophenyl)imino]bisethanol sulfate (4APE) was administered at dietary levels of 0 (control), 300, 1000 and 3000 ppm to groups of 50 male and 50 female Fischer 344/DuCrj rats for 104 wk. As slight body weight retardation was observed in the male 3000 ppm group in the preliminary 13-wk feeding study, this dose was selected for the highest exposure level. Mean body weights of both sexes in the 3000 ppm group were lower than those of the controls from wk 2 to termination. However, there were no treatment-related clinical signs or adverse effects on survival rate, food consumption or haematology data. Very slight but statistically significant increases in relative thyroid weights were found in males of the 3000 ppm group, but there was no significant treatment-related increase in the incidence of any non-neoplastic or neoplastic lesions. Thus, under the experimental conditions used, 4APE was not carcinogenic in Fischer 344 rats of either sex.

Effects of D&C yellow no. 11 ingestion on F344/N rats and B6C3F1 mice.

D&C yellow no. 11 (CAS no. 8003-22-3) was administered in the feed at concentrations of 500-50,000 ppm to groups of F344/N rats and B6C3F1 mice of each sex for 13 wk to determine the toxicity. In addition, a perinatal study was conducted to determine the effects of feeding diets containing D&C yellow no. 11 to female rats during reproduction and to their offspring. Although the estimated intake (g/kg) of D&C yellow no. 11 of mice was more than twice that of rats, the results were generally similar for both rats and mice. In both species, D&C yellow no. 11 caused no mortality, but it did reduce body weight gain slightly in both sexes of rats exposed to 17,000 and 50,000 ppm. Absolute and relative liver weights were significantly increased in all groups of rats and mice administered D&C yellow no. 11 in the feed. There was minimal to mild degeneration of the periportal hepatocytes in rats at doses of 1700 ppm and higher and in mice at 5000 ppm and above. A dose-related yellow-brown pigment was observed in hepatocytes, Kupffer cells, and biliary epithelium of the liver of both sexes of both species and in the renal tubule epithelium in both sexes of rats. In male rats, all treated groups had increased number and size of hyaline droplets in the renal tubule epithelium of the cortex and outer medulla. To determine if these renal and hepatic lesions were reversible, male rats were administered 5000 ppm dietary D&C yellow no. 11 for 70 d and then examined at 3, 14, and 28 d after the chemical was removed from the diet. Pigment persisted in the kidney and liver for as long as 28 d following removal of D&C yellow no. 11 from the diet, but hepatocellular degeneration and cytoplasmic alteration in the kidney completely resolved by d 3 and 14, respectively. In the perinatal toxicity study, body weight gain in rat dams given diets containing as much as 50,000 ppm D&C yellow no. 11 for 4 wk before mating to untreated males was similar to that of controls at the time of mating but was lower at parturition and weaning. However, fertility, gestation length, litter size, and pup birth weights were unaffected by treatment. At weaning, there was a significant dose-related decrease in pup body weights from the 5000, 17,000, and 50,000 ppm groups. At 8 wk of age, pups fed the same dosed-feed concentrations as the dams had depressed body weights in the 17,000 and 50,000 ppm treated groups. Microscopic lesions in the liver and kidney of the pups in all dose groups were similar to those described in the 13-wk study. The results of these studies indicate that compound-related effects occurred at all dietary concentrations of D&C yellow no. 11. Liver weights were increased in dosed rats and mice, minimal to mild hepatocellular degeneration was seen in rats receiving dietary concentrations of 1700 ppm and above and in mice at 5000 ppm and above, and there was an increase in the number and size of hyaline droplets in all dosed groups of male rats. Similar compound-related effects were also seen in all dosed rats in the perinatal toxicity study. With the exception of pigment accumulation, the treatment-related kidney and liver lesions in male rats were reversible by 14 d after chemical was withdrawn from the diet.

Single-dose and 13-week repeated-dose neurotoxicity screening studies of chlorpyrifos insecticide

Chlorpyrifos (CPF), a widely used organophosphate insecticide, was screened for neurotoxic effects in Fischer 344 rats using United States Environmental Protection Agency 1991 guidelines for single-dose and 13-wk repeated dose studies. The studies emphasized a functional observational battery (which included grip performance and hindlimb splay tests), automated motor activity testing and comprehensive neurohistopathology of perfused tissues. Doses of up to 100 mg/kg body weight in corn oil by gavage in the single-dose study and up to 15 mg/kg body weight/day in diet for 13 wk in the repeated dose study were administered. It is known that CPF and other phosphorothionates can be activated to the oxon in local (extrahepatic) tissues. Local activation could possibly cause different effects in different tissues with cholinergic innervation, and thereby create syndromes unique to each phosphorothionate according to their structure. Consequently, the conduct of CPF neurotoxicity screening studies by contemporary guidelines offered an opportunity to characterize the CPF over-exposure syndrome in rats. Single-dose Zhigh levels of oral exposure to CPF caused a range of clinical signs characteristic of cholinergic overstimulation. Although there was no clinical evidence of wide differences in sensitivity of one cholinergic response versus another, motor dysfunction (inco-ordination etc.) was more prominent than other signs, for example soiling. Effects were much more apparent in females and regressed over several days. Effects were minimal in the 13-wk study, and there was no evidence of accumulation of toxicity during the 13 wk of daily dietary exposure. Motor activity was decreased at the high dose in males and females at wk 4, but was not significantly different from controls in subsequent weeks. The ‘normalization’ of motor activity later in the study was interpreted as tolerance to repeated administration of CPF. Comprehensive neuropathological examination revealed no treatment-related lesions in either study

Chemicals associated with decreases in the incidence of mononuclear cell leukemia in the Fischer rat.

A significant treatment-related decrease in the incidence of mononuclear cell leukemia (MCL) was identified in Fischer-344/N rats for 20 chemicals tested in the National Toxicology Program's 2-yr carcinogenicity bioassay. Fourteen of the 20 chemicals caused decreases of MCL in both male and female rats; 6 of the 20 caused a significant decrease only in males and a marginal or no decrease in female rats. Seventeen of the chemicals associated with a decrease in MCL had a free aromatic amine or nitro functional groups that could be metabolized to free amines. With 1 exception, all 14 chemicals causing a decrease in MCL in both sexes produced spleen toxicity in the 13-wk studies. Reduced body weight and decreased survival, related either to toxicity or to an increase in other types of lethal neoplasms, did not contribute to the decreases in MCL observed in chemical exposure groups. Thirteen of the 20 chemicals were positive in Salmonella tests, and 15 were associated with increases in neoplasms at other sites in rats and/or mice, suggesting that different metabolites could be responsible for these varied biological effects.

Evaluation of the safety of sodium pectate as a food ingredient

New potential uses of pectates in food products have recently stimulated interest in re-evaluating the information available concerning the safety of pectins and pectates as food ingredients. Data relevant to this re-evaluation have been obtained in rats in recent 14-day and 13-wk subchronic feeding studies with sodium pectate. Ames tests and other mutagenicity tests have been conducted with sodium pectate, bleached sodium pectate and mixed sodium/calcium pectate salts. These toxicological studies with pectates have provided further evidence of their safety, and support the continued GRAS status of pectins and pectate salts.

Study on the carcinogenicity of tannic acid in F344 rats

The carcinogenicity of tannic acid, a compound that is used as a food additive, a clarifying agent and a refining agent, was examined in F344 rats of both sexes. Tannic acid was dissolved in distilled water at concentrations of 0.25 and 0.5%. The doses were selected on the basis of results from a 13-wk subchronic study. Groups of 50 male and 50 female rats were given one of these solutions ad lib as their drinking water for up to 2 yr. The mean body weights of the treated males were essentially comparable with those of the controls, whereas treated females had lower mean body weights than the control group. A variety of tumours developed in all groups, including the control group, but all the neoplasms were histologically similar to those known to occur spontaneously in this strain of rats, and no statistically significant increase in the incidence of any tumour was found in the treated groups of either sex. Thus, it is concluded that, under the conditions of the experiment, tannic acid has neither carcinogenic potential in F344 rats nor modifying effects on spontaneous tumour development.

Acute, 2-week, and 13-week inhalation toxicity studies on dimethylethoxysilane vapor in Fischer 344 rats.

Dimethylethoxysilane (DMES), a volatile liquid, is used by NASA to waterproof the heat-protective silica tiles and blankets on the Space Shuttle. Acute, 2-wk, and 13-wk inhalation exposures to DMES vapor were conducted in male and female Fischer 344 rats. In the acute study, rats were exposed to 4000, 2000, 1000, 500, or 0 (control) ppm DMES for 4 h and observed for 14 days. There were no deaths. Narcosis and ataxia were observed in rats of the two highest concentrations only. These signs disappeared within 1 h following exposure. There were no DMES-related gross or microscopic tissue lesions in rats of all exposure groups. In the 2-wk study, rats were exposed for 6 h/day, 5 days/wk to 3000, 1000, 300, 100, or 0 ppm DMES. During exposure, narcosis was observed in rats of the 3000 and 1000 ppm groups. There was a mild decrease in body weight gain in rats of the 3000 ppm group. A decrease in platelet count, an increase in bile acids, and reduced weights of the thymus, testis, and liver were observed in rats of the 3000 ppm group. Microscopically, hypospermatogenesis and spermatid giant cells were observed in the seminiferous tubules of the testes of rats exposed to 3000 ppm DMES. In the 13-wk study, rats were exposed 6 h/day, 5 days/wk to 2000, 600, 160, 40, or 0 ppm DMES. During exposure, rats of the 2000 ppm group exhibited mild narcosis and loss of startle reflex. Recovery from these central nervous system signs was rapid. Body weights were mildly decreased for rats of the 2000 ppm group. There were no exposure-related effects in hematology, serum chemistry, or urinalysis. Female rats of the 2000 ppm group had delayed estrous cycles (6 days compared to 5 days in control rats). Noteworthy organ weight changes in rats of the 2000 ppm group included decreases in thymus, liver, and testicular weights; however, pathologic lesions were observed in the testes only. Sperm motility, epididymal sperm count, and testicular spermatid count were dramatically reduced. Microscopic lesions included degeneration of the seminiferous tubular cells, pyknosis or absence of germ cells, and hypospermia in the epididymis. Rats of the 600 ppm group had a slight decrease in thymic weight and a transient decrease in body weight. Results of the acute, 2-wk, and 13-wk inhalation studies indicate DMES concentrations of 1000 ppm and higher produce narcosis that rapidly disappears following exposure. Repeated exposure of rats to DMES at either 3000 ppm for 2 wk or 2000 ppm for 13 wk caused testicular atrophy and hypospermia in male rats. Female rats exposed to 2000 ppm for 13 wk had delayed estrous cycles. Toxicological effects in rats of the 600 ppm group were minimal and equivocal. The 160 ppm concentration was a no-observable-effect level (NOEL) for 13 wk of exposure to DMES.

Toxicity and carcinogenicity of hydroquinone in F344/N rats and B6C3F 1 mice

Toxicology and carcinogenesis studies were conducted by administering hydroquinone (more than 99% pure) by gavage to groups of F344/N rats and B6C3F 1 mice of each sex for 14 days, 13 wk or 2 yr. 14-day studies were conducted by administering hydroquinone in corn oil to rats at doses ranging from 63 to 1000 mg/kg body weight and to mice at doses ranging from 31 to 500 mg/kg, 5 days/wk. In the 13-wk studies, doses for rats and mice ranged from 25 to 400 mg/kg. At those doses showing some indication of toxicity in the 14-day and 13-wk studies, the central nervous system, forestomach and liver were identified as target organs in both species and renal toxicity was observed in rats. Based on these results, 2-yr studies were conducted by administering 0, 25 or 50 mg hydroquinone/kg in deionized water by gavage to groups of 65 rats of each sex, 5 days/wk. Groups of 65 mice of each sex were given 0, 50 or 100 mg/kg on the same schedule. 10 rats and 10 mice from each group were killed and evaluated after 15 months. Mean body weights of high-dose male rats and high-dose mice were approx. 5–14% lower than those of controls during the second half of the study. No differences in survival were observed between dosed and control groups of rats or mice. Nearly all male rats and most female rats in all vehicle control and exposed groups had nephropathy, which was judged to be more severe in high-dose male rats. Hyperplasia of the renal pelvic transitional epithelium and renal cortical cysts were increased in male rats. Tubular cell hyperplasia of the kidney was seen in two high-dose male rats, and renal tubular adenomas were seen in 4/55 low-dose and 8/55 high-dose male rats; none was seen in vehicle controls or in female rats. Mononuclear cell leukaemia in female rats occurred with increased incidences in the dosed groups (vehicle control, 9/55; low dose, 15/55; high dose, 22/55). Compound-related lesions observed in the liver of high-dose male mice included anisokaryosis, syncytial alteration and basophilic foci. The incidences of hepatocellular neoplasms, primarily adenomas, were increased in dosed female mice (3/55; 16/55; 13/55). Follicular cell hyperplasia of the thyroid gland was increased in dosed mice. In summary, there was evidence of hydroquinone-related carcinogenicity in male F344/N rats as indicated by increased incidences of tubular cell adenomas of the kidney, in female rats as shown by increases in mononuclear cell leukaemia, and in female mice based on increases in hepatocellular neoplasms, mainly adenomas. There was no evidence of carcinogenicity in male mice.

Subchronic toxicity study of Caramel Colour II in F344 rats

Caramel Colour II is a distinct type of colourant with a pronounced reddish hue. It is made with sulphite reactants but without ammonia. The red colour and a high alcohol solubility provide functional characteristics that are important in foods or beverages containing natural flavour extractives. Caramel Colour II is widely used in ice creams and liqueurs; however, it represents less than 1% of total caramel colour manufacture. The toxicity of Caramel Colour II was evaluated in a 13-wk study in Fischer-344 (F344) rats. The test material was mixed with demineralized water and the solutions were given to the animals ad lib. in the drinking fluid. The concentrations of caramel colour in the drinking fluid were adjusted periodically to achieve the desired caramel colour intake/kg body weight/day. Groups of 20 rats/sex were given Caramel Colour II at levels of 0, 4, 8, 12 or 16 g/kg for at least 13 wk. There were no deaths in any of the groups fed Caramel Colour II. All rats fed caramel colour had soft faeces. All treated groups also had lower fluid consumption that was attributed to poor palatability of the high concentrations of caramel colour that were fed. A number of changes observed (reduced food consumption in all treatment groups except males given 4 g/kg; significantly lower body weights for males given 12 g/kg or more and for females given 8 g/kg or more; lower urine volume and higher specific gravity) were attributed to the reduced water intake and not considered to be toxicologically significant. There were no consistent treatment-related alterations in haematology or blood chemistry variables, and random changes noted were not associated with macroscopic or microscopic pathological alterations. There were no toxicologically important pathological findings. Based on this study, Caramel Colour II was not toxic in F344 rats treated for 13 wk. The highest dose level tested in this study (16 g/kg) was considered to be the no-observed-adverse-effect level.

Clinical pathology in the National Toxicology Program.

The National Toxicology Program (NTP) developed a standard approach for clinical pathology investigations that was integrated into most toxicity studies designed and conducted after 1986. Protocols for these studies include specific hematology and clinical chemistry analyses at 3 selected time points in 13-wk studies. Requirements concerning the anesthetization of animals, collection and analysis of samples, and reporting of results have been established to control sources of variability within and between contract laboratories that perform these studies for the NTP. Laboratories must meet minimum standards to be approved for participation in the Program. Important areas of consideration for these laboratories to perform clinical pathology investigations include the facility, equipment, personnel, performance, and quality control procedures. Clinical pathology results from approximately 60 13-wk studies that have been conducted by the NTP in 7 laboratories since 1987 are being analyzed to generate a database of control values for the Fischer rat and B6C3F1 mouse and to identify sources of variability. Experimental data from these studies are being analyzed and correlated with histopathologic findings to evaluate the contribution of clinical pathology to the characterization of toxicity and to examine the appropriateness of the current approach. Efforts such as these will provide for the evolution and continued relevance of clinical pathology in toxicity testing.

Toxicity studies of Caramel Colour III and 2-acetyl-4(5)-tetrahydroxybutylimidazole in F344 rats

Caramel Colour III is used as a colour additive in beers and a variety of foods. Beer is the most important single source of Caramel Colour III in the diet although consumption of dark beers has been decreasing in recent years. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has established an acceptable daily intake of 200 mg/kg/day for Caramel Colour III. The safety of Caramel Colour III has been questioned during recent years following feeding studies in the rat that were associated with reduced white cell and lymphocyte counts. These effects have been attributed to the presence of 2-acetyl-4(5)-tetrahydroxybutylimidazole (THI) in this class of caramel colour. Short-term oral toxicity studies were conducted on low-THI and high-THI samples of Caramel Colour III (13 wk) and on a sample of THI (28 days). In both studies, the test materials were mixed with demineralized water and the solutions were given to the animals ad lib. in the drinking fluid. In the 13-wk subchronic toxicity study of Caramel Colour III, groups of 20 rats/sex were given concentrations of caramel colour equivalent to intakes of 0, 10, 15 or 20 g low-THI caramel colour/kg body weight/day or 20 g/kg of a high-THI caramel colour. In the 4-wk toxicity study with THI, groups of 20 rats/sex were given 0, 8 or 64 ppm THI (equivalent to approx. 0, 0.9 or 7.2 mg/kg/day) and 10 rats/sex were given 1, 2, 4, 16 or 32 ppm THI (equivalent to approx. 0.1, 0.2, 0.5, 1.9 or 3.7 mg/kg/day) for 4 wk followed by a 2-wk recovery phase for 10 rats/sex in the 0, 8 and 64 ppm groups. Rats given Caramel Colour III had soft faeces; there were no other treatment-related clinical observations and no treatment-related deaths occurred. All treated groups given Caramel Colour III had lower food and fluid consumption than controls. Males given 15 or 20 g low-THI caramel colour/kg or 20 g high-THI caramel colour/kg and females given 20 g/kg of either type had lower body weights than controls. In the 4-wk toxicity study with THI, there were no treatment-related ante-mortem observations, and no effects on body weights or food consumption. Fluid consumption by males and females treated with 64 ppm THI was lower than that of controls. Haematological changes noted in both studies included lower total white blood cell and lymphocyte (absolute and percentage) counts, and higher absolute and percentage neutrophil counts; these changes returned to normal by study termination in the caramel colour study, and by the third day post-treatment in the THI study. There were no consistent treatment-related alterations in blood chemistry or urinalysis variables for either study, and any changes noted were not associated with macroscopic or microscopic pathological alterations. There were no toxicologically important pathological findings for either study. Based on these studies the no-observed-adverse-effect level (NOAEL) for low-THI Caramel Colour III was considered to be 20 g/kg body weight. The acceptable daily intake for this caramel colour established by JECFA is 200 mg/kg body weight. The NOAEL for THI was found to be 0.38 mg/kg body weight for male rats and 0.12 mg/kg body weight for females.

Glutathione Blood Levels and Other Oxidant Defense Indices in Men Fed Diets Low in Vitamin C

Because ascorbic acid is an important contributor to the oxidant defense system in body tissues, we studied the effects of a low dietary intake of ascorbic acid on various indicators of oxidant defense and oxidant damage. During a 13-wk study eight healthy men (25-43 y), residing in a live-in metabolic unit, were fed controlled diets containing different amounts of ascorbic acid for four consecutive periods: period 1 = 250 mg/d for 4 d; period 2 = 5 mg/d for 32 d; period 3 = 10 or 20 mg/d for 28 d and period 4 = 60 or 250 mg/d for 28 d. Measurements were made at several time intervals of the activities of glutathione peroxidase and superoxide dismutase in RBC, DNA strand breaks in mononuclear leucocytes, glutathione concentrations in plasma and RBC and NAD and NADP in RBC. After 60 d of low ascorbic acid intakes and associated with plasma ascorbic acid levels less than 6 mumol/L, the total glutathione concentration and the reduced glutathione:oxidized glutathione ratio were decreased in plasma. At the same time NAD and NADP levels in RBC were elevated. It seems that chronic marginal vitamin C deficiency states may be associated with selected biochemical changes in oxidant defense indices.

13‐week inhalation toxicity study and fertility assessment in rats exposed to atmospheres containing adiponitrile

Adiponitrile is an aliphatic dinitrile that is used as an intermediate in the synthesis of hexamethylenediamine. In order to asses potential health effects associated with industrial exposure, rats were exposed 6 h/d, 5 d/wk for 4 or 13 wk to atmospheres containing a range of adiponitrile concentrations and observed for signs of toxicity. A fertility assessment was also included as a component of the 13-wk study. Mortality and reduced weight gain were observed within 1 wk only in rats exposed to 493 mg/m3. Evidence of slight anemia was present in rats exposed to 99 mg/m3 and above. There was no histopathological evidence of organ toxicity in about 30 tissues from both sexes exposed up to 99 mg/m3, the highest concentration tested, for 13 wk. In addition, fertility, as monitored by reproductive performance and litter parameters, was normal in both males and females similarly exposed.

Neurotoxicity and carcinogenicity ofN‐methylolacrylamide in F344 rats and B6C3F1 mice

Toxicology and carcinogenicity studies of N-methylolacrylamide were conducted by administering the chemical by gavage in water to both sexes of F344/N rats and B6C3F1 mice 5 times per week for 16 d, 13 wk, or 2 yr. In 16-d studies, rats receiving doses of 200 mg/kg or higher and mice receiving 400 mg/kg died. In 13-wk studies, all rats given 100 mg/kg or higher doses died. Rats receiving 50 mg/kg or higher doses developed hindlimb ataxia progressing to paralysis. In neurobehavioral assessments, decreased forelimb and hindlimb grip strength occurred in rats at doses as low as 12.5 mg/kg. Landing footspread was also increased in dosed rats compared to controls. Axon filament and myelin sheath degeneration in the spinal cord and/or peripheral nerves occurred in rats receiving doses of 25 mg/kg or higher. Necrosis in the granular cell layer of the cerebellum was seen in rats given 200 mg/kg. Mice receiving 200 mg/kg in 13-wk studies died. Decreased grip strength was noted in mice at doses as low as 25 mg/kg, and rotarod performance was also affected by N-methylolacrylamide administration, but no neuropathology was seen microscopically. Testicular weights were decreased at doses as low as 12.5 mg/kg, and hepatocellular necrosis, thymic lymphocyte necrosis, and hemorrhage, necrosis, and mineralization of the zona reticularis of the adrenal gland were seen in mice that died (200 mg/kg). In 2-yr studies, survival and weight gains in male and female rats receiving doses of 6 or 12 mg/kg/d were minimally affected. No biologically important clinical signs or neoplastic or nonneoplastic lesions were attributed to N-methylolacrylamide administration to rats, suggesting that higher doses could have been tolerated. In mice, survival was not different between dosed and control groups (0, 25, or 50 mg/kg/d). Body weights were higher by as much as 25% in dosed compared to control groups. No compound-related clinical signs were observed, but increases in neoplasms of the harderian gland, liver, and lung were clearly related to chemical administration in both sexes of mice. Benign granulosa-cell neoplasms of the ovary were also increased in dosed female mice.

Utilization of Three Maturities of Alfalfa by Dairy Cows Fed Rations that Contain Similar Concentrations of Fiber

The effect of alfalfa maturity on ration utilization and lactation performance by high producing dairy cows was measured in a 13-wk lactation study. Eighteen multiparous Holsteins were fed one of three rations containing first crop alfalfa hay harvested at early vegetative (36.1% NDF), late bud (51.7% NDF), or full bloom (51.7% NDF) maturity. Forage to concentrate ratios were 68:32, 53:47, and 45:55 for early vegetative, late bud, and full bloom rations, respectively. As offered, the late bud ration was higher in fiber (34.6% NDF) than either the early vegetative (31.8% NDF) or full bloom (30.6% NDF) rations. Cows fed the ration with early vegetative hay produced as much 4% FCM (32.5kg) as cows fed the ration containing more fiber (late bud, 32.9kg) or the later maturity hay (full bloom, 32.2kg). Milk fat and protein composition were not affected by ration fiber concentration or forage maturity. Average dry matter intake per day and body weight change were similar across treatments. Time spent ruminating was similar for the three rations (6.8 h/d), but eating time was affected by forage maturity (early vegetative, 4.4 h/d vs. full bloom, 5.7 h/d). Cows receiving the higher fiber diet (late bud, 6.2 h/d) spent more time eating than cows receiving the lower fiber diets. Fiber concentration and forage maturity did not affect milk production, milk composition, or body weight change of high producing dairy cows in early lactation.

Toxicity and carcinogenicity of nitrofurazone in F344/N rats and B6C3F 1 mice

Toxicology and carcinogenesis studies were conducted by feeding diets containing nitrofurazone (99% pure) to groups of F344/N rats and B6C3F 1 mice for 14 days, 13 wk or 2 yr. In the 14-day studies, in which doses ranged from 630 to 10,000 ppm, nitrofurazone was more toxic to mice than to rats. Accordingly, in the 13-wk studies, doses for rats ranged from 150 to 2500 ppm and for mice from 70 to 1250 ppm. At the higher doses, convulsive seizures and gonadal hypoplasia were observed in both species. Evidence of toxicity in rats also included degenerative arthropathy. For the 2-yr studies, rats were exposed to 0, 310 or 620 ppm nitrofurazone and the survival of male rats given 620 ppm was lower than that of controls ( 33 50 , 30 50 and 20 50 in the control, 310- and 620-ppm groups, respectively). Nitrofurazone administration increased the incidences of mammary gland fibroadenomas in female rats ( 8 49 , 36 50 and 36 50 in the control, 310- and 620-ppm groups, respectively). In male rats it was associated with a marginal increase in sebaceous gland adenomas and trichoepitheliomas of the skin, mesotheliomas of the tunica vaginalis, and tumours of the preputial gland. Nitrofurazone caused testicular degeneration (atrophy of germinal epithelium and aspermatogenesis) in rats, and degeneration of vertebral and knee articular cartilage in rats of both sexes. In mice, dietary concentrations of nitrofurazone for the 2-yr studies were 0, 150 or 310 ppm. In mice of each sex, nitrofurazone administration induced stimulus-sensitive convulsive seizures, primarily during the first year of study. In male mice, there was no evidence of any chemically-related carcinogenic effects, but there was a treatment-related decrease in survival ( 39 50 , 31 50 and 27 50 in the control, 150- and 310-ppm groups, respectively). In female mice nitrofurazone induced ovarian lesions with increased incidences of benign mixed tumours ( 0 47 , 17 50 and 20 50 in control, low- and high-dose groups, respectively) and granulosa cell tumours ( 1 47 , 4 50 and 9 50 in control, low- and high-dose groups, respectively).

Evaluation of the subchronic and reproductive effects of a series of chlorinated propanes in the rat. I. Toxicity of 1,2,3-trichloropropane.

Repeated inhalation exposure and 1-generation reproduction studies have been conducted in the rat to address the adequacy of the 10 ppm occupational exposure limit established for 1,2,3-trichloropropane (TriCP). Groups of 5 rats per sex were exposed for 6 h/d, 5 d/wk up to 4 wk to target TriCP concentrations of 0-900 ppm. Nine of 10 rats died after a single exposure at 900 ppm. Additional deaths were seen in the 300 (1 death) and 600 (3 deaths) ppm test groups. Mean body weights for all TriCP-treated groups were lower than control values. Liver weights were increased in animals of both sexes at 600 ppm and lower. For females ovary weights for the 300 and 600 ppm groups and spleen weights for the 300 ppm group were lower than those of controls. Males exhibited decreased testes weights only at the 600 ppm TriCP level (not evaluated at 900 ppm). Results of a 13-wk exposure, 6 h/d, 5 d/wk of 15 rats/sex.group to TriCP target vapor concentrations of 5, 15, or 50 ppm also resulted in liver weight increases at all test levels. Histopathologic examination showed hepatocellular hypertrophy in male rats at all TriCP levels. Other microscopic findings related to treatment in rats exposed to 15 ppm and to 5 ppm TriCP included lung hyperplasia (both sexes) and splenic extramedullary hematopoiesis (females only) and parallel observed organ weight increases. No treatment-related deaths were observed in this study, nor were there apparent effects on the hematology or clinical chemistries. Group mean body weights at 50 ppm (both sexes) and 15 ppm (females only) TriCP were reduced when compared to controls. In a 13-wk follow-up study in rats at 0, 0.5, and 1.5 ppm TriCP, no gross or microscopic findings related to treatment were found. Groups of 10 male and 20 female rats were exposed 6 h/d, 5 d/wk to 0, 5, or 15 ppm TriCP vapor during premating and mating. Females also were exposed during gestation. Low mating performance was observed in all groups of female rats including the controls, although fewer females in the 15-ppm group mated than in other study groups. Mating and fertility indices of male rats in both treated and control groups were generally low. All measured progeny indices appeared unaffected by treatment. A follow-up study of the same design was conducted at levels of 0, 0.5, and 1.5 ppm TriCP.(ABSTRACT TRUNCATED AT 250 WORDS)