Walter H. Hansen

Pathologic changes in rats and dogs from two-year feeding of sodium arsenite or sodium arsenate.

Abstract Feeding of sodium arsenite or sodium arsenate to weanling Osborne-Mendel rats in 2-year experiments resulted in marked enlargement of the common bile duct in a large proportion of the animals on the highest dosage of each compound, namely, 250 and 400 ppm As in the diet, respectively. These levels also caused pronounced weight depression and decreased survival. At the next lower dosages, 125 ppm As as arsenite and 250 ppm As as arsenate in the diet, enlargement of the common bile duct was also present, but was less pronounced. Survival was slightly reduced with the arsenate, and both compounds caused reduced weight, particularly in the females. Levels of 62.5, 31.25, or 15.63 ppm As as arsenite and 125, 62.5, or 31.25 ppm As as arsenate did not cause common bile duct enlargement and did not affect survival. Weight was slightly reduced in both sexes with 125 ppm As as arsenate, and in females only with 62.5 ppm As as arsenite. No carcinogenic effect of the two arsenicals in these experiments could be detected. Histologically, the enlarged common bile ducts showed hyperplasia of the glandular elements, focal necrosis, and fibrosis. Intrahepatic bile ducts were little affected. Young beagle dogs in a 2-year experiment were fed either of the arsenicals at As levels of 125, 50, 25, or 5 ppm in the diet. None of the six on the high level of arsenite survived 2 years; all lost much weight during the feeding period. Only one of six dogs on the high level of arsenate died, and this one dog lost much weight; the survivors showed some weight loss. Gross and microscopic changes in the nonsurviving dogs were essentially those of inanition. Dogs on levels of 50 ppm or less of As as either compound did not differ from the controls. For a given degree of effect in either species (dog survival and weight loss, rat survival and weight depression, and rat common bile duct enlargement), more arsenic in arsenate than in arsenite form was required.

Effects of prolonged ethylene thiourea ingestion on the thyroid of the rat

Abstract Male and female Charles River rats, approximately 5 wk old, were fed ethylene thiourea (ETU) for 2 yr at levels of 0, 5, 25, 125, 250 or 500 ppm in the diet. Body weights, the weights of the thyroid and other organs, thyroidal uptake of 131 I, haematology and histology were the criteria studied. ETU was found to be a thyroid carcinogen for the rat at the 250 and 500 ppm dietary levels and a thyroid tumorigen at the 125 ppm level; it caused slight thyroid hyperplasia at the 5 and 25 ppm feeding levels. Thyroid hyperplasia was not noticeably reversible in those rats in each group that were changed to control diet after 66 wk on ETU diets. ETU ingestion at the 5 and 25 ppm levels was not biologically deleterious to the rat.

Chronic toxicity of 2,4-dichlorophenoxyacetic acid in rats and dogs

Abstract Starting at 3 wk of age, rats (25 female and 25 male per group) were fed for 2 yr either 0, 5, 25, 125, 625, or 1250 ppm of 2,4-dichlorophenoxyacetic acid (2,4-D) in the diet. No significant effect on growth rate, survival rate, organ weights, or hematologic values was noted. Total (male plus female) tumor incidence in the control and experimental groups was 15, 14, 18, 20, 23, and 22, respectively. No target organ tumors were observed; the individual tumor types were randomly and widely distributed and of the type normally found in aging Osborne-Mendel rats. Statistical analysis of the randomly distributed tumor types indicated a tendency for the proportion of females with tumors to increase with 2,4-D dosage, and a trend toward dose related increases in the proportion of males with malignant tumors. These tendencies do not reflect important pathologic differences. Groups of 3 male and 3 female beagle dogs each were fed 0, 10, 50, 100, or 500 ppm of 2,4-D in the diet for 2 yr, starting at 6–8 mo of age. Twenty-eight dogs surviving the 2-yr period were clinically normal, and no 2,4-D related effects were noted. One female at 100 ppm that was emaciated at autopsy at two years showed no significant lesions. Moderate depletion of cellular elements was noted at 10 ppm in 1 male that died after 10 mo. In a 3-generation 6-litter rat reproduction study, no deleterious effect of dietary 2,4-D at 100 or 500 ppm was evident. At 1500 ppm, however, 2,4-D, while apparently affecting neither fertility of either sex nor litter size, did sharply reduce the percent of pups born surviving to weaning and the weights of weanlings.

The effect of carbaryl (Sevin) on reproduction of the rat and the gerbil

Abstract The pesticide carbaryl (Sevin) was fed at dietary levels of 0, 2000, 5000, or 10,000 ppm to Osborne-Mendel rats for 3 generations. At 10,000 ppm, fertility was impaired and no litters were produced for the second mating of the second generation. Marked effects from birth to day 4 were manifested, as seen in the survival index. Dose-related decreases were observed in the averages of litter size, number of liveborn progeny, number of survivors to day 4, and number weaned. In a comparative study, 3 generations of gerbils were fed carbaryl at dietary levels of 0, 2000, 4000, 6000, and 10,000 ppm. No litters were produced for the second mating of the third generation at 10,000 ppm. Gerbils appeared to be more sensitive to carbaryl than rats since significant decreases were found consistently at low dietary levels, but the effects were not so clearly dose-related. As in rats, the survival of liveborn to day 4 was markedly decreased.

Chronic toxicity of two food colors, Brilliant Blue FCF and Indigotine

Abstract Brilliant Blue FCF (FD & C Blue No. 1) and Indigotine (FD & C Blue No. 2) were fed to groups of 24 Osborne-Mendel rats for two years at dietary levels of 0.5, 1.0, 2.0, and 5.0%. Growth inhibition occurred in the male animals fed Indigotine at the 2.0 and 5.0% levels. No effect was observed on survival, hematology, or organ weights. No significant pathologic changes were observed in animals fed either color. Five beagle dogs received a dietary level of 2.0%, and three dogs a level of 1.0% of Brilliant Blue FCF for one year. One dog died after 17 days on the 2% dietary level and another died after 46 weeks on the 1% level. Indigotine was fed to beagle dogs for periods up to two years at dietary levels of 1.0 and 2.0%. Four of the six dogs at the 2.0% level died during the two years, and one of the four at the 1.0% level was sacrificed in a moribund condition. All the deaths were attributed to intercurrent virus infections. No clinical signs, gross lesions, or microscopic pathology were attributed to intercurrent virus infections. No clinical signs, gross lesions, or microscopic pathology were attributed to ingestion of Brilliant Blue FCF or Indigotine; however, because of deaths from intercurrent virus infections a no-effect level has not been established for Indigotine. Subcutaneous injections of 30 mg of Brilliant Blue FCF or 20 mg of Indigotine into rats weekly for two years produced fibrosarcomas at the site of injection. Other than acute convulsive deaths of some of the test mice immediately after injection, no deleterious effects attributable to the weekly subcutaneous injection of 2.5 mg of Indigotine were observed over a two-year period.

Chronic oral toxicity of Ponceau 3R.

The results of two-year oral toxicity studies with Ponceau 3R using two strains of rats have been presented. Dosage levels were 5.0, 2.0, 1.0, and 0.5% for Osborne-Mendel rats and one dosage level of 2.0% for Bethesda Black rats. Preliminary results of the continuing dog and mouse studies are reported. Ponceau 3R at 5.0, 2.0, 1.0, and 0.5% of the diet produced high mortality, growth inhibition, and increased weight of the liver and kidneys. Malignant liver tumors occurred in both strains of rats at all dietary levels of Ponceau 3R, statistically significant (P < 0.01) in comparison with the controls at the 5.0% level of the Osborne-Mendel rats and the 2.0% level of the Bethesda Black animals. Incidence of benign liver tumors was also significant for all test levels. Marked weight loss and high mortality have been observed in dogs fed Ponceau 3R at dosage levels of 2.0% and 1.0%. After a feeding period of 32 weeks, changes in the liver were the most pronounced tissue changes. Increased mortality of C3HeBFe mice has been observed on 2.0% and 1.0% feeding levels. Hepatic adenomas and carcinomas have been observed on the 2.0% level.

CHRONIC AND SUBACUTE TOXICOLOGY AND PATHOLOGY OF METHYL SALICYLATE IN DOGS, RATS, AND RABBITS.

Abstract The feeding of methyl salicylate to rats for 2 years at levels up to 2.0% of the diet produced statistically significant growth retardation in the rats on the 2.0% and 1.0% diets. All rats on the 2.0% diet died by the 50th week. There was an increase in cancellous bone in all bones examined from the rats on the 2.0% methyl salicylate diet; this was less pronounced in degree and incidence in the rats on the 1.0% diet. The oral administration to dogs of capsules containing up to 1200 mg/kg/day of methyl salicylate resulted in weight loss and death in all dogs on the 500 mg/kg/day level and above. The livers of the dogs on the 1200 and 800 mg/kg/day levels had moderate to marked degrees of fatty metamorphosis. Dogs given capsules with 350 or 150 mg/kg/day methyl salicylate for 2 years had enlarged livers and increased size of the hepatic cells. The dogs on the higher level lost weight; those on the next higher level showed growth retardation. Rabbits inuncted with methyl salicylate up to 4.0 ml/kg/day for 90 days showed early deaths and kidney damage in those of the high dose group. The remaining nine rabbits appeared predisposed to “spontaneous” nephritis and mild hepatitis.

Biochemical and toxicologic studies of Rhodamine B and 3,6-Diaminofluoran

Abstract 3,6-Diaminofluoran, the ultimate metabolite of Rhodamine B (tetraethyl-3,6-diaminofluoran) exerted fewer toxic effects in rats than Rhodamine B. The LD50 (intravenous) for Rhodamine B was 89.5 mg/kg compared to and LD50 of 140.0 mg/kg for 3,6-diaminofluoran by the same route. The growth of rats fed 1% Rhodamine B for 90 days was inhibited to a greater extent than that of rats fed 3,6-diaminofluoran at the same dietary level over the same period of time. Liver enlargement, the most significant abnormality at autopsy, occurred to a greater extent in the rats fed Rhodamine B. In vitro incubation studies showed that the metabolism of Rhodamine B occurred in the microsomes of liver cells and duplicated the stepwise de-ethylation process previously observed in the intact animal. Substantially all the orally ingested 3,6-diaminofluoran could be accounted for in the excreta. This indicates that the rat is unable to degrade this metabolite of Rhodamine B.

Studies of the metabolism of Rhodamine B

Rhodamine B (D & C Red No. 19) was metabolized identically in the dog, rat, and rabbit as evidenced by paper chromatograms and paper electrophoretograms of their urines and fecal extracts. The dye was extensively absorbed from the gastrointestinal tract at low dietary levels. At the 1% dietary level, only 3–5% of the ingested material was recovered unchanged. Rhodamine B (tetraethyl-3′,6′-diaminofluoran), was de-ethylated when ingested by animals. Two of three observed metabolites were identified as 3′,6′-diaminofluoran, and N,N′-diethyl-3′,6′-diaminofluoran on the basis of their Rf values, fluorescence, color, diazotization characteristics, and activation and fluorescence spectra when compared to synthetic counterparts. Evidence presented indicates that the third metabolite is the monoethylated analog of Rhodamine B. Feeding studies with synthetic counterparts of the identified metabolites indicated that the basic fluoran structure of the Rhodamine B molecule was not destroyed and strengthened other evidence that no chemical changes other than de-ethylation occurred during the metabolism.

Long-term toxicity studies of erythrosine. I. Effects in rats and dogs.

Abstract Groups of 12 male and 12 female weanling Osborne-Mendel rats were fed diets containing erythrosine (FD & C Red No. 3) at levels of 0, 0·5, 1·0, 2·0 or 5·0% for 2 yr. Growth depression was observed in the rats given the 5·0% level. Spleen weights, expressed as organ-to-body weight ratios, were decreased in the male rats given the 0·5, 2·0 and 5·0% dose levels and in female rats given the 5·0% dose level. Slight caecal distension occurred in rats given erythrosine at 1·0 % and the condition increased with increasing dose levels. The histology of the distended caeca was normal. Chronic nephritis was approximately doubled in incidence in the 0·5 % group compared with that in the control group, but this was probably due to chance variation. Weekly sc injections of erythrosine (approximately 12 mg/rat) were given to a group of 18 rats for 2 yr. Injection-site ulcerations were produced but no tumours were observed. In an acute oral toxicity study in rats, the LD50 of erythrosine was found to be 1840 mg/kg. In another long-term study, erythrosine was fed at dietary levels of 0, 0·5, 1·0 or 2·0% to groups of three male and three female beagle dogs. All dogs survived the 2-yr period and no effects attributable to the ingestion of erythrosine were observed.