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This study was designed to evaluate and characterize any subchronic toxicity of rhamsan gum, a polysaccharide produced from Sphingomonas strain ATCC 31961, when administered to both sexes of Crl:CD(SD)IGS rats at dietary levels of 0 (control), 0.5, 1.5, and 5.0% (10 rats/sex/group). During the study, the treatment had no adverse effects on clinical signs, survival, body weights and food and water consumption, or on findings of urinalysis, ophthalmology, hematology, or blood biochemistry. Examination of gross pathology and histopathology exhibited no differences of toxicological significance between control and treated rats. Increased relative cecum (filled) and cecum (empty) weights, evident in males of 1.5% group and both sexes of the 5.0% group, were considered to be a physiological adaptation. Thus, the results indicated the toxic level of rhamsan gum to be more than 5.0%, and the no-observed-adverse-effect level (NOAEL) was concluded to be 5.0% (3,362 mg/kg body weights/day for males, and 4,304 mg/kg body weights/day for males) from the present study.

This study was designed to evaluate any adverse effect of fermentation-derived cellulose, produced by Acetobacter aceti subspecies xylinum, when administered to both sexes of F344 rats at dietary levels of 0, 1.25, 2.5, and 5.0% for 28 days. The treatment had no adverse effects on clinical signs, mortality, body weights and food and water consumption, or on urinalysis, ophthalmology, hematology, blood biochemistry, and histopathology findings. At necropsy, slight increased absolute and relative cecum weights, evident in females ingesting 2.5% and 5.0% dietary levels, were considered to be a physiological adaptation to the poorly absorbed fermentation-derived cellulose. The non-observed-adverse-effect level (NOAEL) from the present study was concluded to be 5.0% in the diet (5,331 mg/kg body weights/day for males, and 5,230 mg/kg body weights/day for females).

This study was designed to evaluate any adverse effect of a hot water extract of black soybeans (Glycine max (L.) Merr.), when administered to both sexes of Crj:CD(SD)IGS rats at dietary levels of 0 (control), 0.5, 1.5 and 5.0% (6 rats/sex/group). During the study, the treatment had no adverse effects on clinical signs, survival, body weights, and food and water consumption, or on findings of ophthalmology, urinalysis, hematology, or blood biochemistry. Organ weights, gross pathology and histopathology exhibited no differences of toxicological significance between control and treated rats. Thus, the no-observed-adverse-effect level (NOAEL) of black soybean extract was concluded to be 5.0% (3,618 mg/kg body weight/day for males and 4,066 mg/kg body weight/day for females) from the present study.

Ethanol extracts of seeds of Psoralea corylifolia are proposed as food additives for processed food preservation. An extract was administered by admixing into diet at concentrations of 0, 0.375, 0.75, 1.5 or 3.0% to 10 male and 10 female F344 rats each for 90 days to evaluate its toxicity. Body weight gain, food consumption and food conversion efficiency (body weight gain per food consumption) were lower in the extract-treated animals, except for the 0.375% males, as compared to the control animals. Absolute and/or relative testes weights in the 1.5 and 3.0% groups and those of ovaries in the 3.0% group were significantly (p < 0.01) lower than in the control group. On histopathological examination, seminiferous tubular atrophy and Leydig cell atrophy in the testes, and epithelial cell atrophy in the seminal vesicles and prostate were observed in the 1.5 and 3.0% males. Decrease in the number of corpora lutea associated with frequent necrotic follicles in the ovaries in the 1.5 and 3.0% females and less frequent endometrial glands in the uterus in the 3.0% females were also detected. These results might suggest disruption of the hypothalamus-pituitary-gonadal axis in Psoralea corylifolia-treated rats as possible mechanisms underlying this gonadal toxicity.

To investigate the modifying effects of eugenol (EUG), a component of cigarette smoke, on lung carcinogenesis, male and female transgenic mice carrying the human prototype c-Ha-ras gene (rasH2 mice) were given a single intraperitoneal injection of 250 mg/kg urethane (UR) or saline, followed by a diet containing 6,000 ppm EUG or basal diet for 26 weeks. Their non-transgenic CB6F1 littermates (non-Tg mice) were also treated in the same manner. In both male and female rasH2 mice, alveolar/bronchiolar hyperplasias, adenomas and carcinomas were observed in all UR-treated groups. However, there were no significant intergroup differences in the incidences and multiplicities of these lesions between the UR alone and UR + EUG groups. In non-Tg mice, alveolar/bronchiolar hyperplasias, adenomas or carcinomas were sporadically observed in UR-treated groups of both sexes, with no significant differences in the incidences and multiplicities between the UR alone and UR + EUG groups. There were no intergroup differences between them in the PCNA-positive ratios of adenomas or carcinomas and the areas of adenomas or carcinomas to the whole lung area examined. The present results suggest that the EUG treatment does not exert modifying effects on lung carcinogenesis induced by UR in both male and female rasH2 mice and non-Tg mice.

In order to evaluate the threshold dose of thyroid tumor-promoting effects of KA, male F344 rats were initiated with N-bis(2-hydroxypropyl) nitrosamine (DHPN; 2000 mg/kg body wt., single s.c. injection) and, starting 1 week later, received pulverized basal diet containing 0%, 0.002%, 0.008%, 0.03%, 0.125%, 0.5% or 2%KA for 20 weeks. Five rats each in the 0%, 0.125%, 0.5% and 2%KA groups were sacrificed at week 12, and 10 rats each in all groups at week 20. As an additional experiment, three groups without DHPN initiation received basal diet, a diet containing 0.5% or 2%KA for 20 weeks. The serum T4 levels were significantly decreased in the DHPN-initiated groups given 0.125%KA or more at week 12. No significant decreases in serum T3 levels were observed in the groups treated with DHPN + KA and a significant increase was evident in the 2%KA-alone group at week 20. Some rats in the DHPN + 2%KA group at weeks 12 and 20 and the 2%KA-alone group at week 20 showed pronounced elevation of serum TSH. Thyroid weights were significantly increased in the DHPN-initiated groups receiving 0.5% and 2%KA at weeks 12 and 20 and in the 2%KA-alone group at week 20. Histopathologically, the incidences of focal thyroid follicular cell hyperplasias in the DHPN-initiated groups treated with 0.125%, 0.5% and 2%KA at week 20 were 5/10, 10/10 and 8/8 rats, respectively. At week 20, adenomas were observed in 7/10 rats in the DHPN + 0.5%KA group and 8/8 rats in the DHPN + 2%KA group, and carcinomas were observed in 6/8 rats in the DHPN + 2%KA group. In the groups without DHPN initiation, only focal follicular cell hyperplasia was observed in 1/9 rats in the 2%KA-alone group. These results suggest that the no-observed-adverse effect for the thyroid tumor-promoting effect of KA is 0.03% (15.5 mg/kg/day) under the present experimental conditions, and that KA possesses weak tumorigenic activity in rats due to continuous serum TSH stimulation by a non-genotoxic mechanism.

To cast light on whether the carcinogenic risk of 2,6-dimethylaniline (DMA), a metabolite of xylazine, may increase by ingestion of edible tissues from domestic animals treated with xylazine, the following studies of xylazine and DMA were performed. In Experiment I, male F344 rats received a single oral administration of 150 mg/kg of xylazine hydrochloride. Rats showed symptoms suggesting loss of sensation and pain immediately after the treatment. These signs had disappeared after 3 hr, but the animals died of hydrothorax and pulmonary edema by 9 hr. The plasma concentration of xylazine was 2.88 +/- 0.95 micrograms/ml at 15 min, and then decreased to 0.10 +/- 0.01 microgram/ml at 6 hr. The plasma level of DMA remained at 0.03 to 0.04 microgram/ml during the measurement period. In Experiment II, male F344 rats were fed a diet containing 1000 ppm of xylazine hydrochloride, regarded as the maximum tolerated dose, for 4 weeks. No clear clinical signs were evident and the plasma levels of xylazine and DMA were at the detection limit (0.02 microgram/ml) or less, although follicular cell hypertrophy of the thyroid was observed in all the treated animals. In Experiment III, male F344 rats were fed a diet containing 3000 ppm or 300 ppm of DMA for 4 weeks. Histological changes, such as atrophy of Bowman's gland and irregular arrangement of olfactory epithelial cells, were only observed in the olfactory epithelium of the 3000 ppm group. The plasma levels of DMA were 0.20 to 0.36 microgram/ml in the 3000 ppm group, but under the detection limit in the 300 ppm group. These results suggest that the probability of nasal carcinogenic effects of DNA on consumers via ingestion of edible tissues from food-producing animals treated with xylazine is extremely low, since DMA levels in the blood of rats subjected to continuous administration of high doses of xylazine remained under the detection limit.

Time course changes in thyroid proliferative lesions as well as related hormone levels in the blood of male F344 rats given N-bis(2-hydroxypropyl)nitrosamine (DHPN: 2800 mg/kg body weight, single s.c. injection) as an initiation treatment followed by pulverized basal diet containing 0% (Group 2), 2% (Group 3) or 4% (Group 4) kojic acid (KA) were examined at Weeks 1, 2, 4, 8 and 12. As an untreated control group (Group 1), rats were given basal diet for 13 weeks and examined in the same manner. Serum T3/T4 levels in the DHPN + 2% KA and DHPN + 4% KA groups were significantly reduced as compared with the DHPN-alone group at each time point. Serum TSH levels in both DHPN + KA groups were significantly increased at each time point in a treatment period-dependent manner from Weeks 1 to 12, and the extent of elevation was more remarkable in the DHPN + 4% KA group. At Week 2, there were no statistically significant intergroup differences in liver T4-UDP-GT activities on a milligram microsomal protein basis. Histopathologically, no thyroid proliferative lesions were observed in the untreated control group or the DHPN-alone group. However, diffuse follicular cell hypertrophy and decreased colloid in the thyroid were apparent in all rats of the DHPN + KA groups at each time point. In addition, focal follicular cell hyperplasias and adenomas of the thyroid were observed at high incidence in the DHPN + 2% KA group from Week 4 and in the DHPN + 4% KA group from Week 8. Multiplicities of focal follicular cell hyperplasias and adenomas of the thyroid in the DHPN + 2% KA group were significantly greater than those in the DHPN + 4% KA group at Week 8. In the pituitary, an increase in the number of TSH producing cells with expanded cytoplasm was apparent from Weeks 4 to 12 in both DHPN + KA groups. These results strongly suggest that thyroid proliferative lesions were induced by KA administration due to continuous serum TSH stimulation through the negative feedback mechanism of the pituitary-thyroid axis, resulting from depression of serum T3 and T4.

Changes of TSH-producing cells in the pituitary and thyroid expression of the growth factors, transforming growth factor alpha (TGF alpha) and epidermal growth factor receptor (EGFR), as well as cyclin D1, were investigated immunohistochemically in order to clarify their contribution to the enhancing effects of excess vitamin A (VA) on thyroidal carcinogenesis induced by thiourea (TU). Male rats were allocated to 4 groups, control, TU, VA, and TU + VA, respectively, receiving no treatment, water containing 0.2% TU, diet containing 0.1% VA, and both for 10 or 19 weeks after a single s.c. injection of DHPN (2800 mg/kg) for initiation. Immunohistochemistry using antibodies against TSH demonstrated enlargement of TSH-producing cells in the TU + VA group as compared to the TU group, supporting our conclusion that enhanced TSH stimulation is mainly responsible for promoting the effects of excess VA. Since the expression of TGF alpha, EGFR, and cyclin D1 in thyroid proliferative lesions did not exhibit any differences between the TU and TU + VA groups in the present study, these factors are unlikely to participate in VA enhancement of carcinogenesis.

The testicular toxicity of cyclophosphamide (Cp) in rats was evaluated by quantitative morphometry of spermatogenic cycle stages. Nine-week-old male Sprague-Dawley rats in Group 1 were given a single oral administration of 100 mg/kg of Cp, and were sacrificed at 1, 7, 14 and 21 days thereafter. Rats in Group 2 were orally given 100 mg/kg/day of Cp for 2 days, followed by 50 mg/kg/day for the next 3 days, and were sacrificed at 1 and 4 days after the last administration. The numbers of seminiferous epithelia were counted in the seminiferous tubules of stages II, V, VII and XII of the spermatogenic cycle. The data were expressed as numbers of spermatogenic cells per Sertoli cells per seminiferous tubule cross section. Animals in Group 1 showed decreased preleptotene spermatocytes at Day 7, decreased zygotene spermatocytes at Day 14, and decreased pachytene spermatocytes at Day 21. In group 2, testicular toxicity could also be clearly detected by this morphometric approach. The present morphometric study thus indicates that testicular toxicity can be detected from Day 7 even after a single administration of Cp.

To examine changes in serum TSH and determine whether the sustained excess is necessary for the development and/or progression of thyroid tumors, male F344 rats were administered drinking water containing thiourea (TU), at 0.1 or 0.05%, or sulfadimethoxine (SM), at 0.025 or 0.0125%, for one week in Experiment I. All of the treated animals showed decreased serum levels of T3 and T4 and an increased TSH. In Experiment II, male rats were given a s.c. injection of N-bis(2-hydroxypropyl) nitrosamine (DHPN:1500 mg/kg BW) and, starting one week later, received drinking water containing the same doses of TU or SM as in Experiment I for the following 20 weeks. Thyroid follicular proliferative lesions were induced in most rats treated with TU and SM. However, these treated animals did not demonstrate any consistent alterations in serum T3, T4 and TSH levels, except for the high dose TU group. The present studies thus suggest that thyroid tumors can grow even under conditions of fluctuating serum TSH levels during the progression phase, although TSH stimulation might be an absolute requirement in the early phase of tumor development.

A stepwise, simple screening test for skin and eye irritations, suitable for industrial chemicals or pesticides which are not required to be examined for their exact potential irritancy levels, was developed. The efficacy of the test was evaluated using 15 chemicals including typical irritants (acetic acid, ammonia, chloroacetic acid, dioxane, ethanolamine, formaldehyde, formic acid, hydrogen peroxide, phenol, phosphoric acid, propionic acid, sodium hydroxide, sodium hypochlorite, sulfuric acid, and trichloroacetic acid). Chemicals were chosen so as to represent irritants which act by different mechanisms (i.e., strongly acidic, alkaline, reactive to protein, oxidizing etc.). The method consisted of physicochemical tests and animal tests using rats, mice or guinea pigs, namely, a skin irritation test, an intradermal reaction test and an eye irritation test in a sequential manner such that further tests are not required if a positive result is obtained in earlier steps. Results obtained between two laboratories using this method were very similar. Comparison of our results with the data obtained by the conventional method using rabbits showed fairly good coincidence. The method was shown to be useful in assessing skin and eye irritation of chemicals and causes minimal suffering to animals.

The gelatin-starch syrup microencapsulation method was applied to subacute toxicity studies of tribromomethane (TBM), dibromochloromethane (DBCM) and bromodichloromethane (BDCM). Groups of Wistar rats (7 males and 7 females) both sexes were given diet containing microcapsules of each of these trihalomethanes (THMs) at the following concentrations: TBM, 0.068, 0.204 and 0.612% in males, and 0.072, 0.217 and 0.651% in females; DBCM, 0.020, 0.062 and 0.185% in males, and 0.038, 0.113 and 0.338% in females; BDCM, 0.024, 0.072 and 0.215% in males, and 0.024, 0.076 and 0.227% in females. Suppression of body weight gain was seen in each high-dose males fed TBM or BDCM and females fed DBCM or BDCM. Histopathologically, hepatic lesions such as vacuolization and swelling of liver cells were significantly noted in both sexes of all groups fed TBM, in both sexes of the middle- and high-dose groups fed DBCM, and in males of the high-dose group and in females of the middle- and high-dose groups fed BDCM. In addition, single cell necroses were observed in males and females fed DBCM and in males fed BDCM. Hepatic cord abnormalities were also noted in males of the high-dose group fed BDCM. Although no increases in serum transaminase activities (ASAT, ALAT) were evident in either sex fed any of the THMs, decreases in triglyceride content, cholinesterase and lactate dehydrogenase activity were observed. Renal lesions reported to occur in gavage studies were not found in the present feeding study. Lowest-observed-adverse-effect-level (LOAEL) of TBM and no-observed-adverse-effect-level (NOAEL) of DBCM and BDCM were determined to be 56.4 mg/kg, 18.3 mg/kg and 20.6 mg/kg, respectively, under the present experimental conditions.

A chronic feeding study was carried out in Wistar rats using microencapsulated bromodichloromethane. The test substance was administered in the diet at doses of 0, 0.014, 0.055 and 0.22% for 24 months. Rats were sacrificed after 6, 12, 18 and 24 months of continuous dosing. The results showed a suppression of body weight gain in the 0.22% group for both males and females. Dose related changes were clearly observed in the liver with histopathological findings including fatty degeneration in the 0.014% or higher dose male groups, and fatty degeneration and granuloma in the 0.055 and 0.22% group females, as well as bile duct proliferation and cholangiofibrosis in the 0.22% group for both males and females. No significant differences in incidences or numbers of neoplastic changes were seen between control and any of the treatment groups. There were no dose-related non-neoplastic lesions in the kidneys of either sex. Lowest-observed-adverse-effect-level was determined to be 6.1 mg/kg/day under the present experimental conditions.