Toxicity study of a rubber antioxidant, mixture of 2-mercaptomethylbenzimidazoles, by repeated oral administration to rats.

2-Mercaptobenzimidazole (2-MBI), a rubber antioxidant, is known to exhibit potent antithyroid toxicity in rats and is a candidate as an environmental endocrine disrupter. 2-Mercaptomethylbenzimidazoles (a 1:1 mixture of 4-methyl and 5-methyl isomers, MMBIs), are also employed industrially as rubber antioxidants and are suspected to exert antithyroid toxicity such as 2-MBI. In this investigation, acute and subacute oral toxicity studies of MMBIs in Wistar rats were conducted. The clinical signs of acute oral toxicity were observed including decreased spontaneous movement, a paralytic gait, salivation and lacrimation, and adoption of prone and lateral positions. The LD50 was estimated to be 330 mg/kg. In the subacute oral toxicity study, male and female rats were treated with MMBIs by gavage at doses of 0 (corn oil), 4, 20 and 100 mg/kg for 28 consecutive days followed by a 2-week recovery period for the control and highest dose groups. Body weight and food consumption, clinical signs, organ weights, clinical biochemistry and haematological parameters including clotting times and micronuclei induction in bone marrow erythropoeitic cells, and histopathology were examined. Relative organ weights of lung, liver and kidney, and serum cholesterol and phospholipid significantly increased in male rats treated with MMBIs at doses of 20 and 100 mg/kg. Male rats administered 100 mg/kg MMBIs exhibited a 1.8-fold increase in thyroid weight associated with histopathological changes but not altered serum thyroid hormone levels. Female rats administered 100 mg MMBIs/kg exhibited significant increases of liver and kidney but not thyroid weights, and serum cholesterol level. The antithyroid toxicity of MMBIs in rats was estimated to be one-tenth that of 2-MBI. No-observed-effect levels for male and female rats were found to be 4 and 20 mg/kg, respectively, in this subacute oral toxicity study.

Toxicity study of a rubber antioxidant, 2-mercaptobenzimidazole, by repeated oral administration to rats.

The chemical structure of 2-mercaptobenzimidazole (2-MBI), which is widely used as a rubber antioxidant, is partially similar to those of thiourea (TU) and ethylenethiourea (ETU), both potent thyrotoxic compounds. In order to determine the oral toxicity of 2-MBI, a 28-day repeated dose toxicity study in Wistar rats followed by observation over a 14-day recovery period was conducted at dose levels of 2, 10 and 50 mg/kg 2-MBI administered by gavage. No toxic deaths occurred due to 2-MBI treatment. Decreases of body weight gain and food consumption in the 50 mg/kg dose group were observed during the second half of the treatment period. In addition, hematological examination and serum biochemical tests revealed decreased white blood cells and hemoglobin and increased serum urea nitrogen, cholesterol, phospholipid, gamma-glutamyl transpeptidase and the Na+/K+ ratio in the 50 mg/kg dose group. Marked thyroid enlargement (to 10 fold the control weight), histopathologically associated with diffuse hyperplasia of follicles with decreased colloid and thickening of the fibrous capsule, was found. Reduction in thymus weight was also observed in a dose-dependent manner, without significant histopathological alteration. The non-observed effect level (NOEL) of 2-MBI in this gavage study was found to be less than 2 mg/kg/day based on the significant decrease in thymus weight in the 2 mg/kg 2-MBI treatment group. In an ancillary study, measurement of serum levels of T3, T4 and TSH, and thyroid weight after gavage treatment with 0.15 and 0.3 mmol/kg of three antithyroid compounds for 14 days revealed a more potent antithyroid effect for 2-MBI than for TU or ETU.

Evaluation of skin sensitization potential of nickel, chromium, titanium and zirconium salts using guinea-pigs and mice.

Contact sensitization capacity of four metal salts, nickel sulphate (NiSO4), potassium dichromate (K2Cr2O7), titanium chloride (TiCl4) and zirconium chloride (ZrCl4), was evaluated using guinea-pigs and mice. In the guinea-pig sensitization tests, we set up an injection concentration to 1% for all chemicals, and changed the challenge concentration. Guinea-pigs were sensitized with NiSO4, K2Cr2O7 and TiCl4. Among the test metal salts, K2Cr2O7 showed the highest sensitization rate and strongest skin reactions. ZrCl4 did not cause any sensitization responses under our experimental conditions. Minimum challenge concentration to cause a skin response was < 0.25% for K2Cr2O7, 0.5% for NiSO4 and 2% for TiCl4, respectively. A sensitive mouse lymph node assay (SLNA) also determined NiSO4 and K2Cr2O7 as a sensitizer. In the SLNA, TiCl4 caused mild lymph node responses, but was classified as a non-sensitizer as well as ZrCl4. Considering these results, the order of sensitization potential was K2Cr2O7 > NiSO4 > ZrCl4. NiSO4- and K2CrO7-sensitized animals did not show any reactions to ZrCl4 and TiCl4. No cross-reaction among these metal salts was found.

Effects of hydroquinone-type and phenolic antioxidants on calcium signals and degranulation of RBL-2H3 cells.

We previously reported that a hydroquinone-type antioxidant, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ), increases intracellular free Ca2+ concentration ([Ca2+]i), causes degranulation together with a protein kinase C activator, phorbol 12-myristate 13-acetate (TPA), and increases antigen-induced degranulation in rat basophilic leukemia (RBL-2H3) cells. In this study, the effects of five-hydroquinone-type and phenolic antioxidants (2,5-di(tert-amyl)-1,4-hydroquinone [DTAHQ], 2-tert-butyl-1,4-hydroquinone [MTBHQ], 3,5-di(tert-butyl)-4-hydroxytoluene [BHT], 3,5-di(tert-butyl)-4-hydroxyanisole [DTBHA], and 3-tert-butyl-4-hydroxyanisole [MTBHA]) on [ca2+]i and degranulation (beta-hexosaminidase release) were examined and compared with that of DTBHQ. DTAHQ (> or = 3 microM) showed effects similar to those of DTBHQ (10 microM) on [Ca2+]i elevation, induction of degranulation with TPA, and increase of antigen-induced degranulation. BHT (50 microM) and DTBHA (50 microM) caused [Ca2+]i elevation and increased degranulation in the presence of TPA or antigen, but their effects were less than those of DTBHQ and DTAHQ. MTBHQ and MTBHA had no effect on [Ca2+]i and degranulation, even at 50 microM. The degree of Ca2+ response caused by the compounds correlated well with the increase in degranulation, but not with their antioxidant activity estimated with the first oxidation potential. From these results, it is suggested that the increasing effects of six antioxidants on degranulation in the presence of TPA or antigen were dependent on [Ca2+]i increase caused by the compounds, probably through their ability to inhibit endoplasmic reticulum Ca2+-ATPase.

Chronic oral toxicity of a synthetic antioxidant, 2,2'-methylenebis(4-ethyl- 6-tert-butylphenol), in rats.

Groups of 30 Wistar rats of each sex were treated with 2,2'-methylenebis(4-ethyl-6-tert-butylphenol) (MBEBP) in the diet at levels of 0, 0.03, 0.1 and 0.3% for up to 18 months. In both sexes, survival rates of treated groups were similar to those of the controls. Body weight gain was depressed (0.3% group in males, 0.1 and 0.3% groups in females). Slight anaemia (0.3% groups in both sexes) and increase of blood urea nitrogen (0.3% groups in both sexes) were observed. Histopathologically, vacuolization of the parathyroid gland cells (0.3% group in males and all treated groups in females) and degenerative changes of the kidney (0.1 and 0.3% groups in males) were observed. No neoplastic responses following MBEBP administration were noted. From these results, the no-observed-adverse-effect level (NOAEL) for MBEBP toxicity was estimated as 12 mg kg-1 body wt. day-1 in male rats. In female rats, the lowest-observed-adverse-effect level (LOAEL) was estimated as 15 mg kg-1 body wt. day-1.

Effects of 2,5-di(tert-butyl)-1,4-hydroquinone on intracellular free Ca2+ levels and histamine secretion in RBL-2H3 cells.

The effects of 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) on the intracellular free Ca2+ level ([Ca2+]i) and histamine secretion of rat basophilic leukemia (RBL-2H3) cells were examined. DTBHQ (0.1-10 mumol/l) alone induced rapid and sustained increases in [Ca2+]i in a concentration-dependent manner. In cells sensitized with anti-dinitrophenyl IgE, DTBHQ (10 mumol/l) further increased the antigen (dinitrophenylated BSA)-induced Ca2+ response. In the absence of external Ca2+ with addition of 1 mmol/l EGTA, both DTBHQ (10 mumol/l) and the antigen (10 microgram/ml) induced transient increase in [Ca2+]i. In sensitized cells, both DTBHQ (10 mumol/l) and antigen (10 micrograms/ml) elicited histamine secretion, although the response was far stronger in the latter case. The DTBHQ-induced histamine secretion was markedly enhanced by addition of the protein kinase C activator, phorbol 12-myristate 13-acetate (TPA) (10 ng/ml) whereas TPA alone did not cause any increase. Moreover, DTBHQ enhanced the antigen-induced histamine secretion. The results suggest that DTBHQ increases [Ca2+]i and enhances antigen-induced histamine secretion while DTBHQ alone does not cause as much histamine secretion as antigen, which support the idea that calcium signals are necessary but are not sufficient for maximum histamine secretion in RBL-2H3 cells.

[Comparative studies on a single dose toxicity of microsomal Ca(2+)ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone and its related analog, mono(tert-butyl)-1,4-hydroquinone, in rats].

We performed comparative studies to determine an acute toxicity of microsomal Ca(2+)ATPase inhibitor, 2,5-di(tert-butyl)-1,4-hydroquinone (DTBHQ) and its related analog, mono(tert-butyl)-1,4-hydroquinone (MTBHQ), which are both used as antioxodants. Wistar rats, 5 weeks old, male and female, were used. By a single dose of oral administration, DTBHQ-induced LD50 values (obtained by Lorke method) in male and female rats were estimated 295.1 and 234.4 mg/kg BW, respectively, whereas each LD50 value for MTBHQ was 711.6 and 400.0 mg/kg BW, respectively. MTBHQ-induced deaths occurred from 8 to 20 minutes after administration, however, DTBHQ-induced deaths occurred more delayed from 1 to 5 days after administration. The observed toxic signs of DTBHQ included diarrhea (jelly like), prone position, lacrimation, salivation and abnormal gait (such as reluctance to walk, limping). Localized purpura and loss of the tail (perhaps as a result of necrosis) were also observed. In comparison, MTBHQ elicited prone position, panting, staggering gait and spastic gait. Without loss of the tail montioned above, dead and sacrified rats showed no remarkable changes in macroscopic examination due to exposure to both compounds.

Acute, subchronic and chronic toxicity studies of a synthetic antioxidant, 2,2'-methylenebis(4-methyl-6-tert-butylphenol) in rats.

General toxicity studies on 2,2'-methylenebis(4-methyl-6-tert-butylphenol) (MBMBP) were conducted using male and female Wistar rats. LD50 values were greater than 5 g/kg BW by oral administration for both sexes. Diarrhea was observed until 5 days. In the subchronic test, rats were fed diet containing MBMBP at 0, 0.12, 0.6 or 3.0% for 12 weeks. Severe suppression of body weight gain was observed in both sexes of 0.6 and 3.0% groups. Death accompanied by hemorrhage from nasal cavity was observed in 0.6 and 3.0% males and 3.0% females. Dose-dependent toxicity to the liver in both sexes was observed in blood chemical analysis. Histopathologically, testicular atrophy and decrease of spermatogenesis were dose- and time-dependently observed in all treated males. Atrophy of ovaries was evident in 0.6 and 3.0% females. Thymus atrophy and bone marrow hypoplasia were observed in both sexes of 0.6 and 3.0% groups. In the chronic test, rats were fed diet containing MBMBP at 0, 0.01, 0.03 and 0.1% for 18 months. Body weight gain was only suppressed in both sexes receiving 0.1%. Histopathologically, testicular atrophy and decrease of spermatogenesis were apparent in 0.1% males. No neoplastic response by MBMBP administration was noted. NOAEL was concluded to be 0.03% in the diet (12.7 mg/kg BW/day for male rats and 15.1 mg/kg BW/day for female rats).

[A 13-week toxicity study of simultaneous administration of cochineal and aluminum potassium sulfate in rats].

Cochineal (C), a scarlet material extracted from the powdered pregnant insect, Dactylopius Coceus Costa, is used as a color food additive in the form of aluminum lakes. A 13 week subchronic toxicity study was conducted to investigate the effects of simultaneous administration of C and aluminum potassium sulfate (A). Male and female Wistar rats (5-weeks-old, 15 rats/group) were given diets containing 0.75%A and 0.75%C (1.5%AC), 1.5%A and 1.5%C (3%AC), 3%C alone or 3%A alone. The following results were obtained. 1) No toxic symptoms or death occurred in any treated group. Body weight gain in male rats of the 3%A group decreased significantly. 2) Serum levels of phospholipids, triglycerides (TG) and total cholesterol in male rats and TG in female rats fed 3%C, 3%A or 3%AC were significantly decreased at the 13th week. The serum level of glutamate dehydrogenase (GIDH) in male rats treated with 1.5% or 3%AC was increased at the 4th week but no difference from control was observed at the 13th week. 3) No histopathological changes attributable to A and/or C administration were observed. In this 13-week oral toxicity study, no dose-dependent synergistic effects of simultaneous administration of C and A were found except for an increase in serum GIDH.

Chemically induced skin carcinogenesis in a transgenic mouse line (TG.AC) carrying a v-Ha-ras gene.

A transgenic mouse line (TG.AC) created in the FVB/N strain, carries a v-Ha-ras gene fused to a zeta-globin promoter gene. These trangenic mice have the properties of genetically initiated skin and have been shown to be sensitive to 12-O-tetradecanoylphorbol-13-acetate (TPA), a well-described promoter of skin papillomas in the two-stage mouse skin tumorigenesis model. It was of interest to determine whether the TG.AC mouse strain was also responsive to other known promoters. Groups of heterozygous or homozygous TG.AC mice were treated topically, 2x/week, for up to 20 weeks with benzoyl peroxide (BPO), 2-butanol peroxide (2-BUP), phenol (PH), acetic acid (AA), TPA and acetone (ACN), the vehicle control. Skin papillomas were induced in all groups treated with TPA, BPO and 2-BUP. Papillomas were observed in some treatment groups as early as 3 weeks. The relative activity of the promoters was TPA > 2-BUP > BPO > PH = AA = ACN. No papillomas were observed in any of the uninitiated FVB/N mice treated in a similar manner and which served as treatment control groups. Studies to determine the sensitivity of TG.AC mice to TPA, indicated that a total dose of 25-30 micrograms of TPA administered in 3 or 10 applications, was sufficient to induce an average incidence of 11-15 papillomas per mouse. The papilloma incidence continued to increase and was maintained up to 15 weeks after TPA treatment was terminated. The short latency period and high incidence of papilloma induction indicate that TG.AC mice have a high sensitivity to known skin promoters. The TG.AC line should prove to be a sensitive model for identifying putative tumor promoters or complete carcinogens.

[Combined chronic toxicity/carcinogenicity test of tris(2-chloroethyl)phosphate (TCEP) applied to female mouse skin].

Tris(2-chloroethyl)phosphate (TCEP), a widely used flame retardant, was tested for its skin chronic toxicity/carcinogenicity using female Slc: ddY mice. TCEP (5 and 50%) dissolved in ethanol was applied to the shaved skin twice a week for 79 weeks. The control group received ethanol under similar condition. In addition, 5 animals in each group were killed at 6 and 12 months and used for the chronic toxicity study. In body weight, food consumption and survival rate, there was no significant difference between the control and treated groups. Spleen weight was decreased in the 50% group. No significant difference in the incidence of tumors and other non-neoplastic lesions of the skin and other organs was found between the control and treated groups. The results indicate that under the conditions of the present study, TCEP has no carcinogenicity and toxicity for the skin.

[Combined long-term toxicity/carcinogenicity test of alpha-bromocinnamic aldehyde (BCA) applied to female mouse skin].

Bromocinnamic aldehyde (BCA), an antibacterial/antifungal agent, was tested for its chronic toxicity/carcinogenicity in female Slc:ddY mice. The animals received 0.25%, 1.0% and 4.0% of BCA dissolved in olive oil applied to the shaved back skin area twice a week for 79 weeks. The control group received olive oil alone under similar conditions. In addition to these animals, 5 animals in each group were killed at 6 and 12 months for investigation of the time-related toxic effect of BCA. A slight inhibition of body weight increase and a slight decrease in the survival rate were seen in the 4.0% BCA-treated group. No significant changes were observed in hematological parameters at 6, 12 and 18 months. In chemical biochemistry determination in the blood at 6 and 12 months, a significant decrease in non-esterified fatty acid and phospholipid values was observed in the experimental groups. Histopathologically, necrosis, scabbing, cell infiltration and thickening of the epidermis were noted at the site of application in the 4.0% BCA group. Moreover, extramedullary hematopoiesis and amyloid degeneration were detected in the spleen, while the incidence of adenomas in the lung decreased with a dose-response. These changes seemed to be the result of a non-specific inflammatory reaction to the irritation effects of the agent on the skin. No significant differences in the incidence of tumors were found between the control and experimental groups, not only at the site of application but also in other organs, although lung adenomas decreased dose-dependently in all treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)