Long-term in vivo carcinogenicity tests of potassium bromate, sodium hypochlorite, and sodium chlorite conducted in Japan.

Long-term in vivo carcinogenicity tests of potassium bromate (KBrO3), sodium hypochlorite (NaClO), and sodium chlorite (NaClO2) have been conducted in Japan from 1977 to 1985. In these investigations, groups of approximately 50 male and 50 female F344 rats or B6C3F1 mice were given solutions of the compounds as their drinking water ad libitum at two dose levels determined on the basis of preliminary 13-week tests. Control animals were given distilled water. The carcinogenic potential of KBrO3 was tested by administering doses of 500 or 250 ppm to rats for 110 weeks. Significantly elevated incidences of renal cell tumors in males and females and mesotheliomas of the peritoneum in males as compared to controls were observed. When female mice were given KBrO3 at doses of 1000 or 500 ppm for 78 weeks, no significant differences in tumor incidences between experimental and control groups were apparent. NaClO was administered to male and female rats, respectively, at doses of 1000 or 500 ppm and 2000 or 1000 ppm for 104 weeks. In mice, NaClO was given at doses of 1000 or 500 ppm to either sex for 103 weeks. The incidences of tumors in NaClO-treated and control animals of both sexes were not significantly different in both rat and mouse studies. NaClO2 was given to rats of both sexes at a dose of 600 or 300 ppm for 85 weeks. No statistically significant differences were observed in the incidences of tumor formation between NaClO2-treated and control groups of both sexes. NaClO2 was administered to mice at a concentration of 500 or 250 ppm for 85 weeks. In males, the combined incidences of hyperplastic nodules and hepatocellular carcinomas of the liver in a low-dose group, and adenomas and adenocarcinomas of the lung in a high-dose group, were marginally increased compared to controls (p less than 0.05). However, these incidences in treated males were within the range of values of historical control data in our program. We concluded that KBrO3 was carcinogenic in rats of both sexes. NaClO was not carcinogenic in either rats and or mice under the conditions of the present studies. Although NaClO2 was shown to be noncarcinogenic in rats, the results for mice were evaluated as inconclusive. Also the results of two-stage mouse skin carcinogenesis using KBrO3, NaClO, and NaClO2 are presented. The necessity for further testing of oxidant chemicals to determine potential carcinogenic and/or promoting effects is suggested in view of the recently proposed role of active oxygen species in carcinogenesis.


Introduction
The cooperative program in Japan for long-term assays of carcinogenicity was begun in 1974 with a subsidy for cancer research from the Ministry of Health and Welfare of Japan (1,2). The primary purpose of these
Sodium chlorite, NaClO2 (CAS.7758- , is used as a bleaching agent. It was acquired from Wako Pure Chemical Industries Ltd., Osaka, at a purity of 99.4%.

Protocol for Carcinogenicity Testing
The experimental design of the long-term carcinogenicity tests was essentially similar to that published in the guidelines of the National Cancer Institute (3). Details are described in the bioassay of KBrO3 in rats (4).
The test animals used were 4to 6-week-old F344 rats or B6C3F1 mice (specific-pathogen-free) of both sexes, purchased from Charles River Japan Inc., Kanagawa.
Groups of approximately 50 male and 50 female rats or mice were treated with the maximum tolerated dose (MTD) or 1/2 MTD, determined on the basis of 13-week toxicity tests. KBrO3 and NaCl02 were dissolved, and NaCIO was diluted, in distilled water and administered orally to animals as their drinking water ad libitum. The doses chosen for the carcinogenicity tests were as follows: KBrO3: 500 or 250 ppm for rats of both sexes and 1000 or 500 ppm for female mice. NaCIO: 1000 or 500 ppm for male and 2000 or 1000 ppm for female rats and 1000 or 500 ppm for mice of both sexes. NaCIO2: 600 or 300 ppm for rats and 500 or 250 ppm for mice, of both sexes. Control animals were given distilled water, and all animals were allowed free access to basal diet (CRF1, Charles River). All three compounds were given continuously to rats and mice for at least 104 and 78 weeks, respectively, except in the case of the test for NaCIO2 in rats, which was terminated prematurely in week 85. The animals were observed daily. Dead animals and those found moribund were autopsied immediately. Consumption of water and body weights were recorded. At termination of the tests, all surviving animals were anesthetized by ether inhalation to collect blood for hematologic and serum biochemical analysis. At necropsy, organs were grossly examined, excised, and weighed. They were stained with hematoxylin and eosin after routine histological processing. After detailed microscopic examination, the data were statistically analyzed using either the chi-square test, Fisher's exact probability test, or Student's t-test.

Carcinogenicity Tests on KBrO3
Rat. In males given 500 ppm of KBrO3 (4), the dose was reduced to 400 ppm at week 60 because of a marked inhibition of body weight increase (Fig. 1). No apparent inhibition of body weight increase occurred in either sex given 250 ppm. The survival curves of male and female rats given KBrO3 for 110 weeks are shown in Figure 2. Dead or moribund rats appeared earlier among males given 500 ppm. For females, the survival curves of treated and control groups were very similar. All surviving animals were sacrificed in week 111. No significant differences in water intake were apparent between treated and control groups. Daily intakes of KBrO3 (mg/ kg body weight/day) were, in males, 27.7 and 12.5 and, in females, 25.5 and 12.5, in highand low-dose groups, respectively. For detailed histopathologic examination for kidney, 10-15 step-serial sections were made per kidney in this study. As a result, high incide nces of renal cell tumors in KBrO3-treated rats of both sexes were noted. Most renal cell tumors were microscopic in size, although some were visible as round yellowish-white or grayish projections from the renal cortex (Fig. 3). Histologically, renal cell tumors were classified as adenocarcimale nomas and adenomas, the former appearing irregularly -~contoured as if several small nodules were aggregated.
--/ They were mostly localized in the cortical areas and exhibited solid growth patterns, although in some cases trabecular, tubular, or papillary patterns were also observed (Fig. 4). Apparent infiltrative growth was seen in cases with grossly large tumors. The tumor cells were _______ found to have either clear cytoplasm (clear cell), eosi-----00--SOOppM nophilic granular cytoplasm (granular cell), or homog--OOOppm enously basophilic cytoplasm (dark cell). Adenomas appeared as oval, solitary nodules consisting of closely packed polygonal cells. They were well circumscribed so 80 100 with thin fibrous capsules. Besides these distinct neoplastic lesions, dysplastic foci, considered preneoplastic mice given KBrO3. changes, were frequently observed.    Table 2). The incidences of mesotheliomas in male rats given 500 or 250 ppm were significantly different from that in controls, respectively, at p<0.001 and p<0.05 levels. Table 3 summarizes the histopathologic diagnosis of tumors occurring at relatively high incidences in various organs other than in the kidney and peritoneum. The incidences of tumor-bearing animals were very high in both control and treated groups of both sexes. This phenomenon resulted from the high incidences of tumors of kidney, testis, peritoneum, thyroid, pituitary, mammary gland, and spleen.
Nonneoplastic renal tubular lesions occurring in KBrO3-treated rats included various degenerative, necrotic, and regenerative changes. Hyaline cast in the tubular lumen, and hyaline droplets, eosinophilic bodies, and brown pigments in the tubular epithelium were also commonly observed. Biochemical analysis of the   serum revealed significant decreases in glutamic-pyruvic transaminase, the albumin-to-globulin ratio, serum potassium, and cholinesterase in females given 500 ppm KBrO3. Also, slightly increased levels of blood urea nitrogen were observed in males and females treated with KBrO3. Mouse. Treatment of male mice was discontinued during this study (S. Takayama, unpublished data) because of severe fighting phenomena. Female mice were given KBrO3 at doses of 1000 or 500 ppm for 78 weeks and thereafter given tap water for 26 weeks. All survivors were sacrificed at week 104. Although body weight increase was markedly inhibited in the high-dose group (Fig. 5), the survival curves were comparable, and sufficient numbers of animals survived for 104 weeks in all three groups (Fig.6). Daily intakes of KBrO3 (mg/kg body weight/day) were 119.8 and 56.5 given at 1000 and 500 ppm, respectively.  Results of histologic examination of tumors are shown in Table 4. No significant differences in the incidences of tumor-bearing animals were apparent between treated and control groups. Although relatively high incidences of lung, liver and lymph node tumors were observed in the high-dose group, the incidences of these tumors were not significantly different from those of controls.

Carcinogenicity Tests on NaCIO
Rat. In this test (M. Takahashi, unpublished data), NaClO was given orally at concentrations of 1000 or 500 ppm and 2000 or 1000 ppm, respectively, to males and females for 104 weeks. The rats were thereafter given distilled water, and all survivors were sacrificed at week 112. The survival ratios at week 104 were very high, ranging from 80% to 70%, in all groups of both sexes (Fig. 7). Dose-dependent inhibition of body weight increase was observed in both male and female rats (Fig.  8). Drinking water intakes were comparable among all groups.
All three groups demonstrated relatively high incidences of tumors of the testis, pituitary, thyroid, lung, pancreas, uterus, mammary gland, spleen, and subcutaneous tissue (Table 5). Histologically, chromophobic adenomas of the pituitary, adenomas and adenocarcinomas of C-cells of the thyroid, adenomas of the lung, insulomas of the pancreas, fibroadenomas of the mammary gland, and mononuclear cell leukemias were identified in both males and females. Also, high incidences of interstitial cell tumors of the testis and fibromas in subcutaneous tissue in males and endometrial polyps of the uterus in females were observed. However, significant compound-related increases in tumor incidences were not found in any organs oftreated animals of either sex. Incidences of nonneoplastic lesions, such as chronic nephropathy in treated males and granulomatous changes in the liver of treated females, were significantly decreased. Hematologic and serum biochemical analyses did not reveal significant dose-related changes in any parameters in either sex treated with NaCIO.
Mouse. Groups of 50 male and 50 female mice were given NaClO at doses of 1000 or 500 ppm for 103 weeks (S. Asahina, unpublished data). However, control groups consisted or 73 males and 72 females in this test. All surviving mice were sacrificed at week 106, at which time the survival rates were 74%, 78%, and 66% in males and 78%, 80%, and 78% in females, respectively, for high-dose, low-dose, and control groups (Fig. 9). Doserelated reductions in body weight increase occurred in both sexes (Fig. 10). Total intakes (g/mouse/103 wks) of NaClO were 3.02 and 1.81 in males and 2.86 and 1.47 in females, respectively, in high-and low-dose groups.
Tumors occurring with relatively high incidences are listed in Table 6. Combined incidences of leukemias and malignant lymphomas and of adenomas and adenocarcinomas of the lung were very high in all groups for both sexes. Also, high incidences of hyperplastic nodules and hepatocellular carcinomas of the liver in males of all groups were noteworthy. However, statistically significant differences were not observed in tumor incidences for any organs of treated animals.

Carcinogenicity Tests on NaC102
Rat. This study (Y. Hiasa, unpublished data) was prematurely terminated at week 85 because of widespread Sendai viral infection in all groups, necessitating immediate sacrifice of all survivors. At necropsy, pneumonias were found in all animals, and an abscess of the lung had developed in some cases. Percentages of survivors at week 85 were 86%, 60%, and 68% in males and 100%, 88%, and 94% in females, respectively, in high-dose (600 ppm), low-dose (300 ppm), and control groups (Fig. 11). Body weight increase was inhibited in a dose-dependent manner in both males and females (Fig. 12). Drinking water intake in treated animals was slightly lower than that in control animals of both sexes. Daily consumption of NaCl02 (mg/kg body weight/day) was 32.1 and 18.0 in males and 40.9 and 28.3 in females, respectively, for highand low-dose groups.
No statistically significant differences in the incidence of tumor-bearing animals were observed between treatment and control groups of either sex. Incidences of tumors in several organs were appreciable (Table 7), i.e., C-cell adenomas of the thyroid, pheochromocytomas of the adrenal, and interstitial cell tumors of the testis in males, and chromophobic adenomas of the pituitary and endometrial polyps of the uterus in females. However, no statistically significant differences in the rates of tumor development in any organs were observed between NaCIO2-treated and control animals of 80 Female Table 5. Histopathologic diagnoses of tumors occurring at relatively high incidence in rats given NaClO.   either sex. Serum biochemistry analysis revealed that levels of glutamic oxaloacetic transaminase in the liver were significantly decreased in the high-dose males. Hematalysis and urinalysis revealed no significant changes in blood or urine. Mouse. Mice in treatment groups ofboth sexes were given NaClO2 at concentrations of 500 or 250 ppm for 85 weeks, at which time all survivors were sacrificed (Y. Konishi, unpublished data). Dead or moribund male mice were found during the experiment earlier in control groups than in the treated groups (Fig. 13) because of severe fighting. Survival percentages at the end of the study were 86%, 94%, and 70% in males and 100%, 100%, and 94% in females, respectively, in high-dose, low-dose, and control groups. However, body weight increases were comparable among all groups of either sex (Fig. 14).
As shown in Table 8, the incidences of liver tumors were higher in treated males than in control males. These tumors were histologically diagnosed as hyperplastic nodules or hepatocellular carcinomas. The combined incidences of these tumors were significantly different in males of the low-dose group (p < 0.05). The incidences of hyperplastic nodules of the liver in males were significantly higher in both highand low-dose groups (p < 0.05), although the incidences did not exhibit a dose-related effect. Also, the combined incidences of adenomas and adenocarcinomas and that of   adenomas of the lung were significantly higher in males of the high-dose group (p < 0.05). Relatively high tumor rates were observed for malignant lymphomas and/or leukemias and adenomas of the Harderian gland in both sexes, and for tumors of the liver and the lung in females of all groups. Of these, the incidences of malignant lym-phomas and/or leukemias in the high-dose female group were smaller by a statistically significant margin.

Discussion
The design and conduct of the long-term assays described in this paper were considered adequate according to recent guidelines for carcinogenicity testing and evaluation of data (1-3, 5, 6), although the test on KBrO3 in mice was carried out only in females, and that on NaClO2 in rats of both sexes was stopped at week 85 because of infection. As a result, KBrO3 was shown to have carcinogenic effects in rats of both sexes. NaC1O2 was negative in rats, although it generated equivocal data in male mice. However, no evidence of carcinogenicity was apparent for NaClO in both rats and mice of either sex.
KBrO3 induced renal cell tumors in both male and female rats and mesotheliomas of the peritoneum in male rats when it was given orally at doses of 500 or 250 ppm for 110 weeks (4,7). The incidences of both tumors were significantly elevated and showed doseresponse relationships. In the study of KBrO3 using female mice, on the other hand, no statistically significant differences in the incidences of any tumors were observed between treatment and control goups. Kidney tumors found in this study were one adenocarcinoma and one adenoma in the high-dose group and one adenoma in the control group. Long-term oral administration of KBrO3 to male mice of three strains (BDF1, CDF1 and B6C3F1) is currently underway at a dose of 750 ppm, based on the fact that kidney tumor incidence in mice is higher in males than in females (8).  Long-term feeding studies for KBrO3 in Great Britain have been reported (9,10). A basal bread diet prepared from untreated flour or from flour treated with 50 or 75 ppm of KBrO3 was given to rats (9) and mice (10) for 104 and 80 weeks, respectively. No evidence of carcinogenicity or chronic toxicity was observed in either experiment. The discrepancy in results can be explained by the finding that almost all KBrO3 added to flour at normal food additive levels is converted to potassium bromide (KBr) during the normal British baking process, and the amount of KBrO3 is negligible in the final products (11,12). Therefore, commercial bread supplemented with KBrO3 was found to be noncarcinogenic to rats and mice in the British studies. In contrast, our test demonstrated positive potential carcinogenicity of KBrO3, which itself is very stable in water, in rats of both sexes when administered in their drinking water.
Recently, long-term dose-response studies on this chemical have been completed (Y. Kurokawa, unpublished data). Male F344 rats were given KBrO3 orally for 104 weeks at concentrations of 500, 250, 125, 60, 30, or 15 ppm (Table 9). Statistically significant increases were observed in the incidences of renal cell tumors in rats treated with > 125 ppm. In addition, incidences of dysplastic foci, considered to be preneoplastic lesions of renal cell tumors (13,14), were significantly higher in rats treated with > 30 ppm. The virtually safe dose (VSD) of KBrO3 for renal cell tumors was estimated by the probit model with independent background (S. Aoki, unpublished data). The VSD at risk levels of 106 and 10-8 was 0.950 and 0.302 ppm, respectively (p = 0.877).
To determine further species differences in the carcinogenicity of KBrO3, male Syrian golden hamsters were treated orally for 89 weeks (Y. Kurokawa, unpublished data). In this study, no statistically significant differences were observed in the incidences of tumors in any organs. However, the rates of renal cell tumors were 11% (2/19), 20% (4/20), 6% (1/17), 0% (0/19), and 0% (0/20), respectively, in groups given 2000, 500, 250, 125, or 0 ppm. Although the incidences were relatively low and were not statistically different, the results seemed to indicate the potential for KBrO3 to induce renal cell tumors in male hamsters, inasmuch as the incidences of spontaneous kidney tumors were extremely low in this rodent species (15)(16)(17).
The promoting effect of KBrO3 was also investigated. KBrO3 given orally at a concentration of 500 ppm was found to have a promoting effect on renal, but not on liver, tumorigenesis, when initiated with N-ethyl-N-hydroxyethylnitrosamine (EHEN) in male F344 rats (14). Subsequently, dose-response studies in two-stage renal carcinogenesis were undertaken to ascertain whether a threshold level of KBrO3 treatment exists for its promoting activity (18). Male F344 rats were treated orally at doses of 500, 250, 125, 60, 30, or 15 ppm for 24 weeks after initiation with EHEN. The results showed that the mean numbers of kidney dysplastic foci were significantly increased in a dose-related manner when KBrO3 was given at doses of > 30 ppm. Therefore, the threshold level for enhancing renal tumorigenesis seemed to lie between 30 and 15 ppm. The potential enhancing effect of KBr was also tested in this study, with the result that it was considered negative for renal carcinogenesis (18).
From studies on carcinogenicity and on promoting effects, we concluded that KBrO3 possessed both initiating and promoting activities, the characteristics of a complete carcinogen, for development of renal cell tumors in rats. In contrast, KBrO3 exhibited neither promoting nor complete carcinogenic activities for skin carcinogenesis (19).
Several experiments were conducted to elucidate the mechanism of carcinogenic action of KBrO3 (Y. Kurokawa, unpublished data). In acute toxicity studies, KBrO3 induced a dose-dependent increase in lipid peroxidation in the kidney. Survival times after a single intravenous administration of KBrO3 were extended by pretreatment with glutathione in male F344 rats. Ultrastructurally, extensive damage to the mitochondria of the renal tubular epithelium was observed. The appearance ofmultiple eosinophilic bodies in the cytoplasm of proximal renal tubules in KBrO3-treated rats was noteworthy as a nonneoplastic change in the kidney. These droplets were strongly stained with eosin and were positive with the Azan stain, but were negative for the periodic-acid Schiff stain (20).
In the test of NaClO2, no evidence of carcinogenicity was apparent in rats of either sex. On the other hand, marginal increases were observed in the incidences of liver and lung tumors in male mice given NaClO2. Namely, statistically significant increases were found in the combined incidences of hyperplastic nodules (HN) and hepatocellular carcinomas (HCC) of the liver in a low-dose group, and adenomas and adenocarcinomas of the lung in a high-dose group in males (both p,<0.05). It should be mentioned, however, that males in the control group died spontaneously, earlier than treated males in this study because of severe fighting (Fig. 13) and were not included in the effective numbers for histopathologic examination. On the other hand, respective incidences of HN and HCC of the liver were 27.8% (range, 22-33%) and 20.1% (range, 8-23%) in 194 untreated B6C3F1 males in earlier bioassays in our program (S. Fukushima, unpublished data). Also, the incidences of adenomas and adenocarcinomas of the lung were 9.8% (range, 6-16%) and 5.7% (range, 2-8%), respectively, in the same controls. Note that, although statistically elevated over those of concurrent controls, the rates of HN of the liver in the high-dose (25.6%) and low-dose (29.8%) groups and that of lung adenomas in the high-dose group (11.6%) were all within the range of historical control data for our program as well as that of the National Toxicology Program (21). We concluded, threrefore, especially since significant increases were observed only in the incidences of benign tumors, that the results of the carcinogenicity test on NaClO2 in mice were inconclusive under the conditions of this study.
Several toxicological studies have reported results of tests on chlorine and chlorite because of the unavoidable human exposure to these substances in drinking water after disinfection (22)(23)(24)(25)(26). The potential toxicity of hypochlorous acid (HOCl) when orally administered to male Sprague-Dawley rats for 12 months at concentrations of 100, 10, or 1 ppm was investigated (26). Significant increases were observed in glutathione levels and osmotic fragility of the erythrocytes. Red blood cell count and hematocrit were significantly decreased. Also, incorporation of [3H]thymidine into kidney and testis nuclei was increased. The results of long-term oral treatment with highly chlorinated water containing 100 ppm of free chlorine was reported (27). BDll rats were given chlorine over their life span in seven consecutive generations. No compound-related toxicity or carcinogenicity was observed. NaClO2 was given to male Sprague-Dawley rats in their drinking water for 12 months at levels of 100 or 10 ppm (25). Various he-matologic parameters were affected in the treatment group similarly to HOCl-treated rats (26). However, such hematologic changes were not observed in rats and mice of both sexes given NaClO or NaClO2 in our carcinogenicity tests and in a subchronic study (28). Although positive promoting activity has been reported for the ddN-strain mouse skin with application of a 10% solution of NaClO (29), this effect was not found when a 1% NaCIO solution was applied to SENCAR mouse skin (19). NaCIO2 painted at a concentration of 100 ppm on SENCAR mouse skin was suspected of exerting skin promoting activity (19).
Results of mutagenicity testing of the three compounds are summarized in Table 10 (30-32); all three gave positive results in three out of the four mutagenicity tests conducted. Therefore, these compounds could be classified as highly mutagenic compounds. Nonetheless, neither NaClO nor NaClO2 appeared carcinogenic in our studies, although the carcinogenicity of KBrO3 was clearly demonstrated.
KBrO3, NaClO, and NaCl02 have been used for a variety of purposes using their oxidizing properties, a characteristic common to all three compounds. With respect to the in vivo carcinogenic and promoting actions of oxidant chemicals, recent reports on mouse skin carcinogenesis deserve mention.
Benzoyl peroxide was shown to be a potent promoter (33) and a possible complete carcinogen (19) in this system and was suspected as a causative agent for skin cancer in humans (34). Hydrogen peroxide, lauroyl peroxide, decanoyl peroxide, cumene peroxide (35,36), NaClO (29), and NaClO2 (19) were demonstrated to be relatively weak as promoters, whereas KBrO3 and ammonium persulfate were inactive either as complete carcinogens or as promoters in mouse skin carcinogenesis (19). In systems other than the skin, hydrogen peroxide given orally was found to have carcinogenic potential in mice by inducing duodenal tumors (37,38) and to have promoting action by increasing intestinal tumor development in rats (39). Although KBrO3 showed a promoting effect in two stage rat renal carcinogenesis (14,18), no such influence was evident for potassium chlorate and sodium chlorate given orally at a level of 1% for 24 weeks after initiation with EHEN (Y. Kurokawa, unpublished data).
It seems generally accepted that carcinogenic and promoting action of these compounds both in vivo and in vitro are caused by various kinds of active oxygen species, e.g., superoxide, singlet oxygen, hydroxy radical, and hydrogen peroxide (40)(41)(42). From extensive toxicological studies on oxidant chemicals such as paraquat (43), ozone (44), and NO. (45), it was revealed that these chemicals could induce lipid peroxidation in their target organs. Induction of lipid peroxidation and clastogenic actions are now considered to be the underlying mechanisms of carcinogenic and/or promoting effects shown by the oxidant chemicals (40,46). Indeed KBrO3 increased the levels of lipid peroxide in the kid-ney (Y. Kurokawa, unpublished data) and was clastogenic in Chinese hamster cells (31). In spite of the accumulating evidence of the role of free oxygen radicals in in vitro promotion and cell transformation, the number of in vivo studies available to support in vitro data remains insufficient (47). Although the skin seems to be a suitable target organ for detecting potential carcinogenic and promoting effects of oxidants (36), the effects of these compounds on gastrointestinal organs should also be tested in view of their local action as skin irritants. The fact that humans are inevitably exposed orally to food additives and to by-products of water disinfection having oxidizing characters also emphasizes the need to test these chemicals for carcinogenic and promoting effects in organs other than skin.
We thank Dr. Fukushima of the Nagoya City Medical School for kindly supplying data on spontaneous incidences of tumors in B6C3F1 mice, Dr. Aoki of the University of Gumma Medical School for estimation of virtually safe dose, and Dr. Ishidate of the National Institute of Hygienic Sciences for information on mutagenicity of the compounds. We are also indebted to Dr. Moore and Miss Hattori for editing and typing the manuscript.