Lack of oncogenicity of wood creosote, the principal active ingredient of Seirogan, an herbal antidiarrheal medication, in Sprague-Dawley rats.

Seirogan, an herbal medicine containing wood creosote (tablets, 10.0% w/w), has been developed and marketed for almost a century in various countries for the control of acute diarrhea and treatment of associated symptoms, such as abdominal cramping. Wood creosote (CAS no. 8021-39-4) is a mixture of simple phenolic compounds, including guaiacol and creosol and related compounds, and is chemically distinct from, and should not be confused with, coal tar creosote, a known carcinogen. In the current study, the oncogenic potential of wood creosote was assessed in a 96/103-week oral gavage study in Sprague-Dawley rats. Groups of 60 rats/sex received wood creosote at dose levels of 20, 50, or 200 mg/kg body weight [bw]/day. An additional group of rats received the vehicle, 0.5% carboxymethylcellulose in deionized, distilled water, at the same dose volume as the treatment groups (10 ml/kg) and served as the controls. Treatment-related decreases in survival, body weight, and food consumption, as well as increased incidences of clinical signs that included rales, decreased activity, and salivation, were noted at 200 mg/kg bw/day when compared with the control group. There was an increased incidence of reddened and edematous lungs in rats from the 200 mg/kg bw/day group that died during the study. The lung findings were suggestive of test article aspiration during dose administration or agonal aspiration preceding and possibly resulting in death, especially because these observations were not seen in animals that survived to scheduled sacrifice. Additionally, phenols are generally recognized as having corrosive properties. There were no changes in clinical pathology and no increases in neoplastic or non-neoplastic lesions, excluding the lung findings, related to treatment with wood creosote at any dose level. Although the results of this study indicate that the maximum tolerated dose of wood creosote was met or exceeded at 200 mg/kg bw/day, there was no evidence of oncogenicity at any dose level. The lack of any evidence of oncogenicity supports the safety profile of the active ingredient in Seirogan, wood creosote.

Review of the toxicologic properties of medium-chain triglycerides.

Medium chain triglycerides (MCTs) are a family of triglycerides, containing predominantly, caprylic (C(8)) and capric (C(10)) fatty acids with lesser amounts of caproic (C(6)) and lauric (C(12)) fatty acids. MCTs are widely used for parenteral nutrition in individuals requiring supplemental nutrition and are being more widely used in foods, drugs and cosmetics. MCTs are essentially non-toxic in acute toxicity tests conducted in several species of animals. In ocular and dermal irritation testing MCTs exhibit virtually no potential as ocular or dermal irritants, even with prolonged eye or skin exposure. MCTs exhibit no capacity for induction of hypersensitivity. Ninety-day toxicity tests did not result in notable toxicity, whether the product was administered in the diet up to 9375mg/kg body weight/day or by intramuscular (im) injection (up to 0. 5ml/kg/day, rabbits). There was no evidence that intravenous (iv) or dietary administration of MCTs adversely affected the reproductive performance of rats or resulted in maternal toxicity, foetal toxicity or teratogenic effects at doses up to 4.28g/kg body weight/day (iv) or 12,500mg/kg body weight/day (dietary). There was no evidence that dietary administration of MCTs adversely affected the reproductive performance of pigs or resulted in maternal toxicity, foetal toxicity or teratogenic effects at doses up to 4000mg/kg body weight/day in the diet. In rabbits, following iv administration, the maternal and foetal no-observed-adverse-effect levels (NOAELs) were between 1.0 and 4.28g/kg body weight/ day. A 2-year study in rats, conducted with a closely related compound (tricaprylin, a triglyceride with C(8) fatty acids), provided no evidence of a carcinogenic effect when the material was administered by oral gavage at levels up to 10ml/kg (9.54g/kg) per day. Although tricaprylin was found to be positive in one of five strains of Salmonella typhimurium in the presence of metabolic activation in an Ames mutagenicity assay, the results of the carcinogenicity test with tricaprylin and mutagenicity tests with caprylic acid indicate that MCTs do not have the potential to be carcinogenic or mutagenic. The safety of human dietary consumption of MCTs, up to levels of 1g/kg, has been confirmed in several clinical trials.

Evaluation of coal liquids derived from the EDS process in carcinogenesis screening tests.

Four process streams derived from the EDS not equal to direct coal liquefaction process were evaluated in two in vitro assays to screen for carcinogenic potential: the Salmonella/mammalian microsome mutagenicity assay and the Syrian hamster embryo morphologic transformation assay. Three high boiling liquids (two recycle solvents, nominal boiling range 200-425 degrees C; and a fuel oil blend, nominal boiling range 200-538 degrees C) were active in both assays. A hydrotreated naphtha sample (< 200 degrees C) was not active in either. The Salmonella data agreed qualitatively with results of dermal carcinogenesis studies; however, quantitative differences as measured by the estimation of mutagenic potency were apparent. The lack of quantitative agreement may have been related to the fact that the dermal carcinogenic activity of coal-derived synthetic fuels is predominantly associated with neutral polycyclic aromatic hydrocarbons, whereas activity in the Salmonella assay is strongly influenced by the presence of aromatic amines and nitroaromatic compounds. Two modifications of the Salmonella assay--detergent dispersion and hamster S9 activation--were examined. These techniques improved assay performance for some but not all of the coal liquids. The differences in response may have been related to compositional differences in the various liquids.

Evaluation of the teratogenic potential of octyl acetate in rats.

Octyl acetate (CAS RN 108419-32-5) was administered via oral gavage to pregnant Sprague-Dawley rats on Gestation Days 6 through 15 at dose levels of 0, 0.1, 0.5, and 1.0 g/kg. The dams were weighed and observed for clinical signs of toxicity during pregnancy, and food consumption was measured. On Gestation Day 20 the dams were sacrificed and the fetuses were examined for external, visceral, and skeletal malformations and variations. The mid- and high-dose levels resulted in maternal toxicity as evidenced by reductions in body weight gain and food consumption. There were no statistically significant effects on embryo-fetal lethality or fetal growth for any treatment group. The number of litters with at least one malformed fetus and the mean percentage of the litter malformed were significantly (p less than 0.05) elevated in the high-dose group only. The results of the present study demonstrate that octyl acetate produced some evidence of developmental toxicity at a dose (1.0 g/kg) that was maternally toxic. Developmental toxicity was not observed at the maternally toxic 0.5 g/kg dose level or the maternally nontoxic dose level (0.1 g/kg). Therefore, these data indicate that octyl acetate is not a selective developmental toxicant in the rat.

The genetic toxicity of 1,2-dibromo-3-chloropropane, 1,2-dibromo-3-chloro-2-methylpropane, and 1,2,3-tribromo-2-methylpropane.

1,2-Dibromo-3-chloro-2-methylpropane (DBCMP) and 1,2,3-tribromo-2-methylpropane (TBMP) are contaminants formed during the manufacture of bromobutyl rubber. These chemicals are structurally similar to 1,2-dibromo-3-chloropropane (DBCP), a known genotoxin and rodent carcinogen. The present study compared the genotoxic properties of DBCMP and TBMP to those of DBCP. In the Salmonella assay, DBCP was positive in strains TA-98, TA-100 and TA-1535 in the presence of exogenous activation; DBCP was weakly active in TA-1535 in the absence of activation. Neither DBCMP nor TBMP produced reproducible evidence of mutagenic activity in the Salmonella assay despite the use of several different variations of this test. In the mouse lymphoma gene mutation assay, DBCP and TBMP were positive in the presence and absence of activation, while DBCMP was positive only in the absence of activation. All three test compounds were active in the Syrian hamster embryo morphologic transformation assay. The results indicated that both DBCMP and TBMP exhibited some genotoxic activity as did DBCP. The presence of the methyl group on the 2-carbon position essentially eliminated the mutagenicity of DBCMP and TBMP in the Salmonella assay.

Assessment of the potential reproductive and subchronic toxicity of EDS coal liquids in Sprague-Dawley rats.

The EDS direct coal liquefaction process is one of several methods of producing liquid fuels from coal which have reached the pilot or demonstration stage of development. Relatively high levels of polycyclic aromatic hydrocarbons are present in distillate fractions boiling above approximately 370 degrees C, and unrefined coal-derived liquids which contain substantial amounts of material from this boiling range are relatively potent dermal carcinogens. Because coal-derived liquids containing high boiling (i.e., greater than 370 degrees C) material may pose a variety of toxic hazards, efforts have been made to evaluate the potential effects on biological endpoints other than cancer. The present studies assessed the potential for reproductive and subchronic toxicity following repeated oral administration of 2 coal-derived liquids, recycle solvent and fuel oil, which contained substantial amounts of high boiling material. Few biologically important differences were found in any of the experimental parameters. In the reproductive toxicity study, frequency of fertilization and implantation, mean number of live births, fraction of litter surviving through the lactation period and mean weight gain of the litters during the lactation period were not affected by treatment; in addition, there was no evidence of increased frequency of malformation. In the subchronic toxicity study, weight gain was reduced in animals from the high dose groups, but was not significantly different from controls. Liver weights were significantly elevated, but there was no microscopic evidence of pathologic changes. Erythrocyte counts, hemoglobin levels and hematocrits were significantly reduced suggesting a tendency towards anemia. These findings suggested that repeated exposure to EDS recycle solvent and fuel oil at levels of up to 0.5 g/kg per day had no detectable effect on reproductive capacity or performance and did not induce substantial systemic toxicity.

Developmental toxicity of EDS recycle solvent and fuel oil.

Direct coal liquefaction is one of several technologies currently under development as alternative means to produce liquid fuels. Relatively high levels of polycyclic aromatic hydrocarbons are present in distillate fractions boiling above approximately 370 degrees C. Coal-derived liquids containing substantial amounts of material from this boiling range were genotoxic in in vitro tests and carcinogenic in mouse skin. Some of the liquids were also teratogenic in rodents. The present report describes studies which assessed the potential effects of 2 coal-derived liquids, recycle solvent (nominal boiling range 200-427 degrees C) and an experimental industrial fuel oil (nominal boiling range 204-538 degrees C) on prenatal development in the rat. The test materials were produced by the EDS direct coal liquefaction process and contained substantial amounts of material boiling above 370 degrees C. Test materials were administered by gavage to pregnant female Sprague-Dawley rats from days 6 to 19 of gestation (G). Animals were sacrificed on day 20G and the uterine contents were removed and examined. Results of both studies were similar. The number of live fetuses declined in a dose-related manner, and there was evidence of intrauterine growth retardation in fetuses which survived to day 20G. Statistically significant effects were noted at doses which did not appear to be maternally toxic. The frequency of malformation was not significantly elevated in either study; however, a thorough evaluation of this endpoint was precluded by embryo lethality at the high doses. It was apparent that both of the EDS liquids examined affected prenatal survival and growth. However, in contrast to studies of other coal-derived liquids, there was no evidence of teratogenic effects at non-toxic doses.

A comparison of developmental toxicity evident at term to postnatal growth and survival using ethylene glycol monoethyl ether, ethylene glycol monobutyl ether and ethanol.

This study was designed to compare an abbreviated evaluation of uterine contents at term (teratology probe) with a modified Chernoff-Kavlock assay (postnatal study), [Chernoff N, Kavlock RJ: J Toxicol Environ Health 10:541-550, 1982]. Mice were intubated during gestation and were evaluated for signs of toxicity. In the teratology probe, uterine contents were examined at term. In the postnatal study, offspring were examined and weighed through day 22 postpartum. Ethylene glycol monoethyl ether (EGEE) produced embryo lethality and malformations, and decreased fetal weight at a dose level which was not maternally toxic in the teratology probe. In the postnatal study, EGEE decreased litter size and neonatal body weight; while litter size continued to decrease beyond the neonatal period, body weights of surviving pups were not significantly different from control. Pups exposed prenatally to EGEE developed kinked tail which was not apparent in fetuses or neonates. Maternally toxic dose levels of ethylene glycol monobutyl ether and ethanol were associated with increased embryo lethality in teratology probe studies. In postnatal studies, there were no significant effects on pup growth or survival at maternally toxic dose levels. Preliminary conclusions regarding maternal and developmental toxicity were comparable based on the teratology probe or postnatal study. Both assays measure litter size and offspring weight, but the teratology probe measures resorption incidence which may be a more sensitive index of prenatal death than number of live born. Neither fetal weight nor neonatal weight reliably predict permanent alteration of growth. A postnatal study permits detection of internal malformations or functional defects which reduce postnatal survival and gross abnormalities which appear postnatally.

Evaluation of the teratogenic potential and reproductive toxicity of coal-derived naphtha.

Liquids which are derived from coal liquefaction processes and boil above approximately 250 degrees C have induced terata in rats. However, few studies have addressed the teratogenic potential of coal liquids which boil below 250 degrees C. The present studies evaluated the reproductive and teratogenic potential of EDS hydrotreated naphtha, a refined coal liquid boiling below 177 degrees C. These studies were conducted by inhalation exposures with Sprague-Dawley rats at target vapor concentrations of 0.2, 1.0, and 5.0 g/m3. The first study assessed teratogenesis. There was no evidence that inhalation exposures for 6 hr per day between Days 6 and 19 of gestation induced maternal toxicity, fetal toxicity, or malformation. In a second study, rats were exposed for 6 hr per day, 5 days per week for 13 weeks, and then mated to assess reproductive toxicity. There was little evidence that inhalation exposure to EDS hydrotreated naphtha adversely affected reproductive performance or fetal development in Sprague-Dawley rats. A low incidence of malformations was observed in treated groups, but these malformations were probably not treatment related.

An evaluation of the genotoxic potential of di-isononyl adipate.

Di-isononyl adipate (DINA) is one of a group of adipates used primarily as plasticizers. Concern over the mutagenic and carcinogenic potential of these materials was stimulated by the finding that one member of this class, di-(2-ethylhexyl) adipate (DEHA), induced liver tumors in female mice in a chronic feeding study. Accordingly, the genotoxic potential of DINA was evaluated in a battery of in vitro tests including the Salmonella/mammalian microsome mutagenicity assay, the mouse lymphoma TK +/-assay, and two tests of morphologic transforming ability, the BALB 3T3 and the Syrian hamster embryo in vitro transformation assays. DINA did not exhibit any evidence of mutagenic or transforming potential in any of the assays utilized.

Balbc/3T3 cell transformation response to extracts of organic air samples as seen by their survival in aggregate form.

Extracts of organic matter from samples of airborne particulate matter have been shown to possess components capable of transforming mammalian cells. This study was done to determine if Balbc/3T3 cells exposed to extracts of air samples could, unlike their normal counterparts, in the absence of a surface for attachment, divide on agar to form aggregates, and if these cells would demonstrate a dose-response phenomenon. Untreated and solvent treated control cells failed to form large aggregates and showed a decline in viable cell number over a 6-day period. Cells treated with either cyclohexane or acetone extracts of airborne particulate matter showed a dose-response increase in cell number along with the formation of progressively larger aggregates, findings similar to those seen with the positive, benzo[a]pyrene (BaP), control. Furthermore, these findings are in direct agreement with those in the simultaneously performed standard cell transformation assay which required 21 days to perform. Results show that survival in aggregate form is a rapid in vitro test system capable of detecting potentially carcinogenic activity in complex environmental mixtures.

Effects of incubation in an atmosphere of 20% CO2 in air on the Syrian hamster embryo clonal transformation assay.

An atmosphere containing 10% CO2 has been generally accepted as optimal for the growth of Syrian hamster embryo cells in a clonal transformation assay. Data presented in this paper show that 10% CO2 may not be the optimum environment for this assay. Using 10 or 20% (analytically measured) CO2 in air (1 atm pressure), hamster embryo cell pools were examined for clonal growth characteristics and transformability using five known carcinogens and a single noncarcinogenic compound. At 10% CO2, only 2 of 11 pools were transformed by the five carcinogens but not by the noncarcinogen. At 20% CO2, six of seven pools were transformed by the five carcinogens and not by the noncarcinogen. Further, the transformation frequencies were found to be greater in cultures incubated in an atmosphere consisting of 20% CO2 in air. The data also show that 20% CO2 increased the cloning efficiency of these cells. A comparison of the 10 and 20% CO2 data to results reported from other laboratories suggests that conflicting interlaboratory results with this assay system may be due, in part, to variations of CO2 concentrations. In some instances, the CO2 levels indicated by incubator flow meters vary considerably from analytically determined CO2 values. To prevent these CO2 discrepancies and their resultant effects on transformation and cloning efficiency, methods for monitoring the CO2 environment other than flow meters are recommended. The observation of increased cloning efficiencies and transformation rates strongly suggests that culture incubation at 20% CO2 is a preferred environment for the conduct of this assay.

Comparative neoplastic transformation responses of Balb/3T3 cells, Syrian hamster embryo cells, and Rauscher murine leukemia virus-infected Fischer 344 rat embryo cells to chemical compounds.

This study provides a preliminary comparative evaluation of the responses to a series of 49 chemicals, in in vitro transformation assays, of Balb/3T3 cells, Syrian hamster embryo cells, and Fischer 344 rat embryo cells infected with Rauscher murine leukemia virus. The chemicals assayed included aromatic amines; polycyclic aromatic hydrocarbons; alkylating agents; nitrosamines, hydrazines, and related compounds; heterocyclic compounds; amides, ureas, and acylating agents; inorganic compounds; and hormones. In all three assays 37 of the chemicals were tested. The most uniform test responses were obtained with the polycyclic aromatic hydrocarbons and inorganic compounds With the other groups of chemicals, more variation in response was observed. This study expands the base of information on the potential of these in vitro transformation systems, and the lack of responses with some of the chemicals underscores the need for incorporation of exogenous metabolic activating systems into these assay systems.

Chemical carcinogenesis in vitro: an improved method for chemical transformation in Rauscher leukemia virus-infected rat embryo cells.

In vitro assay for carcinogenesis, using mammalian cells, provide opportunity for rapid and inexpensive means, compared to in vivo assays, for studying carcinogenesis and for identifying potential carcinogens. These assays must, however, be shown to be reproducible, reliable and able to detect a variety of known carcinogens before they can be recommended for general use. We have, independently, reproduced a transformation assay which utilizes murine leukemia virus-infected rat embryo cells as targets. In the process a new culture, designated 2FR4(50), was generated to replace the F1706 line, of Freeman, which is no longer available. Through careful control of the assay parameters a readily reproducible test has been developed. In 2-4 culture passages after chemical treatment, morphologically transformed foci of cells are observed while no such foci are found in noncarcinogen treated or control cultures. Over 75 compounds have been tested in this assay; 20 of these are detailed here as representative of the chemically diverse types of carcinogens detected.

Two-stage carcinogenesis in vitro: transformation of 3-methylcholanthrene-initiated Rauscher murine leukemia virus-infected rat embryo cells by diverse tumor promoters.

A modified in vitro transformation assay demonstrated potential for use in the identification and study of tumor promoters. Rauscher murine leukemia virus-infected F344 rat embryo cells were transformable by various chemical carcinogens when they were administered at appropriate doses. Cells treated with subeffective doses of 3-methylcholanthrene did not transform. However, when these cells were regularly treated with 12-O-tetradecanoylphorbol 13-acetate, sodium phenobarbital, limonene, oleic acid, lauric acid, or saccharin, transformation was observed. Thus several dhemically diverse in vivo tumor promoters behaved as promoters in this in vitro system. This assay appeared to be useful for in vitro efforts to identify promoters and may be of value in studies on the mechanisms of action of these cocarcinogens.

Infection of rhesus monkeys and chimpanzees with Epstein-Barr virus.

Seventy-two nonhuman primates were entered into a long-term study to evaluate the pathogenicity of Epstein-Barr virus (EBV). Infectious virus was inoculated into 42 rhesus monkeys, 4 chimpanzees and 1 cynomolgus monkey. Immunostimulation or immunosuppression was attempted in 34 of these animals to enhance the oncogenic potential of the virus. Eleven inoculated animals were followed for more than 3 years and two were observed for 8 years. No tumors were observed in any of the animals; however, serological evaluation of the 47 inoculated primates and 25 matched controls indicated that at least 14 rhesus monkeys and the cynomolgus monkey were successfully infected with EBV. The potential use of rhesus monkeys as a model for EBV-induced disease in humans is discussed.

Productive Epstein-Barr viral infection of the human lymphoblastoid cell line 6410 with release of early antigen inducing and transforming virus.

The human lymphoblastoid cell line 6410 was superinfected with P3HR-1 derived Epstein-Barr virus. Subsequent to the first cycle of infection, characterized by early (EA) and virus capsid (VCA) antigen synthesis, the culture, designated 6410-EBV, continued to synthesize VCA. Further immunofluorescence and electron microscopic examination over 18-24 months showed the 6410-EBV culture to be productively infected with EBV. Virus was recovered, in quantity, from the culture fluids and assayed for ability to induce EA synthesis in Raji cells and to transform human umbilical cord lymphocytes. The virus was found to possess both properties, in contrast to P3HR-1 virus which only induces EA. HLA and karyologic analyses of P3HR-1, 6410 and 6410-EBV cultures showed relatedness of 6410-EBV to 6410 cells and dissimilarity to P3HR-1. The biologic activity data coupled with the cytologic analyses confirm a productive infection of the target cells. The reason for differences in biologic activity between the infecting (P3HR-1) and progeny virus is unresolved. It may be speculated that the endogenous EBV genome of 6410 cells was activated and gave rise to a different strain of EBV or to a mixed progeny-parent population as a result of dual infection. Alternatively, the parent strain of virus may have been modfied as a result of interaction with the new host cell.