Skin carcinogenicity of condensed asphalt roofing fumes and their fractions following dermal application to mice.

Condensed roofing asphalt fumes, generated at 316 degrees C, were collected by cold trap condensation and fractionated by preparative high performance liquid chromatography. Chemical classes in each of the fractions (A-E) were identified by gas chromatography/mass spectroscopy. The fractions, various combinations of fractions, the raw and heated asphalt, the neat asphalt fume and the reconstituted asphalt were tested for carcinogenicity, and three fractions were tested for cocarcinogenicity and tumor promotion with benzo[a]pyrene (BaP). The skin application carcinogenesis bioassay was conducted by twice weekly application of test materials in 0.05 ml of acetone/cyclohexane (1:1) for 104 weeks to 40 groups of male C3H/HeJ mice (30/group). Fractions were applied at a mass in proportion to their amount in the neat asphalt fumes. In addition, the neat asphalt fume was tested on Sencar mice to determine if this strain was more susceptible to the carcinogenic effects of the fumes. Condensed neat asphalt fumes produced similar and statistically significant increased tumor yields of papillomas and carcinomas in both strains as compared to respective vehicle controls. Recombination of all fractions resulted in a tumor response similar to neat asphalt fumes. Among individual fractions, C was most potent, followed by B. The other single fractions were without significant tumorigenic activity. Combinations containing fractions B and C were most active among the mixtures that were assayed and no evidence of enhancement of tumorigenesis in the mixtures was found. No significant cocarcinogenic or tumor promoting activity was observed with fractions A, D, or E and BaP. Raw unheated asphalt produced a few tumors in C3H mice, but no tumors were seen when raw asphalt heated to 316 degrees C, with the fumes permitted to escape, was applied.

Coteratogenic effects of caffeine.

Coteratogenicity studies have been carried out using various physical and chemical agents along with caffeine. For ionizing radiation in mice, enhancement of teratogenic responses (cleft palate, limb malformations) was noted with single systemic bolus doses of 50 to 200 mg/kg. Studies in rats with ethanol or nicotine reveal only an additive effect with caffeine. There are mixed results with chemical carcinogens and caffeine with some studies showing enhancement and others showing that caffeine inhibits the teratogenic effect of some carcinogens. The time of treatment, at the time of carcinogen exposure for the inhibition and later in the gestation period for the enhancement, appears to be the critical factor. For a variety of pharmaceutical agents (acetazolamide, mitomycin C, hydroxyurea, 5-fluorouracil), caffeine was shown to enhance the teratogenic effect of the agent. With 5-azacytidine in rats, caffeine suppressed limb malformations. Administration of inhibitors of beta-adrenergic function reduces the teratogenic effect of caffeine in mice. The interpretation of the experimental studies in terms of human hazard is complicated by the general use of high-dose bolus exposures which are not typical of human exposures, and the use of test systems that are not readily applicable to humans. The studies in human populations show clearly that caffeine itself has no link to negative birth outcome, and in the few instances where it has been examined there appears to be no interaction between coffee consumption and either alcohol consumption or smoking on pregnancy outcome.

International Commission for Protection Against Environmental Mutagens and Carcinogens. Power frequency electric and magnetic fields: a review of genetic toxicology.

Epidemiologic studies have reported a modestly increased risk of childhood leukemia associated with certain electric power wire configurations. Since cancer likely involves DNA damage, this review discusses the evidence of direct and indirect genetic toxicity effects for both electric and magnetic fields at 50- and 60-Hz and miscellaneous pulsed exposures. Exposure conditions vary greatly among different end points measured, making comparisons and conclusions among experiments difficult. Although most of the available evidence does not suggest that electric and/or magnetic fields cause DNA damage, the existence of some positive findings and limitations in the set of studies carried out suggest a need for additional work.

Interlaboratory evaluation of the C3H/10T1/2 cell transformation assay.

The C3H/10T1/2 transformation assay was evaluated for its responsiveness and interlaboratory reproducibility. Two laboratories participated in this study and tested a series of 46 chemicals. The majority of these chemicals were tested under code. Of the 46 chemicals tested, seven were determined to be active in both laboratories, and 14 were determined to be inactive. When the total number of chemicals is adjusted for assays considered "no test" in either one or both laboratories as well as for tests of chemicals yielding positive results in only one laboratory, reproducible responses were obtained for 21/35, or 60%, of the chemicals tested.

Rat liver foci and in vitro assays to detect initiating and promoting effects of chlorinated ethanes and ethylenes.

Nine chlorinated aliphatics (CAs) were examined in a rat liver foci assay for tumor initiating and promoting activities. In this model, young adult male Osborne Mendel rats were first subjected to a partial hepatectomy, the test chemical was then administered at the maximum tolerated dose in the initiation or promotion phase in conjunction with diethylnitrosamine (DEN; 30 mg/kg b.w.) or phenobarbital (PB; 0.05 percent, w/w, in the diet), and gamma glutamyltranspeptidase (GGT) was used as a putative preneoplastic indicator. When administered in the promotion protocol after initiation with DEN, 1,1-dichloroethane, 1,1,2-trichloroethane (1,1,2-TCE), 1,1,2,2-tetrachloroethane (1,1,2,2-TTCE), tetrachloroethylene (TTCY), and hexachloroethane induced significant increases in GGT+-foci above control levels. 1,1,2,2-TTCE, TTCY, and 1,1,2-TCE also induced significant increases in GGT+-foci when administered in the promotion protocol without DEN initiation. Two variants of GGT+-foci were observed: the classical type associated with PB promotion, and the other, which was more diffuse, less intensely stained, resembling foci undergoing redifferentiation and associated with CAs. A number of CAs were also genotoxic in short-term in vitro tests. Taken together, the studies suggest that CAs may be complete carcinogens in vivo with weak initiating activity and stronger promoting activity.

An interlaboratory evaluation of the Syrian hamster embryo cell transformation assay using eighteen coded chemicals.

Eighteen coded chemicals were evaluated in the Syrian hamster embryo (SHE) cell transformation assay in three different laboratories using the same basic experimental protocol with minor modifications. In addition, individual cell and serum sources were selected. Major factors influencing intra-and interlaboratory reproducibility were the source of cells and serum, the toxicity of the chemicals, and the dose-range selected for transformation evaluation. Two or three assays from each laboratory were required to determine the transformation-inducing potential of a chemical because of the low number of transformants scored in any single assay and the difficulty of interpreting morphological variations. Rodent carcinogenicity data were available for 16 of the 18 chemicals tested and the transformation response of 14 of those chemicals was in agreement with the rodent carcinogenicity data (if the positive results are adopted for the four chemicals that produced contradictory results). Four rodent carcinogens, di-(2-ethylhexyl) phthalate, diphenylhydantoin, methapyrilene hydrochloride and o-toluidine hydrochloride, that were negative in the Salmonella/microsome assay, induced morphological transformation in the SHE assay. Although the labour, cost and lack of reproducibility might preclude application of this transformation assay for routine screening, it might, nevertheless, prove valuable for distinguishing between non-mutagenic carcinogens and non-carcinogens.

An interlaboratory comparison of transformation in Syrian hamster embryo cells with model and coded chemicals.

Three independent laboratories tested eight "model" and five coded chemicals in the Syrian hamster embryo clonal transformation assay system to establish the intra- and interlaboratory reproducibility of the system and to identify sources of variability. When a common cell pool and the same lot of fetal calf serum were used, the three laboratories obtained consensus on the activity of eight model chemicals: five chemicals (benzo(a)pyrene, 7,12-dimethylbenz(a)anthracene, N-methyl-N'-nitro-N-nitrosoguanidine, nitroquinoline-N-oxide, and lead chromate) induced morphological transformation without exogenous metabolic activation and three (N-2-fluorenylacetamide, pyrene, and anthracene) produced no transformation response. Five coded chemicals (2,6-dichloro p-phenylenediamine, 4,4'-oxydianiline, cinnamyl anthranilate, dichlorvos, and reserpine), representative of environmental chemical classes, but not necessarily strong carcinogens, produced more equivocal responses in this interlaboratory study. Thus, while the assay can be used to distinguish between transforming and nontransforming chemicals in some cases, the intrinsic limitations in low transformation frequency and in achieving any dose-response results are major constraints to the use of this system in a routine testing program at the present time. Efforts to increase the transformation frequency or to amplify the expression of the transformed phenotype constitute some of the approaches which should be explored in order to overcome these limitations.

In vitro transformation of BALB/c-3T3 cells by chlorinated ethanes and ethylenes.

Eight chlorinated ethanes and 3 chlorinated ethylenes were tested in the BALB/c-3T3 cell transformation assay. Under the conditions of the assay, vinyl chloride and 1,1,1-trichloroethane induced a clear positive transformation response while 1,1,2-trichloroethane and trichloroethylene were weakly positive. Chloroethane, 1,1- and 1,2-dichloroethane, 1,1,1,2- and 1,1,2,2-tetrachloroethane, hexachloroethane and tetrachloroethylene were all negative in the assay conducted in the absence of an exogenous metabolic activation system. These results suggest that the BALB/c-3T3 cells possess capability to activate some, but not all, of the chlorinated hydrocarbons which exhibit species specificity in producing carcinogenicity in mice but not in rats.

Use of rodent hepatocytes for metabolic activation in transformation assays.

Although the evidence is not definitive, available information suggests that hepatocytes from appropriate rodent, and possibly other mammalian species, can be used to obtain metabolic activation in transformation assays that utilize cells with limited endogenous metabolic capabilities. On the basis of the available information, it appears that rat hepatocytes may be less suitable than hepatocytes from either mice or Syrian hamsters. In order to resolve these issues, it would be desirable to conduct a set of validation assays using: hepatocytes from several species, and possibly different mouse strains, a small selection of test chemicals from key chemical classes, and a reasonably facile and responsive assay system (BALB/c 3T3). This would provide the data needed to design one or more metabolic activation protocols for general use in screening transformation programs. In addition, the metabolic capacity of BALB/c 3T3 (3T3) cells to convert aniline-based amines to active carcinogens suggests that the application of genetic means to enhance the endogenous metabolic breadth of this or other cell strains, either by modulating control processes or adding genetic information, is a reasonable avenue of research.

Syrian hamster embryo cell transformation as a screening assay: sources of variability.

Our inter- and intralaboratory results showed that the SHE transformation assay is not yet sufficiently developed for a large scale screening program. The major source of variability is largely due to the low number of transformed colonies induced by test chemicals in any one experiment. Although this limitation presumably can be overcome by scoring a larger number of colonies, we feel such an approach would defeat the practical purpose of a screening assay. Of the experimental variables examined, we believe that medium supplements other than fetal calf serum and alternative detection/selection methods for transformation are two areas that need further development in order to improve the reproducibility of the SHE assay system for testing diverse chemicals. Until then, this system under defined conditions should remain a useful tool for research purposes.

Comparison of primary hepatocytes and S9 metabolic activation systems for the C3H-10T 1/2 cell transformation assay.

Three metabolic activation systems, primary rat hepatocytes, primary mouse hepatocytes and Aroclor 1254-induced rat liver S9 fraction were examined as exogenous activation systems for the C3H-10T 1/2 cell transformation assay. Under the conditions of the assay the primary mouse hepatocytes were more effective than the rat S9 fraction in mediating the transformation of C3H-10T 1/2 cells by the antineoplastic drug cyclophosphamide. However, the S9 fraction was more consistent than the mouse hepatocytes in the activation of dimethylnitrosamine. The primary rat hepatocytes were ineffective for activating either cyclophosphamide or dimethylnitrosamine in the transformation of C3H-10T 1/2 cells. The presence of mouse hepatocytes, but not the S9 fraction, inhibited transformation of C3H-10T 1/2 cells by 3-methylcholanthrene. These results demonstrate that the three systems were differentially effective in the activation of procarcinogens.

Cell transformation by chemical agents--a review and analysis of the literature. A report of the U.S. Environmental Protection Agency Gene-Tox Program.

The literature on cell transformation by chemical carcinogens has been critically reviewed. This subject is highly relevant to carcinogenesis in vivo, because the phenotypic changes that are collectively referred to as cell transformation usually involve the acquisition of tumorigenicity on inoculation into suitable rodent hosts. The systems chosen for review fall into 3 categories: cell strains (cells with a limited lifespan); cell lines (cells with an unlimited lifespan); and oncogenic viral-chemical interactions involving cells (Fischer rat embryo cells expressing an endogenous retrovirus, mouse embryo cells expressing the AKR leukemia virus, chemical enhancement of a simian adenovirus, SA7 transformation of Syrian hamster or rat embryo cells). Of the entire literature reviewed, 117 papers have been accepted for data abstraction by pre-defined criteria; these include 41 references to cell strains, 40 in cell lines, and 38 in viral-chemical interactions including cells. Because different systems have been reviewed, it would be meaningless to group all the compounds. The overall summary of the systems is as follows (many compounds have been tested in more than one system and, hence, are duplicated in these totals). (Chart: see text) In general, there is a reasonably good correlation between the results of the cell transformation systems and in vivo carcinogenesis. However, the many deficiencies of the EPA Merged Carcinogen List preclude definitive comparisons. Moreover, a number of 'false negatives' were obtained in systems that did not employ external metabolic activation. Further validation of all systems is required, but it seems very probable that several cell transformation systems will become valuable in assaying (with reasonable time and cost) the carcinogenic potential of environmental chemicals.

Variations among species and cell types in the effects of caffeine on mutagen-induced cytotoxicity and postreplication repair of DNA.

The influence of caffeine on cytotoxicity and postreplication repair of DNA was examined following exposure of several cell types to physical and chemical agents known to damage DNA. The cell types used in this study were normal human fibroblasts (HS-WP), human xeroderma pigmentosum fibroblasts (SGL), Chinese hamster V79 cells, mouse BALB/c-3T3 cells, and secondary Syrian hamster embryo cells. The DNA damaging agents were ultraviolet light (UV), N-2-acetoxy-fluorenylacetamide (AFAA), nitroquinoline-N-oxide (NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Induction of cytotoxicity in Chinese hamster V79 cells due to ultraviolet light or AFAA exposure was enhanced by caffeine at a concentration of 1.0 mM in the culture medium, but not at 0.2 or 0.05 mM. Caffeine also inhibited postreplication repair in these cells at the same concentrations. In contrast, postreplication repair was not affected by caffeine at concentrations up to 1.0 mM in normal human fibroblasts (HS-WP), human xeroderma pigmentosum fibroblasts (SGL), secondary Syrian hamster embryo cells, and mouse BALB/c-3T3 cells following treatment with ultraviolet light, AFAA, NQO, or MNNG. Cytotoxicity in BALB/c-3T3 cells following exposure to ultraviolet light or AFAA was enhanced in the presence of caffeine at 1.0 or 0.2 mM, although these concentrations of caffeine had no effect on postreplication repair in these cells. The inhibitory effect of caffeine on postreplication repair was found only in Chinese hamster V79 cells among the five cell types used in this study. Both normal and xeroderma pigmentosum human cells repaired mutagen-induced DNA damage equally well in the absence or presence of caffeine at concentrations of 1.0 mM or less.

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.

Cell culture tumor promotion experiments with saccharin, phorbol myristate acetate and several common food materials.

The BALB/c-3T3 cell neoplastic transformation system was modified to examine the tumor promoting activity of a set of substances. Following initiation of the target cells with 3-methylcholanthrene, treatment of the cultures with phorbol myristate acetate (0.01 microgram/ml; 1.5 X 10(-8) M) during the remainder of the 4-week assay interval resulted in a marked increase in both spontaneous and initiated Type III transformed foci. In contrast, a similar treatment with saccharin at 20, 100 or 500 microgram/ml (0.08, 0.4 or 2.1 X 10(-3) M) did not influence the occurrence of Type III transformed foci and did not result in a promoting response. Sodium ascorbate (2.53 X 10(-3) M) and L-tryptophan (2.45 X 10(-3) M) almost completely inhibited both spontaneous and initiated Type III transformed foci. Calcium pantothenate (2.10 X 10(-3) M) exhibited a marginal promoting effect. Under the conditions of this study in which the classical tumor promoter phorbol myristate acetate was highly active in promoting Type III transformed foci, saccharin was not active as either a direct transforming or promoting agent at doses up to 5 orders of magnitude higher.

Overview and status of in vitro transformation.

The development of standardized assay procedures has permitted the exploitation of cell culture systems as bioassay tools for the detection of chemical carcinogens. These systems fall generally into 3 classes: diploid cell strains, Syrian hamster embryo cells; cell lines, mouse BALB/c-3T3 and mouse C3H-10T1/2; and cells+virus, Fischer rat cells infected with Rauscher leukemia virus and Syrian hamster embryo cells infected with adenovirus. The results accumulated to date show a good correlation between transformation response in cell culture and carcinogenicity of chemicals in whole animal studies. The major advantages of these systems are their relative brevity (10 days-6 weeks) and resultant low costs, their agreement with whole animal bioassays, and their direct biological relevance to the carcinogenic process. The present major disadvantages are the uncertain nature of the metabolic capabilities of the target cells and the lack of a metabolic activation system that is reliable and adaptable for routine bioassays. The development of epithelial cell systems such as breast, liver, lung, and skin may solve the problem of carcinogen metabolism as well as provide target cells that are representative of major organ sites for cancer in man. The rational use of cell culture bioassays for neoplastic transformation is a valuable component of the toxicological armamentarium to assess risk to humans from exposure to chemicals.