Application of process chemistry and SAR modelling to the evaluation of health findings of lower olefins.

Epidemiology studies show increased leukemia mortality among styrene butadiene rubber (SBR) workers but not among butadiene monomer production employees. A detailed review of the SBR manufacturing process indicates that sodium dimethyldithiocarbamate (DMDTC) introduced into the SBR manufacturing process for a period in the 1950s coincides with increased leukemia mortality. Using the Computer-Optimized Molecular Parametric Analysis of Chemical Toxicity (COMPACT), we assessed the enzyme (cytochrome P450) substrate specificity of an olefin series including 1,3-butadiene (BD) and also modeled its interaction with DMDTC. These analyses showed correlation of a structural/electronic parameter--the COMPACT radius--with the presence or absence of cytogenetic activity and also found that DMDTC would inhibit the oxidative metabolism of BD at least at high concentrations. Both DMDTC and its diethyl analog (DEDTC) bind with CYP 2E1 and CYP 2A6. Both of these isoforms are important in the initial oxidative metabolism of butadiene and other olefins. In co-exposure studies in mice of DMDTC with BD or with epoxybutene (EB), we found that there was a reduced increase in genotoxic activity based on micronuclei induction compared with BD or EB exposure alone. Treatment with DMDTC significantly increased the protein carbonyl contents of hepatic microsomes compared with that of controls, a finding that may be related to DMDTC's activity as a prooxidant. Co-exposure with DMDTC and EB increased hepatic microsomal carbonyls to levels significantly greater than those of DMDTC-treated mice, while EB administration in the absence of DMDTC did not change protein carbonyls relative to those of controls. The increase in hepatic microsomal protein carbonyls suggests that DMDTC may modulate EB metabolism towards the formation of reactive intermediates that react with proteins. The present molecular modeling and mechanistic studies suggest that co-exposure of BD and DMDTC is a plausible biological hypothesis regarding increased leukemia risk among SBR workers.

Results of the L5178Y mouse lymphoma assay and the Balb/3t3 cell in vitro transformation assay for eight phthalate esters.

Eight phthalate esters, with alcohol chain lengths of 1-11 carbon atoms and with various degrees of branching, were tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay and in the Balb/3T3 cell transformation assay. The tests were performed as part of a voluntary testing agreement between the Chemical Manufacturers Association's Phthalate Esters Panel and the United States Environmental Protection Agency (US EPA). The esters tested were: dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), di-¿n-hexyl, n-octyl, n-decyl¿ phthalate (610P), di-isononyl phthalate (DINP), di-¿heptyl, nonyl, undecyl¿ phthalate (711P), di-isodecyl phthalate (DIDP) and di-undecyl phthalate (DUP). Both DMP and DBP were found to produce significant increases in the mutant frequency in the mouse lymphoma assay in the presence but not in the absence of an Aroclor-induced rat liver activation system (S-9). Ester 610P gave equivocal results in the mouse lymphoma assay in the presence and absence of rat liver S-9. There was no indication of mutagenic potential for any of the other test materials in the mouse lymphoma assay, and none of the test materials increased transformation frequency in the Balb/3T3 cell transformation assay. Aldehyde metabolites of the de-esterified alcohols are postulated to play a role in the positive results for DMP and DBP.

Di(isononyl) phthalate (DINP) and di(isodecyl) phthalate (DIDP) are not mutagenic.

Recently there have been reports of liver and kidney tumors in rodents following long-term exposure to di(isononyl) phthalate (DINP). Mechanistic studies suggested that the liver tumors were a consequence of peroxisomal proliferation, whereas the kidney tumors (found only in male rats) were associated with induction of alpha(2u)-globulin. Because both peroxisomal proliferation and alpha(2u)-globulin are considered to be non-genotoxic carcinogenic processes, it seemed appropriate to investigate the genotoxic potential of DINP. Additional studies were also conducted on di(isodecyl) phthalate (DIDP), a structurally related substance that also induces peroxisomal proliferation, although it has not been tested in a carcinogenicity bioassay. The DINP was tested in Salmonella, in vitro cytogenetics and mouse micronucleus assays, whereas DIDP was evaluated in a mouse micronucleus test. All of these tests produced negative results, i.e. neither phthalate was mutagenic in any of the test systems. These data are consistent with results of other published and unpublished genotoxicity tests and provide support for the hypothesis that the liver and kidney tumors induced by DINP were the result of non-genotoxic processes.

Assessment of the utility of the micronucleus test for petroleum-derived materials.

The micronucleus test is a commonly used in vivo assay for chromosomal damage and is an integral part of many mutagenicity testing strategies. The present report describes an assessment of the micronucleus test for the detection of mutagenic potential of petroleum-derived materials. To this end, studies were conducted with catalytically cracked clarified oil (CCCO). This material contains high levels of polycyclic aromatic constituents (PAC) and is a very potent inducer of mouse skin tumors. CCCO is also active in the Salmonella assay and other in vitro tests. As CCCO is the most potent of the various petroleum-derived materials in other assays, it was assumed to be the most easily detectable in the micronucleus test. CCCO was tested in standard mouse micronucleus tests utilizing oral and intraperitoneal injection for test material administration. All of these studies were negative, although DMBA, tested at roughly equivalent levels based on potency in the Salmonella assay, produced statistically significant increases in micronucleus frequency. In a second series of studies, aromatic fractions of CCCO were prepared and tested at up to acutely toxic levels. Results of these studies were also negative. Finally, another petroleum-derived material which is carcinogenic and contained PAC was tested in the micronucleus assay. It also produced negative results. Thus, it was concluded that petroleum-derived materials do not produce clastogenic effects in vivo in the mouse micronucleus test, despite the fact that some pure polycyclic aromatic hydrocarbons are quite active in this assay.

Toxicology of n-pentane (CAS no. 109-66-0).

n-Pentane (CAS no. 109-6-0) is a hydrocarbon solvent with an estimated production volume of 50000 metric tons in Europe. The present work was carried out to strengthen the toxicological information available for regulatory decision-making, particularly product classification, occupational exposure limits and risk assessment. The work described in this report was sponsored either by the Pentane Special Interest Group under the auspices of the Hydrocarbon Solvent Producers Association of CEFIC (European Chemical Industry Council) or by Exxon Chemical Europe. The following results were obtained: n-pentane was not acutely toxic by oral or inhalation routes, it was not a skin or eye irritant and did not induce skin sensitization. It did not exhibit cumulative toxicity at levels up to 20000 mg m(-3), which is approximately one-half of the lower explosive limit and the highest level considered safe to test. It did not induce developmental toxicity and was not mutagenic. From this and other existing information, it is concluded that n-pentane does not require classification for potential toxic hazards (as defined by Annex VI of the EU Dangerous Substance Directive, 1993), although the physical properties indicate that it would be appropriate to warn for the potential for aspiration. Further, there is no reason to reduce the current occupational exposure recommendations. Finally, although there are some safety issues relating to the flammability of n-pentane, the absence of any demonstrable toxicity at high treatment levels indicates that the risk of adverse health effects is minimal for all segments of the population.

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.

Increased frequency of resistance to terminal differentiation in C3H mouse cells produced by genotoxic but not nongenotoxic carcinogens.

Certain cells present in mouse skin are resistant to calcium-induced terminal differentiation. It is believed that these calcium-resistant cells (CRCs) represent an early stage in the carcinogenic process, in part, because frequency increases after treatment with mutagens. The frequency of CRCs in C3H mouse skin was measured before and after treatment with certain petroleum-derived materials. One objective was to determine whether this assay could differentiate between genotoxic and nongenotoxic mouse skin carcinogens. An additional objective was to determine whether CRCs are an important factor in the tumorigenicity of petroleum middle distillates (PMDs), a class of apparently nongenotoxic mateials. Three petroleum-derived materials were tested: mineral oil (MO), a noncarcinogenic product used as the negative control; catalytically cracked clarified oil (CCCO), a highly carcinogenic and mutagenic material; and a lightly paraffinic (LRPO), a PMD which has produced tumors when repeatedly applied, but is not mutagenic and does not initiate most skin tumors. The CRC frequency was not increased by LRPO treatment; however, a statistically significant and dose-related increase was produced by CCCO. These results are consistent with observations that genotoxic, petroleum-derived liquids are capable of tumor initiation in mouse skin, whereas PMDs which are not genotoxic do not initiate skin tumors. The number of CRCs in untreated and MO-treated mice was approximately twice the tumor frequency measured in bioassays of PMDs. Thus, tumor production associated with these products could be due to promotion of preexisting, spontaneously initiated cells.

Evaluation of the genetic toxicity of middle distillate fuels.

Petroleum middle distillate (PMD) fuels are mixtures of hydrocarbons that distill between approximately 170-370 degrees C. Commercial products that fall into this category include kerosine, diesel fuel, jet fuel, and home heating oil. These products contain both saturated (paraffins and cycloparaffins) and aromatic species, but because of the boiling range normally contain very small amounts of the 3-6 ring polycyclic aromatic hydrocarbon (PAH) constituents, which are considered to be carcinogenic. Nevertheless, there is evidence of weak tumorigenic activity when these materials are repeatedly applied to mouse skin. In the current studies representative products were tested in two commonly used, short-term assays for genetic toxicity, the Salmonella/mammalian microsome mutagenicity assay and the mouse bone marrow micronucleus test. All samples were inactive in the micronucleus assay, and three were clearly inactive in the Salmonella test. Of the remaining two, one was marginally active in the Salmonella assay, and one was equivocal. The marginally active sample contained detectable levels of PAH due to the use of catalytically cracked materials as blending stocks. The results indicated that PMDs that do not contain cracked material were not mutagenic. Thus they may produce tumors via nongenotoxic processes. Those products that do contain cracked stocks may have sufficient PAH to be mutagenic in the Salmonella assay, and in those cases the PAH might also contribute to tumor formation.

The carcinogenic initiating and promoting properties of a lightly refined paraffinic oil.

The dermal carcinogenic potential of some petroleum-derived liquids is related to the polycyclic aromatic hydrocarbon (PAH) content. However, repeated application of middle distillates (MDs), e.g., kerosene, diesel fuel, and heating oil, produced tumors in mouse skin. This result was unexpected since the MDs typically contain very low levels of biologically active PAHs. The present study examined the tumorigenic mechanism of a lightly refined paraffinic oil (LRPO), an MD shown to be active in mouse skin. The LRPO was separated into saturated and aromatic fractions. Whole LRPO and various fractions were tested for mutagenic activity in the Salmonella assay and for carcinogenic initiating and promoting activity. There was no evidence that any of the samples examined were mutagenic in bacteria or carcinogenic initiating agents in mouse skin. Thus no support was provided for the hypothesis that the complete tumorigenic activity of LRPO was in any way related to the presence of low levels of PAHs or to an interaction between initiating and promoting constituents. There was evidence that LRPO was a weak promoter of dimethylbenzanthracene (DMBA)-initiated mouse skin. It was also found that repeated application of LRPO produced chronic irritation and hyperplasia, and this may have been responsible for the promotional effects. Based on these data, it seemed likely that the complete carcinogenic activity of this class of products is also the result of an epigenetic process related to skin irritation.

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.