Results of chronic dietary toxicity studies of high viscosity (P70H and P100H) white mineral oils in Fischer 344 rats.

Two-year dietary studies were conducted to determine the chronic toxicity and its reversibility, and the carcinogenicity of P70(H) and P100(H) white mineral oils in Fischer-344 rats (F-344). The studies were identical in design and followed the Organization for Economic Cooperation and Development, Guidelines for Testing Chemicals, Guideline 453, 1981. Additional endpoints evaluated were: (1) extent of mineral hydrocarbon deposition in liver, kidneys, mesenteric lymph nodes, and spleen of female rats at 3, 6, 12, 18 and 24 months, and (2) reversibility of effects following cessation of exposure. Dietary concentration were 60, 120, 240, and 1,200 mg/kg/day, adjusted periodically to account for bodyweight changes. Study results were consistent with preceding subchronic studies. No treatment-related mortality, neoplastic lesions, or changes in clinical health, hematology, serum chemistry, or urine chemistry were evident in any group administered either white oil. Statistically significant higher food consumption was noted in the 1,200 mg/kg group males and females exposed to either white oil and statistically significant higher body weights were noted in the 1,200-mg/kg males during the latter portion of the P100(H) study. Higher mesenteric lymph node weights were accompanied by increased severity of infiltrating histiocytes. This occurred to a greater extent with the P70(H) than the P100(H) oil. No other histopathology of significance was observed. Mineral hydrocarbons were detected in the liver following exposure to either oil. Maximal concentrations of mineral hydrocarbons in the liver were similar with both oils but occurred more rapidly with the P70(H) oil. Liver mineral hydrocarbon content returned to near-background levels during the reversibility phase. In conclusion, lifetime exposer of F344 rats to P70(H) and P100(H) white oils resulted in only minimal findings and with no consequence to clinical health. Thus, under the conditions of these studies, the No Observable Adverse Effect Level (NOAEL) for these studies was considered to be 1,200 mg/kg/day.

Assessment of bioconcentration and secondary poisoning of surfactants.

The relevance of the bioconcentration behaviour of surfactants for the secondary poisoning assessment and for the risk characterisation in the bird and mammalian food chain has been investigated. The approach used is described in the recently revised EU Technical Guidance Document for the Risk Assessment of Substances. The results demonstrate that, based on experimentally derived bioconcentration factors, environmental concentrations and effects in animals, there is a clear level of safety for both linear alkylbenzene sulphonate (LAS) and alcohol ethoxylates (AE), the most important surfactants by volume. To assess other surfactants used in detergents, a bioconcentration factor that would need to be attained for secondary poisoning to be of concern has been estimated from predicted environmental concentrations and known long-term effects data in animals. Based on the known structural similarity of these surfactants to LAS and AE and the ubiquitous nature of the enzymatic systems that are present in biotransformation processes in organisms, it is concluded that bioconcentration of these surfactants to these levels is highly unlikely. Therefore the potential for secondary poisoning effects of these surfactants is extremely low.

Two-generation reproduction toxicity studies of di-(C(7)-C(9) alkyl) phthalate and di-(C(9)-C(11) alkyl) phthalate in the rat.

Di-(C(7)-C(9) alkyl) phthalate (D79P) and di-(C(9)-C(11) alkyl) phthalate (D911P), based on high-normality linear oxo-alcohols, have been assessed for their impact upon reproductive performance in Sprague-Dawley rats. Rats were continuously exposed to either D79P or D911P at dietary levels of 0%, 0.1%, 0.5%, or 1.0% over two generations. Selected F(0) offspring (F(1) generation) were exposed to the same dietary concentration of D79P or D911P as the respective F(0) animals, and were mated to produce F(1) offspring. Both D79P and D911P markedly reduced body weight gain in F(0) and F(1) adult males at the highest dose, but females were affected to a lesser extent. There was no impairment of fertility, fecundity, or development in either generation, but body weights of offspring in the 1.0% D79P and 1.0% D911P groups were slightly and transiently reduced over the weaning period. Although decreases in the weight of several organs were accounted for by depressed body weight, ovary weights were reduced in both generations exposed to 1.0% D79P, and epididymidal weights were slightly reduced in adults of both generations exposed to 1.0% D911P. However, ovarian function-assessed by the oestrus cycle and mating behaviour-and epididymidal sperm concentration, motility, and morphology were unaffected by either substance. Treatment resulted in liver changes, particularly in males, characterised by increased liver weight in young animals, histopathologic changes and reduced organ weight in mature animals, and an increase in palmitoyl CoA oxidase activity. In conclusion, neither D79P nor D911P impaired reproductive function in rats when administered in the diet at levels that induce systemic toxicity, and the NOAEL for effects on reproduction in the rat is 0.5% for both D79P and D911P.

Assessment in rats of the reproductive toxicity of gasoline from a gasoline vapor recovery unit.

Gasoline (CAS 86290-81-5) is one of the world's largest volume commercial products. Although numerous toxicology studies have been conducted, the potential for reproductive toxicity has not been directly assessed. Accordingly, a two-generation reproductive toxicity study in rats was conducted to provide base data for hazard assessment and risk characterization. The test material, vapor recovery unit gasoline (68514-15-8), is the volatile fraction of formulated gasoline and the material with which humans are most likely to come in contact. The study was of standard design. Exposures were by inhalation at target concentrations of 5000, 10 000, and 20 000 mg/m(3). The highest exposure concentration was approximately 50% of the lower explosive limit and several orders of magnitude above anticipated exposure during refueling. There were no treatment-related clinical or systemic effects in the parental animals, and no microscopic changes other than hyaline droplet nephropathy in the kidneys of the male rats. None of the reproductive parameters were affected, and there were no deleterious effects on offspring survival and growth. The potential for endocrine modulation was also assessed by analysis of sperm count and quality as well as time to onset of developmental landmarks. No toxicologically important differences were found. Therefore, the NOAEL for reproductive toxicity in this study was > or =20 000 mg/m(3). The only systemic effects, in the kidneys of the male rats, were consistent with an alpha-2 u-globulin-mediated process. This is a male rat-specific effect and not relevant to human health risk assessment.

Studies on the dermal and systemic bioavailability of polycyclic aromatic compounds in high viscosity oil products.

The assessment of skin penetration by viscous oil products is an important element in the risk assessment of these materials where skin contact is likely. Systemic bioavailability (body uptake) is viewed as a good indicator of skin penetration following cutaneous exposures. The results of this study provide quantitative information on the influence of viscosity on the bioavailability of a specific polycyclic aromatic compound (benzo(a)pyrene) in base oils, residual aromatic extracts and bitumens following skin exposures to mice. The materials studied were a base mineral oil (viscosity 32 cSt at 35 degrees C), a 1:1 blend of the mineral base oil and a residual aromatic extract (198 cSt), several residual aromatic extracts (ca. 5000 cSt, 35 degrees C) and a range of bitumens (0.65-69 x 10(6) cSt, 35 degrees C). These were each spiked with 0.1% radiolabelled benzo(a)pyrene, as a representative carcinogenic polycyclic aromatic compound, then used for cutaneous exposures to mice. The results indicate that as viscosity increased in the range ca. 30 to 5000 cSt (base oil to residual aromatic extract) the uptake of the radiolabelled benzo(a)pyrene into blood was reduced by ca. fivefold. Further increases in viscosity from ca. 5000 to 69 x 10(6) cSt (i.e. residual aromatic extract to bitumen) resulted in a further but smaller (ca. twofold) reduction in uptake. The relationship between the amounts of free benzo(a)pyrene measured in blood and viscosity showed the same trend. This trend was also mirrored by the degree of binding of benzo(a)pyrene metabolites to DNA in skin. The findings in mouse skin in vivo indicate that viscosity can significantly affect skin penetration and systemic bioavailability of polycyclic aromatic compound components of oil products. Results obtained with viable human skin in vitro also showed that the bioavailability of benzo(a)pyrene was reduced by the viscosity of the oil product matrix. It is thus necessary to take account of physical properties such as viscosity in the overall risk assessment of viscous oil products, particularly in the case of very viscous materials such as bitumens. The significantly reduced bioavailability of hazardous compounds from undiluted materials is thus an important factor to consider when assessing the risks from dermal exposures.

A comprehensive evaluation of the mechanism of skin tumorigenesis by straight-run and cracked petroleum middle distillates.

The role of skin irritation and other factors on the tumorigenic activity of petroleum middle distillates (PMDs) in mice was examined in a comprehensive research program. The program culminated in a 2-year dermal carcinogenicity study which compared the effects of equal weekly doses of irritating and nonirritating PMDs. Modified Ames mutagenicity studies and three- to seven-ring polycyclic aromatic compound (PAC) analyses indicated that the mutagenic activity of PMDs was correlated to PAC content. In subchronic and subacute studies, PMDs produced marked skin irritation which was ameliorated if the test samples were diluted in mineral oil. The reduction in irritation level was not a result of reduced dermal absorption. Straight-run kerosine (SRK), straight-run gas oil (SRGO), and catalytically cracked light cycle oil (LCO) were evaluated in the dermal carcinogenicity study. Test materials were applied either undiluted (2x/week) or as 28.5% (7x/week) or 50% (4x/week) concentrations in mineral oil for a total weekly dose of 100 microliters PMD per animal. All three materials produced moderate to marked skin irritation and increased tumor frequency when applied undiluted. When diluted, the irritant effects of SRK and SRGO, which contain low levels of PACs, were ameliorated, and there were no significant increases in tumors relative to controls. LCO, containing 8.7% three- to seven-ring PACs, increased tumor frequency when diluted, even when skin irritation was limited. These data indicate that the tumorigenic activity of straight-run MDs is likely a consequence of a nongenotoxic process, associated with frequent cell damage and repair. PMDs which contain low levels of three- to seven-ring PACs are unlikely to cause tumors in the absence of prolonged skin irritation. In addition, genotoxic mechanisms may also contribute to tumor formation for other PMDs containing higher levels of PACs, e.g., products blended with cracked stocks.

Ninety-day feeding study in Fischer-344 rats of highly refined petroleum-derived food-grade white oils and waxes.

Subchronic 90-day feeding studies were conducted in male and female Fischer-344 (F-344) rats on highly refined white mineral oils and waxes representative of those used for food applications. The goal was to help clarify the mixed results found in other toxicity studies with laboratory animals. Seven white oils and 5 waxes were fed at dietary doses of 20,000, 2,000, 200, and 20 ppm and compared with control groups on untreated diet; toxicity was assessed at 90 days and also after a reversal period of 28 days and/or 85 days. Higher molecular-sized hydrocarbons (microcrystalline waxes and the higher viscosity oils) were without biological effects. Paraffin waxes and low- to midviscosity oils produced biological effects that were inversely related to molecular weight, viscosity, and melting point; oil type and processing did not appear to be determinants. Biological effects were more pronounced in females than in males. Effects occurred mainly in the liver and mesenteric lymph nodes and included increased organ weights, microscopic inflammatory changes, and evidence for the presence of saturated mineral hydrocarbons in affected tissues. Inflammation of the cardiac mitral valve was also observed at high doses in rats treated with paraffin waxes. Further studies are required to elucidate the mechanism for the responses observed and the relevance of these inflammatory responses in the F-344 rat to other species, including humans.

Evaluation of modified bacterial mutagenicity assays for the genotoxicity testing of mineral oils.

A modified bacterial mutagenicity assay based on the Ames Salmonella/mammalian microsome test has been developed for application in the genotoxicity testing of mineral oils. The assay uses washed microsomes from rat liver in place of S9 fraction in order to increase the sensitivity of detection of genotoxicity. The modified assay was used to test a series of oils for which skin carcinogenicity bioassay data in mice were available. Oils were tested as emulsions in water using Tween 80 as a dispersant. A mutagenicity index for each oil was obtained using non-linear regression analysis of data from the dose-response curve. The results showed an empirical correlation between increasing mutagenicity index, carcinogenicity and the polycyclic aromatic hydrocarbon content of the oils. The washed-microsome assay was also compared with modified Ames assays developed by Blackburn et al. (Cell Biol. Toxicol., 1, 40, 1984; Cell Biol. Toxicol., 2, 63, 1986) which employed increased levels of S9 (rat and hamster liver) to test dimethyl sulphoxide extracts of oils. The washed-microsome assay can be used for the testing of whole oils rather than extracts which are necessary for the modified Ames assay. It is recognised that the determinants of carcinogenic activity in vivo include promoting activity which such assays are unable to detect. Nevertheless, such modified bacterial assays may be a useful prescreen since genotoxicity is recognised as a key initial step in carcinogenesis.

Feeding studies in rats with mineral hydrocarbon food grade white oils.

This investigation compared the effects of feeding rats diets containing food grade white oil processed by either conventional oleum treatment or the more modern method of catalytic hydrogenation. In two separate experiments, male or female Fischer-344 rats were given free access for 90 days to diets containing 0, 10, 100, 500, 5,000, 10,000, or 20,000 ppm of either oleum-treated white oil (OTWO) or hydrotreated white oil (HTWO). There were no mortalities and no adverse clinical signs associated with feeding either white oil. Treatment-related effects evidenced by hematological, clinical chemical, and pathological changes were generally dose-related and more marked in female than in male rats, and the OTWO caused a greater pathological response than the HTWO. Tissue residues of saturated hydrocarbons were up to 5.2 times higher in female rats than in males. Rats fed 5,000 ppm or more of either white oil showed dose-related alterations in several hematological and clinical chemistry variates associated mainly with hepatic damage or functional alteration. At necropsy, mesenteric lymph nodes were enlarged, and increases in weight of liver, kidney, and spleen were significant. Microscopic changes were characterized by multifocal lipogranulomata in mesenteric lymph node and liver. No changes were observed in rats fed OTWO or HTWO for 90 days at dietary concentrations of 10 or 100 ppm, equivalent to a minimum intake of 0.65 and 6.4 mg/kg/day, respectively. Differences in degree of pathological response associated with each oil may have been due to their differences in specification rather than processing method.

Assessment of the potential germ cell mutagenicity of industrial and plant protection chemicals as part of an integrated study of genotoxicity in vitro and in vivo.

An approach is described that enables the germ cell mutagenicity of chemicals to be assessed as part of an integrated assessment of genotoxic potential. It is recommended, first, that the genotoxicity of a chemical be defined by appropriate studies in vitro. This should involve use of the Salmonella mutation assay and an assay for the induction of chromosomal aberrations, but supplementary assays may be indicated in specific instances. If negative results are obtained from these 2 tests there is no need for the conduct of additional tests. Agents considered to be genotoxic in vitro should then be assessed for genotoxicity to rodents. This will usually involve the conduct of a bone marrow cytogenetic assay, and in the case of negative results, a genotoxicity test in an independent tissue. Agents found to be non-genotoxic in vivo are regarded as having no potential for germ cell mutagenicity. Agents found to be genotoxic in vivo may either be assumed to have potential as germ cell mutagens, or their status in this respect may be defined by appropriate germ cell mutagenicity studies. The basis of the approach, which is supported by the available experimental data, is that germ cell mutagens will be evident as somatic cell genotoxins in vivo, and that these will be detected as genotoxins in vitro given appropriate experimentation. The conduct of appropriate and adequate studies is suggested to be of more value than the conduct of a rigid set of prescribed tests.

Effect of mutation on the virulence in mice of a strain of foot-and-mouth disease virus.

Twenty-eight mutations, representing mutation in five different polypeptide-coding regions of the foot-and-mouth disease genome, were examined for their effect on the virulence of the virus for suckling mice. Five types of mutation were examined: temperature-sensitive (ts), electrophoretic (e), co-variant temperature-sensitive and electrophoretic (ts/e), guanidine-resistant (gs+) and putative co-variant guanidine-resistant and electrophoretic (gs+/e). All the ts mutations and three out of the 11 non-ts mutations produced some reductions in virulence. In the majority of cases this reduction in virulence was shown to co-vary with the mutation. No correlation was observed between the site of a mutation or its 'cut-off' temperature and the extent of the reduction in virulence. Studies of the growth in vivo of a small selection of ts mutants suggested that for most mutants their reduced virulence was a trivial effect of their slow growth rate. With one exception they all eventually grew to parental virus levels, the resulting virus being temperature-sensitive and the disease indistinguishable from that caused by the parental virus. The one exception was an avirulent ts mutant which only grew to one-thousandth the titre of the parent virus. This mutant did not cause disease and was therefore considered to be the only avirulent mutant. Its mutation was in the coat protein-coding region of the genome, probably the region coding for VP3.

An extended genetic recombination map for foot-and-mouth diseases virus.

The original foot-and-mouth diseases virus recombination map (Lake, Priston & Slade, 1975), which include 35 mutagen-induced ts mutants, has been extended both in detail and size by the mapping of a further 33 ts mutants (9 mutagen-induced and 24 spontaneous). The size increase from 0.57% to 3.27% maximum recombination fequency was principally due to the use of a new standardization technique for recombination fequencies but, in addition, the original map distance was increased by approx. 30% due to the mapping of new mutations. As in the original map, there was a marked concentration of mutations near the guanidine (gs) locus, i.e. 83% of the mutants had mutations in the third of the map adjacent to the gs locus.

Temperature-sensitive mutants of foot-and-mouth disease virus: the isolation of mutants and observations on their properties and genetic recombination.

A number of temperature-sensitive mutants were isolated from two strains of foot-and-mouth disease virus (FMDV). Various properties of the mutants were examined including comparative growth curves at permissive and restrictive temperatures, cut-off temperatures, thermal lability and pH sensitivity. Recombination was observed between various pairs of mutants of FMDV strain Pacheco. It occurred early in the growth cycle and the proportion of recombinants remained constant thereafter. Maximum recombination was achieved if the input multiplicity of each virus was 6 p.f.u./cell or greater, provided the ratio of the input multiplicities did not vary by more than a factor of two. Day-to-day variations could be substantially reduced by normalizing recombination frequencies in terms of a standard cross. The results suggested that genetic mapping was possible with two-or three-factor crosses.