Classification of skin sensitizing substances: a comparison between approaches used by the DFG-MAK Commission and the European Union legislation.

A systematic classification of substances (or mixtures of substances) with regard to various toxicological endpoints is a prerequisite for the implementation of occupational safety strategies. As its principal task the "Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area" of the "Deutsche Forschungsgemeinschaft" (DFG-MAK Commission) derives and recommends maximum workplace concentrations and biological tolerance values (MAK and BAT values) based exclusively on scientific arguments. Several endpoints are evaluated separately in detail, e.g. carcinogenicity, risks during pregnancy, germ cell mutagenicity or contribution to systemic toxicity after cutaneous absorption. Skin- and airway sensitization is also considered; the present paper focuses on these two endpoints.

A toxicologic and dermatologic assessment of cyclic acetates when used as fragrance ingredients.

An evaluation and review of a structurally related group of fragrance materials.

Report of an Expert Panel on the reanalysis by of a 90-day study conducted by Monsanto in support of the safety of a genetically modified corn variety (MON 863).

MON 863, a genetically engineered corn variety that contains the gene for modified Bacillus thuringiensis Cry3Bb1 protein to protect against corn rootworm, was tested in a 90-day toxicity study as part of the process to gain regulatory approval. This study was reanalyzed by Séralini et al. who contended that the study showed possible hepatorenal effects of MON 863. An Expert Panel was convened to assess the original study results as analyzed by the Monsanto Company and the reanalysis conducted by Séralini et al. The Expert Panel concludes that the Séralini et al. reanalysis provided no evidence to indicate that MON 863 was associated with adverse effects in the 90-day rat study. In each case, statistical findings reported by both Monsanto and Séralini et al. were considered to be unrelated to treatment or of no biological or clinical importance because they failed to demonstrate a dose-response relationship, reproducibility over time, association with other relevant changes (e.g., histopathology), occurrence in both sexes, difference outside the normal range of variation, or biological plausibility with respect to cause-and-effect. The Séralini et al. reanalysis does not advance any new scientific data to indicate that MON 863 caused adverse effects in the 90-day rat study.

Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients.

The threshold of toxicological concern (TTC) has been used for the safety assessment of packaging migrants and flavouring agents that occur in food. The approach compares the estimated oral intake with a TTC value derived from chronic oral toxicity data for structurally-related compounds. Application of the TTC approach to cosmetic ingredients and impurities requires consideration of whether route-dependent differences in first-pass metabolism could affect the applicability of TTC values derived from oral data to the topical route. The physicochemical characteristics of the chemical and the pattern of cosmetic use would affect the long-term average internal dose that is compared with the relevant TTC value. Analysis has shown that the oral TTC values are valid for topical exposures and that the relationship between the external topical dose and the internal dose can be taken into account by conservative default adjustment factors. The TTC approach relates to systemic effects, and use of the proposed procedure would not provide an assessment of any local effects at the site of application. Overall the TTC approach provides a useful additional tool for the safety evaluation of cosmetic ingredients and impurities of known chemical structure in the absence of chemical-specific toxicology data.

A toxicologic and dermatologic assessment of related esters and alcohols of cinnamic acid and cinnamyl alcohol when used as fragrance ingredients.

An evaluation and review of a structurally related group of fragrance materials.

A toxicologic and dermatologic assessment of ionones when used as fragrance ingredients.

An evaluation and review of a structurally related group of fragrance materials.

A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients.

An evaluation and review of a structurally related group of fragrance materials.

The K-region trans-8,9-diol does not significantly contribute as an intermediate in the metabolic activation of dibenzo[a,l]pyrene to DNA-binding metabolites by human cytochrome P450 1A1 or 1B1.

Metabolic activation of the K-region trans-8,9-diol of the highly carcinogenic hexacyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) by human cytochrome P-450 (P450) 1A1 and 1B1 was investigated in Chinese hamster V79 cell lines expressing human P450 1A1 or 1B1. P450 1A1 and 1B1 are the major P450s involved in metabolic activation of polycyclic aromatic hydrocarbons in human cells. The major DNA adducts formed by metabolism of DB[a,l]P in cultures expressing P450 1A1 or 1B1 resulted mainly from the fjord region (-)-anti-DB[a,l]P-11,12-diol 13,14-epoxide [(-)-anti-DB[a,l]PDE] and, to a lesser extent, (+)-syn-DB[a,l]PDE. In V79 cells expressing human P450 1A1, high amounts of as yet unidentified highly polar DNA adducts are formed in addition to the DNA adducts derived from DB[a,l]PDEs. Human P450 1A1 has been found to metabolize DB[a,l]P on its K-region to the trans-8,9-diol, and it has been proposed that the DNA binding of the parent compound in P450 1A1-expressing tissues may be partially mediated by activation of the K-region trans-8,9-diol to form bis-diol epoxides. V79 cells expressing human P450 1A1 or 1B1 formed only low amounts of DNA adducts after treatment with high doses of the K-region trans-8,9-diol. None of the adducts formed were identical to the main adducts formed in the same cell lines by metabolic activation of DB[a,l]P or (-)-DB[a,l]P-trans-11,12-diol. These results demonstrate that the K-region trans-8,9-diol does not significantly contribute to the genotoxicity of the very potent carcinogen DB[a,l]P in human cells or tissues expressing P450 1A1 or 1B1.

Cytochrome P450 mediated reactions studied in genetically engineered V79 Chinese hamster cells.

V79 Chinese hamster cells genetically engineered for stable expression of rat and human CYP have been shown to serve as analytical tools for studying metabolism related problems in toxicology and pharmacology. Here, the application of rat and human CYP1A1 and CYP1A2 is demonstrated for comparative studies on the oxidation of polycyclic aromatic hydrocarbons, such as phenanthrene, benz[a]anthracene, and benzo[a]pyrene. Live cells were cultivated for 2 days in the presence of these chemicals. Thereafter, the supernatant medium was checked for metabolites by gas chromatography and mass spectrometry. Marked cytochromes P450 and species dependent differences in the metabolite profiles were observed. Most important was the finding, that human cytochrome P450 1A1 almost exclusively oxidized benzo[a]pyrene in the 7,8,9,10-position, yielding the ultimate carcinogen 7,8-dihydroxy-9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene whereas the rat cytochrome P450 1A1 oxidized benzo[]pyrene in the 4,5-position and 7,8,9,10-position. The importance of this finding is underlined by results from cytotoxicity studies. Benzo[a]pyrene was twice as cytotoxic in the human cytochrome P450 1A1 than in the rat cytochrome P450 1A1 expressing V79 cells. Species and cytochrome P450 specific metabolite profiles were also observed for phenanthrene and benz[a]anthracene.

Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells.

A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells. Expression was followed by Western analyses using three different NO synthase antisera. Activity remained stable during 4 months of continued cultivation. Activities were 25 pmol min-1 mg-1 cytosolic protein with L-arginine and 47 pmol min-1 mg-1 cytosolic protein with NG-hydroxy-L-arginine as substrates. Activity was concentration-dependently inhibited by inhibitors such as NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea. The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS. Parental V79 cells do not express and cannot be induced for NO synthase activity. Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase. The new cell line may serve as a useful tool to study human inducible NO synthase.

Strategies for setting occupational exposure limits for particles.

To set occupational exposure limits (OELs) for aerosol particles, dusts, or chemicals, one has to evaluate whether mechanistic considerations permit identification of a no observed effect level (NOEL). In the case of carcinogenic effects, this can be assumed if no genotoxicity is involved, and exposure is considered safe if it does not exceed the NOEL. If tumor induction is associated with genotoxicity, any exposure is considered to be of risk, although a NOEL may be identified in the animal or human exposure studies. This must also be assumed when no information on the carcinogenic mechanism, including genotoxicity, is available. Aerosol particles, especially fibrous dusts, which include man-made mineral fiber(s) (MMMF), present a challenge for toxicological evaluation. Many MMMF that have been investigated have induced tumors in animals and genotoxicity in vitro. Since these effects have been associated with long-thin fiber geometry and high durability in vivo, all fibers meeting such criteria are considered carcinogenic unless the opposite has been demonstrated. This approach is practicable. Investigations on fiber tumorigenicity/genotoxicity should include information on dose response, pathobiochemistry, particle clearance, and persistence of the material in the target organ. Such information will introduce quantitative aspects into the qualitative approach that has so far been used to classify fibrous dusts as carcinogens. The rationales for classifying the potential carcinogenicity of MMMF and for setting OELs used by the different European committees and regulatory agencies are described.

Maximum workplace concentration values and carcinogenicity classification for mixtures.

In Germany, the Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) generally sets maximum workplace concentration values (i.e., a proposed occupational exposure level [OEL]) for single substances, not for mixtures. For mixtures containing substances with a genotoxic and carcinogenic potential, the commission considered it scientifically inappropriate to establish a safe threshold. This approach is currently under discussion. Carcinogenic mixtures are categorized according to either the carcinogenicity of the mixture or the classification of the carcinogenic substances included. In regulating exposure to mixtures, an approach similar to that used by the American Conference of Governmental Hygienists is proposed: For components with the same target organ and mode of action or interfering metabolism, synergistic effects must be expected and the respective OELs must be lowered. However, if there is proof that the components act independently, the OELs of the individual compounds are not considered to be modified. In the view of the commission, calculating OELs for solvent mixtures according to their liquid phase composition is not justified, and the setting of scientifically based OELs for complex mixtures is not possible.

Age-, sex-, and strain-dependent differences in the induction of enzyme-altered islands in rat liver by diethylnitrosamine.

The formation of foci with loss of ATPase and emergence of gamma-GTase was studied histochemically in livers of male and female Wistar and Sprague-Dawley rats of 3--4 and 6--7 weeks of age, respectively, after application of diethylnitrosamine. A single dose of 8 mg/kg body weight induced a considerable island formation in weanlings of both sexes. Island induction in adults was observed only after repeated application. No difference in island size and number was observed with the exception of greater island size and Sprague-Dawley females. Sex-dependent differences in susceptibility to island induction were observed in weanlings, females being more sensitivity than males and Sprague-Dawley females being the most sensitive of all. No correlation was seen between monooxygenase activity and the extent of island formation. The coincidence of ATPase-deficiency and emergence of gamma-GTase was highest in Sprague-Dawley females. The importance of this result in respect to cancer formation is discussed. Weanling Sprague-Dawley females seemed to be the most suitable for use in a screening test system for chemical carcinogenicity, especially for testing low doses or weak carcinogens.

Inhaled ethylene oxide induces preneoplastic foci in rat liver.

The metabolite of E, EO, has been shown to be an extrahepatic carcinogen in rats in long-term studies. By means of a rat liver foci bioassay with 3 to 4 days old Sprague-Dawley rats, EO showed an initiating capacity in the livers of female, but not of male rats, measured as incidence of foci deficient in ATPase. After inhalation of 55 and 100 ppm EO, 8 h daily, 5 days weekly, and over 3 weeks, 1 week of pause, and another 8 weeks of promotion with polychlorinated biphenyls, foci incidence was generally low. But it was concentration dependently higher than in controls 12 weeks after starting the experiment. A linear concentration-effect relationship existed with a correlation coefficient of r = 0.991. With 33 ppm EO the number of foci was not enhanced significantly. The administration of 10,000 ppm E did not result in an enhanced foci incidence. In general the carcinogenic potential of EO, which has not been shown so far to cause hepatic tumors in rats, could be demonstrated in rat liver using a sensitive rat liver foci bioassay.

Changes in the classification of carcinogenic chemicals in the work area. (Section III of the German List of MAK and BAT values).

Carcinogenic chemicals in the work area were previously classified into three categories in section III of the German List of MAK and BAT values (the list of values on maximum workplace concentrations and biological tolerance for occupational exposures). This classification was based on qualitative criteria and reflected essentially the weight of evidence available for judging the carcinogenic potential of the chemicals. In the new classification scheme the former sections IIIA1, IIIA2, and IIIB are retained as categories 1, 2, and 3, to correspond with European Union regulations. On the basis of our advancing knowledge of reaction mechanisms and the potency of carcinogens, these three categories are supplemented with two additional categories. The essential feature of substances classified in the new categories is that exposure to these chemicals does not contribute significantly to the risk of cancer to man, provided that an appropriate exposure limit (MAK value) is observed. Chemicals known to act typically by non-genotoxic mechanisms, and for which information is available that allows evaluation of the effects of low-dose exposures, are classified in category 4. Genotoxic chemicals for which low carcinogenic potency can be expected on the basis of dose/response relationships and toxicokinetics and for which risk at low doses can be assessed are classified in category 5. The basis for a better differentiation of carcinogens is discussed, the new categories are defined, and possible criteria for classification are described. Examples for category 4 (1,4-dioxane) and category 5 (styrene) are presented.

Stable expression of human cytochrome P450 1A1 cDNA in V79 Chinese hamster cells and metabolic activation of benzo[a]pyrene.

A V79 Chinese hamster cell line stably expressing human cytochrome P450 1A1 (CYP1A1) was obtained by chromosomal integration of the human CYP1A1 cDNA under the control of the SV40 early promoter. Chromosomal integration was verified by Southern analysis, and effective transcription of the human CYP1A1 cDNA was demonstrated by Northern analysis. The CYP1A1 cDNA-encoded protein was characterized by Western analysis using anti-rat CYP1A1. Intracellular association of CYP1A1 with the endoplasmic reticulum could be visualized by in situ immunofluorescence. Crude cell lysates of the V79 derived cell line was able to catalyze 7-ethoxyresorufin-O-deethylation (EROD) with an activity of about 50 pmol min-1 mg-1 total protein, and an aryl hydrocarbon hydroxylase activity (AHH) of 25 pmol min-1 mg-1. CYP1A1 dependent cytotoxicity, measured by neutral red uptake, and genotoxicity, determined by the frequency of micronucleus formation, of benzo[a]pyrene (B[a]P) and trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) could be demonstrated at substrate concentrations as low as 10 nM. Thus, this cell line presents a sensitive tool for studying CYP1A1 mediated metabolism of polycyclic aromatic hydrocarbons (PAH). B[a]P and the purified (+)- and (-)-enantiomers of B[a]P-7,8-diol were compared for their mutagenicity. The (-)-enantiomer was found to be 3-5-fold more mutagenic than the (+)-enantiomer.

Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells.

A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg-1 total protein x min-1. CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.

Genetically engineered V79 Chinese hamster cells for stable expression of human cytochrome P450IA2.

V79 Chinese hamster cells were genetically engineered for stable expression of human P450IA2. Full length cDNA, encoding human P450IA2, was inserted into an SV40 early promoter containing eukaryotic expression vector and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to the neomycin derivative G418) into V79 Chinese hamster cells. The recombinant expression vector was introduced into two different V79 sublines, one expressing an endogenous acetyltransferase (V79-NH), the other not (V79-MZ). The presence of human cytochrome CYP1A2 cDNA in the G418 resistant V79 cell clones was confirmed by Southern blotting. The transcription of the cDNA into mRNA was detected by Northern blotting and the translation into an authentic cytochrome P450IA2 protein was shown by Western blotting. The enzymatic activity in these cells was determined by the cytochrome P450IA2-dependent methoxy-, ethoxy-, benzoxy-, and pentoxyresorufin dealkylation activity.

Taurolithocholate inhibits taurocholate uptake by isolated hepatocytes at low concentrations.

The cholestatic bile acid taurolithocholate inhibits taurocholate uptake by isolated liver cells non-competitively. Inhibition is instantaneous and inversely related to the cell number in the incubate. The Ki amounts to 7 micron in the presence of 2 mg cellular protein per ml. Secretion of taurocholate by isolated liver cells is not affected by taurolithocholate up to a concentration of 50 micron. This indicates a difference between the carrier for taurocholate uptake and the carrier for taurocholate secretion. Inhibition of bile acid uptake of liver cells may be involved in the pathogenesis of lithocholate-induced cholestasis.

Classification of carcinogenic chemicals in the work area by the German MAK Commission: current examples for the new categories.

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area (MAK Commission) introduced an extended classification scheme in 1998. In addition to the traditional three categories still used to date, now called: Category 1 (human carcinogen); Category 2 (animal carcinogen); and Category 3 (suspected carcinogen), two new Categories (4 and 5) were added. Classification of substances into the new Categories 4 and 5 is based on the knowledge of mode of action and the potency of carcinogens. The essential feature of substances classified in the new Categories 4 and 5 is that exposure to these chemicals does not contribute significantly to the risk of cancer to man, provided that an appropriate exposure limit (MAK value) is observed. Chemicals known to act typically by non-genotoxic mechanisms are classified in Category 4. Genotoxic chemicals for which low carcinogenic potency can be assessed on the basis of dose-response relationships and toxicokinetics are classified in Category 5. Since the use of this scheme for 3 years, various chemicals have been classified in one of the new categories. However, in several cases data to sufficiently substantiate a MAK value are missing. Such substances are now classified in a subcategory of Category 3, called Category 3 A, which indicates that further data are required for final classification. Examples are given for classification of dichloromethane into Category 3 A, chloroform and sulfuric acid into Category 4 and ethanol into Category 5.