In vivo cytogenetic effects of natural humic acid.

As humic compounds are naturally widespread in the environment and present in surface water, studies on their genotoxicity are justified. Humic acid (HA) has not been demonstrated to be genotoxic either in vitro or in vivo. In the present paper we investigated its activity both in intestinal and bone marrow cells following a single dose (100 mg/kg b.w. corresponding to 0.5 ml per animal of an aqueous solution of 4 g/l) of HA administered to mice by gastric intubation, to mimic the most likely route of human exposure. HA induced structural and, in particular, numerical chromosome abnormalities in intestinal cells. A marginal, non-significant induction of aneuploidy was also found in bone marrow cells.

Mutagenic activity of some coal-derived humic compounds evaluated by the Ames test.

Two coal-derived humic substances (Sulcis and South Africa, Eniricerche, Italy) have been evaluated for their mutagenic activity on TA98 and TA100 Salmonella typhimurium strains, either in presence or in absence of metabolic activation (S9). Both compounds showed no effect on the two strains, as observed with natural humic acid (Fluka). After chlorination, coal-derived humic acids induced a strong dose-related increase in the number of revertants on TA100 without S9, whose extent was directly proportional to the chlorination ratios. Such effect was completely suppressed when a sodium thiosulfate solution (10%) was added at the end of the chlorination period (about 90 h). The analogies with natural humic acid mutagenicity are discussed.

Cytogenetic monitoring of a group of Italian floriculturists: no evidence of DNA damage related to pesticide exposure.

The induction of sister chromatid exchanges (SCE), structural chromosome aberrations (CA) or micronuclei (MN) was investigated in peripheral lymphocytes of a group of Italian floriculturists exposed to a mixture of pesticides. No statistically significant difference in the frequencies of cytogenetic damage was detected between exposed and control subjects. Assessment of the effect of confounding factors indicated that smoking affected both SCE and CA frequencies. Multiple regression analysis showed that in heavy smokers (> or = 20 cigarettes/day), SCE and CA levels increased significantly by 17% and 54%, respectively, as compared to non-smokers.

Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects.

The comet test (single cell gel electrophoresis, SCGE) appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. Previously, we analyzed the degree of DNA damage in peripheral human lymphocytes from 100 healthy subjects living in Pisa (Italy) taking into account age, gender and smoking habit, and we also reported some results aiming at the assessment of the comet test (Betti el al., 1994). In addition, SCE analysis was carried out in order to compare the two endpoints. Because of the interesting results obtained, the present study was extended to 200 individuals, and data analyzed included information concerning number of cigarettes smoked a day, tar/cigarette and job. Data obtained confirmed that the SCGE is more sensitive than SCE in revealing smoking habit effects but comet induction did not seem to be related to the amount of cigarette tar inhaled. Moreover, sampling time was found to play a greater role in the comet assay as compared to SCE. Job position did not significantly influence SCE mean/subject or comet length mean/subject.

Alternatives in cosmetics testing.

This paper represents a summary of presentations made during a round-table discussion at the ECVAM Opening Symposium. After introductory comments on the cosmetic industry's use of alternative methods in the safety assessment process, the use of alternative methods by L'Oréal and by the Japanese cosmetic industry is outlined, current validation studies in Japan are noted, and the involvement of COLIPA, the European Cosmetic, Toiletry and Perfumery Association, in promoting the use of alternative methods is discussed. Two final sections deal with the effect of data variability on the performance of alternative methods in validation studies and on the integrated use of quantitative structure-activity relationship (QSAR) analysis with other approaches in the safety assessment process.

In vivo genotoxic interactions among three phenolic benzene metabolites.

Three benzene metabolites, hydroquinone (HQ), cathecol (CAT) and phenol (PHE) were studied to define their possible interaction in inducing micronuclei (Mn) in mouse bone marrow polychromatic erythrocytes (PCEs). HQ and CAT, administered separately, induced Mn while PHE showed no genotoxic effects. Binary and ternary mixtures of two or three metabolites gave different results, causing considerable increase or decrease in Mn induction. HQ and PHE, in binary mixtures, as well as PHE and CAT, increased Mn synergistically, while HQ and CAT interacted negatively. The genotoxicity of ternary mixtures was mainly the consequence of two metabolites: HQ and CAT. The maximal effect obtained is far below the induction of Mn consequent to benzene treatment. These data suggest that toxic and genotoxic effects of benzene alone could be the result of more complex interactions among these and other metabolites.

Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects.

Microscopic examination of individual human lymphocytes embedded in agarose, subjected to electrophoresis and stained with a fluorescent DNA-binding dye, provides a novel way of measuring DNA damage as extent of migration of DNA fragments, mainly single-strand breaks. With this relatively simple method, DNA damage arising as a consequence of smoking, age and other factors was examined in peripheral human lymphocytes from 100 healthy individuals living in Pisa (Italy). The extent of DNA migration was found to be significantly increased by smoking. It is noteworthy that the effect of smoking was more significant in men than in women and that DNA migration was similar in the young and in the older people. SCE analysis did not reveal any significant effect of smoking, sex or age in the same population, suggesting a higher responsiveness of the comet test to DNA-damaging agents.

Two years' air mutagenesis monitoring in a northwestern rural area of Italy with an industrial plant.

The mutagenicity of organic extracts from inhalable airborne particles, collected in a northwestern rural area of Italy in which an industrial plant producing chemical intermediates is present, was assessed during the years 1989 and 1990. The Ames plate test with Salmonella strains TA98 and TA100 with and without metabolic activation was used. Eight sites in the first and three sites in the second year were monitored once and twice a month respectively. Results show that the mutagenicity of air particulate matter reaches maximum values in the cold months and is not dependent on plant activities. In addition, a correlation analysis between mutagenicity data and number of vehicles seems to indicate traffic emissions as the main source of mutagens.

Cytogenetic effects of benzimidazoles in mouse bone marrow.

The cytogenetic effects of three benzimidazoles, i.e., benomyl, methyl thiophanate and methyl 2-benzimidazolecarbamate (MBC), were studied in mouse bone marrow cells by analyzing three genetic endpoints: micronuclei, structural chromosome aberrations plus or minus gaps, and aneugenic effects (hyperdiploidy or polyploidy). In general, the effects were small, but it was observed that benomyl and MBC significantly induced micronuclei as well as aneugenic effects, hyperdiploidy (no metaphases with more than one or two extra chromosomes, 2n + 1 or 2n + 2, were observed) and polyploidy (4n). The induction of chromosome gaps and breaks was less evident. Methyl thiophanate significantly induced micronuclei, but it was less effective than benomyl and MBC. Our results showed that micronuclei are a good indicator of aneugenic effects in mouse bone marrow cells. A curvilinear trend test has been devised to fit the curves originating from the time-dependent responses.

Guidelines for safety evaluation of cosmetics ingredients in the EC countries.

The Scientific Committee on Cosmetology (SCC) of the Commission of the European Communities was established in 1978 to assist the Commission in the application of the 76/768 Directive, which regulates the production and marketing of cosmetics products. The Committee has been asked to update the general guidelines, defined in 1982, for testing cosmetics ingredients with the aim of ensuring consumers' safety. In the present paper the full document approved by the SCC in October 1990 is reported. This new document is based on the experience of the Committee over the last 10 years, during which more than 400 cosmetics ingredients have been evaluated. The document also highlights the need to proceed to define standard methods to be used to assess dermal absorption and phototoxicity--areas in which international guidelines have not yet been approved. The document also includes some comments made by the author in order to explain better the position of the Committee in relation to certain items.

Genotoxicity studies in vitro and in vivo on carminic acid (natural red 4).

The potential genotoxic activity of carminic acid (CAS no. 1260-17-9; EINECS no. 215-023-3; C.I. no. 75410), a component of natural red colouring products (cochineal: CAS no. 1343-78-8; EINECS no. 215-680-6; C.I. no. 75470), used in food, cosmetics and drugs, has been evaluated by means of a series of short-term tests in vitro and in vivo, namely Salmonella reverse mutation, chromosome aberrations and sister chromatid exchanges in vitro on Chinese hamster ovary cells, and the mouse micronucleus test. All studies have produced negative results. The data obtained strongly support the non-mutagenic/non-carcinogenic activity of this compound. Genotoxicity data previously obtained for carminic acid, concerning the induction of a series of other genetic endpoints in different test systems, have also been considered, as have recent findings that indicate lack of carcinogenic activity in the cochineal preparation containing 29.8% carminic acid.

The analysis of the joint effect of substances on reversion systems and the assessment of antimutagenicity.

The statistical methods for the analysis of mutagenicity and carcinogenicity underwent considerable theoretical-practical development following the need for assessing the mutagenic and carcinogenic potential of substances. Antimutagenicity is investigated through the analysis of respondents in dose-response assays, when two different molecules are administered separately and as a mixture to a respondent system. When the number of respondent units is high, and doses are orthogonal, it is possible to apply simple models such as analysis of variance. This is not always possible or common, and alternative approaches have been developed, based on multiple regression and on tables of proportions. In this work, some of the most frequently used methods for the assessment of joint responses are reviewed, particularly those based on multiple regression, such as the method of Shaeffer et al. and the method of Hass et al. In order to illustrate these methods, joint responses of perylene and cyclopentapyrene, of N-acetylcysteine and dinitropyrene, and of N-acetylcysteine and extracts from diesel exhausts were analyzed. An antagonistic effect of perylene on the action of CPP was detected by the algorithm of Shaeffer et al. The effect is not multiplicative, i.e., it is not proportional to the product of doses. The antimutagenic effect of N-acetylcysteine on dinitropyrene is multiplicative, as detected by the method of Hass et al. The latter reveals that the inhibition by N-acetylcysteine on the mutagenic effect of extracts from diesel exhausts is also multiplicative.

Skin cytogenetic assay for the detection of clastogens-carcinogens topically administered to mice.

A method for assessing the effect of clastogens on mouse skin epidermal cells was devised and applied. Toxic and mutagenic responses in epidermal cells were tested using two known mutagens and carcinogens, urethane (URE) and 7,12-dimethylbenz[a]anthracene (DMBA). Cell generation time, sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) after topical and intraperitoneal (i.p.) treatment were measured in epidermal and bone marrow cells. After topical administration both tissues responded similarly, whereas after i.p. treatment skin cells were less responsive than bone marrow cells. However, the results indicate the validity of this new cytogenetic approach for the assessment of the genotoxicity of compounds applied directly to skin.

Data selection and treatment of chemicals tested for genotoxicity and carcinogenicity.

A database containing qualitative and quantitative results of experimental studies in the fields of genotoxicity and carcinogenicity has been developed. By analyzing results of the studies performed by the U.S. National Toxicology Program, or by a similar program developed in Japan, or reported in the scientific literature, as well performed by private organizations, information has been collected relating to 3389 chemicals, identified by their CAS number. The studies considered for the database include three genotoxicity/mutagenicity short-term test (STTs), namely, two in vitro (Salmonella, gene mutation assay, and mammalian cells/human lymphocytes chromosome aberration assay) and one in vivo, the rodent bone marrow micronucleus assay. To investigate the possible predictive value of these STT assays for carcinogenicity, the results of animal long-term bioassays have also been collected. We have re-evaluated all the genotoxicity studies and the majority of those cases studied in different laboratories with contrasting results has been resolved; a small proportion of questionable cases is, however, still present in the database. In total, 2898 (85.5%) of the chemicals have been tested in the Salmonella assay; 1399 (41.3%) have been tested in the in vitro chromosome aberration assay; 319 (9.4%) have been tested in the in vivo rodent bone marrow cell micronucleus assay; 716 (21.2%) of the chemicals have been tested in the in vivo animal long-term bioassay. For 1118 chemicals tested in the Salmonella assay, 30,650 quantitative studies have been included in the database, thus allowing a possible classification of mutagenic chemicals according to their mutagenic potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light--I. Assay development.

Two complementary assay systems have been adapted for the detection of compounds which may form mutagenic photoproducts during exposure to UV light from an Osram Ultra-Vitalux sunlamp as used in the evaluation of the effectiveness of sun filters. The effects of UVA and of UVB were evaluated. A bacterial plate test using Escherichia coli strain WP2 allowed the bacteria, co-plated with test chemical in soft agar, to be irradiated with various doses of UV light. Mutagenesis was assessed by scoring numbers of tryptophan-independent colonies. The chosen reference compound was 8-methoxypsoralen (8-MOP) which was non-mutagenic alone at the highest dose tested (1000 micrograms/plate). Following simultaneous exposure of bacteria to 8-MOP and doses of UV light which alone had little effect, large numbers of revertants were scored. Numbers of mutants were dependent upon the doses of both 8-MOP and of UV light. The second test system involved the exposure of Chinese hamster ovary cells to UV light in the presence of test chemical to determine the clastogenic effects of photoproducts. Treatment with 8-MOP alone up to 50 micrograms/ml was not clastogenic but concomitant exposure to non-damaging doses of UV light caused large increases in the incidence of chromosome aberrations of all types. Damage was again dependent on the doses of both components. Two additional photoactive compounds, para-aminobenzoic acid and chlorpromazine both show photoclastogenic but not photomutagenic properties. These two complementary assay systems take advantage of using no-effect levels of UV light as a baseline against which photomutagenicity readily can be compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlations between 15 polycyclic aromatic hydrocarbons (PAH) and the mutagenicity of the total PAH fraction in ambient air particles in La Spezia (Italy).

Airborne particulate matter has been monitored 4 times a month for 1 year (1988) in the city of La Spezia (Italy). The polycyclic aromatic hydrocarbon (PAH) fractions were extracted, purified and characterized for the content of 15 individual PAH. In general when concentrations of individual PAH were compared statistical correlation was obtained. Mutagenicity studies were performed by the use of the Ames plate test with the Salmonella strains TA98, TA100, TA98NR and TA98DNP6 with and without metabolic activation (S9 mix). The TA98 strain was by far the most responsive and the S9 mix was absolutely required as expected when PAH are assayed. Besides mutagenicity, toxicity was also considered and it proved to be correlated with mutagenicity in TA98, +S9. The TA98NR and TA98DNP6 strains showed no appreciable differences from the parental strain TA98 indicating the absence of significant amounts of direct-acting nitro derivatives in our PAH samples. Of the 15 PAH considered in this study the amounts of cyclopental[c,d]pyrene (CPP) correlated best with mutagenicity. The role of CPP in contributing to the indirect mutagenicity of urban air PAH samples is discussed.

An experimental approach to identifying the genotoxic risk from cooked meat mutagens.

In order to define the toxicological risk to the human population from the chemical compounds formed during the process of cooking animal meat, which have been described as possessing mutagenic, genotoxic and carcinogenic activities, an extensive study was undertaken of cooked meat extract and two cooked meat mutagens, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) and 2-amino-3,8-dimethylimidazo(4,5-f)quinoxaline (MeIQx). The study involved toxicokinetics and mouse-tissue distribution studies of the two chemicals, in vitro and in vivo mutagenicity/genotoxicity analyses (i.e. the detection of gene mutations, chromosome aberrations and micronuclei in mouse bone marrow cells, and mouse urine and faeces mutagenicity tests), as well as in vivo protein and DNA binding assays. IQ and MeIQx were found to be positive for the induction of gene mutations in Salmonella typhimurium TA98, but not in Chinese hamster V79 cells; IQ only was found to be positive for the induction of chromosome aberrations in Chinese hamster ovary cells and cultured human lymphocytes. IQ and MeIQx were negative for the induction of micronuclei in mice treated with 40 mg chemical/kg body weight; the lowest effective dose administered to the mice that produced mutagenic urine was 0.4 mg IQ/kg body weight and 0.04 mg MeIQx/kg. A dose of 40 mg IQ/kg, given orally by gavage to mice, produced an excretion of 1-4% of the applied dose in the urine and 0.1-2% of the applied dose in the faeces, when evaluated chemically or mutagenically. The number of DNA adducts in the liver correlated with the dose of IQ or MeIQx administered to the mice. All the data have been used for defining a possible risk estimate to the human population as a consequence of a cooked meat diet.

Induction of chromosomal aberrations and SCE by chloramphenicol.

The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with chloramphenicol (CAP), an antimicrobial agent acting by inhibiting protein synthesis. Moreover chromosomal aberrations and sister-chromatid exchanges were studied in bone marrow cells of treated mice and in Chinese hamster cell cultures (V79) respectively. While no aberrations were induced by short treatments in human lymphocytes exposed in G1 and G2 phases, high frequencies of aberrations, exclusively of the chromatid type, were induced when the drug was administered during a whole cell cycle. Aberrant metaphases were detected only at the end and a few hours after the end of treatment; at later times aberrant cells reached control values. Doses producing aberrations only slightly increased SCE both in human lymphocytes and in V79 cells. In mouse bone marrow cells CAP induced a high mitotic delay and few structural aberrations; intrachromosomal vacuoles were observed.

An experimental approach to identifying the genotoxic risk by cooked meat mutagens.

In order to define the toxicological risk for the human population derived from the chemical compounds formed during the process of cooking animal meat, which have been described to possess a mutagenic, genotoxic, and carcinogenic activity, an extensive study has been developed on cooked meat extract and two cooked meat mutagens, IQ and MeIQx. The study has been based on toxicokinetics and mouse tissue distribution of the two chemicals, on in vitro and in vivo mutagenicity/genotoxicity analyses (gene mutation, chromosome aberration, micronuclea in mouse bone marrow cells, mice urine and faeces mutagenicity test), as well as in vivo protein and DNA binding. The two chemicals have been found positive for the induction of gene mutation on Salmonella, but not in V-79 Chinese hamster cells; IQ only has been found positive for the induction of chromosome aberrations on CHO cells and cultured human lymphocytes. IQ and MeIQx were negative for the induction of micronuclea in mice treated with 40 mg/kg of the chemicals; the lowest effective administered dose to the mice which produced mutagenic urine was 0.4 mg/kg of IQ and 0.04 mg/kg of MeIQx. A dose of 40 mg/kg of IQ given by gavage to mice produced an excretion of 1-4% of the applied dose in the urine and 0.1-2% of the applied dose in the faeces, when evaluated chemically or mutagenically. The DNA adducts for the liver were correlated with the dose of the IQ and MeIQx administered to the mice. All the data have been used for defining a possible risk estimate derived to the human population as a consequence of a cooked meat diet.