Nonlinear dose-response relationship for the binding of the carcinogen benzo(a)pyrene to rat liver DNA in vivo.

With radioactive compounds of high specific activity, the binding of carcinogens to DNA can be measured with doses that are ineffective in long-term studies. The binding of tritiated benzo(a)pyrene to liver DNA of adult male rats has been determined 50 hr after a single i.p. injection of doses between 40 microgram/kg and 4 mg/kg. The dose-response relationship is linear up to 1 mg/kg, shows a step towards 2 mg/kg, and gives a shallow linear slope above that value. The observed binding ranges from 1.7 to 180 nmoles benzo(a)pyrene per mole DNA phosphate. The nonlinearity could be due to an induction of metabolizing enzymes. The microsomal aryl hydrocarbon hydroxylase activity increases significantly 24 hr after a single dose of 4 mg/kg and 48 hr after doses of 2 and 4 mg/kg, but no induction is found with 1 mg/kg. The binding from an equimolar dose is 35 times lower than the one found on mouse skin DNA and 300 times lower than that of N,N-dimethylnitrosamine in rat liver. A good correlation exists to the respective tumor formation in long-term studies.

The real role of risk assessment in cancer risk management.

Regulatory actions taken to reduce the risk of harmful effects of exposure to chemicals often are not commensurate with the toxicological risk assessment. A number of factors relating to psychology, sociology, economics and politics rather than science and medicine affect the final decision. Werner Lutz and colleagues illustrate the situation using the leukemia-inducing chemical benzene as an example.

[Adnexitis and pelvic inflammatory disease]. / Adnexitis und "Pelvic Inflammatory Disease".

Pelvic inflammatory disease and upper genital tract infection describe inflammatory changes in the upper female genital tract of any combination: endometritis, salpingitis, tubo-ovarian abscess, peritonitis in the small pelvis. The International Infectious Disease Society for Obstetrics and Gynecology recommends a revision of the CDC guidelines taking into account the type of germ or the triggering agent and the seriousness of the disease. Infections with Chlamydia trachomatis and Neisseria gonorrhoeae are increasing worldwide. They are one of the main causes of tubal sterility, chronic abdominal pain and ectopic pregnancies. More than 30% of the infections are subclinical and asymptomatic. Therefore it is most recommendable to generally screen young, sexually active women with any of the risks mentioned above. Antibiotic therapy should be started as early as possible, in case of doubt even probatively, and should cover a broad spectrum of germs. C. trachomatis and N. gonorrhoeae should be treated according to resistance testing. In uncomplicated cases, hospitalization is unnecessary, ambulant therapy is sufficient.

DNA methylation in the digestive tract of F344 rats during chronic exposure to N-methyl-N-nitrosourea.

The formation of O6-methyldeoxyguanosine (O6-MedGuo) was determined by an immuno-slot-blot assay in DNA of various tissues of F344 rats exposed to N-methyl-N-nitrosourea (MNU) in the drinking water at 400 ppm for 2 weeks. Although the pyloric region of the glandular stomach is a target organ under these experimental conditions, the extent of DNA methylation was highest in the forestomach (185 mumol O6-MedGuo/mol guanine). Fundus (91 mumol/mol guanine) and pylorus (105 mumol/mol guanine) of the glandular stomach, oesophagus (124 mumol/mol guanine) and duodenum (109 mumol/mol guanine) showed lower levels of O6-MedGuo but differed little between each other. Thus, no correlation was observed between target organ specificity and the extent of DNA methylation. This is in contrast to the gastric carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which preferentially alkylates DNA of the pylorus, the main site of induction of gastric carcinomas by this chemical. In contrast to MNU, the non-enzymic decomposition of MNNG is accelerated by thiol compounds (reduced glutathione, L-cysteine), which are present at much higher concentrations in the glandular stomach than in the forestomach and oesophagus. During chronic exposure to MNNG (80 ppm), mucosal cells immunoreactive to O6-MedGuo are limited to the luminal surface [Kobori et al. (1988) Carcinogenesis 9:2271-2274]. Although MNU (400 ppm) produced similar levels of O6-MedGuo in the pylorus, no cells containing methylpurines were detectable by immunohistochemistry, suggesting a more uniform methylation of mucosal cells by MNU than by MNNG. After a single oral dose of MNU (90 mg/kg) cells containing methyl-purines were unequivocally identified using antibodies to O6-MedGuo and the imidazole-ring-opened product of 7-methyldeoxyguanosine. In the gastric fundus, their distribution was similar to those methylated by exposure to MNNG, whereas the pyloric region contained immunoreactive cells also in the deeper mucosal layers. After a 2-week MNU treatment, the rate of cell proliferation, as determined by bromodeoxyuridine immunoreactivity, was only slightly enhanced in the oesophagus and in the fundus, but markedly in the forestomach and the pyloric region of the glandular stomach. It is concluded that the overall extent of DNA methylation, the distribution of alkylated cells within the mucosa and the proliferative response all contribute to the organ-specific carcinogenicity of MNU.

Interaction of cations and chelators with the intestinal absorption of tetracycline.

The influence of certain compounds on intestinal absorption of tetracycline was tested, using two different in vivo techniques: a) Disappearence of the drug from perfused intestinal segments of the rabbit; b) Measuring urinary excretion in the rat after oral dosage. Ca2+ and Fe2+ ions both reduced the permeation rate of tetracycline by about 30%. In presence of Ca2+ and salicylate ions, leading to ion-pair formation with the antibiotic, intestinal uptake was significantly increased in comparison to experiments where only calcium was present. A more pronounced increase was observed with EDTA. Phosphate ions had different effects on the uptake of the antibiotic in both experimental techniques. Possible mechanisms are discussed.

In vivo covalent binding of retronecine-labelled [3H]seneciphylline and [3H]senecionine to DNA of rat liver, lung and kidney.

Retronecine-labelled [3H]seneciphylline ([3H]SPH) and [3H]senecionine ([3H]SON) of high specific radioactivity (22 and 49 mCi/mmol, respectively) were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine as precursor. [2,3-3H]Putrescine was synthesized by Gabriel synthesis of 1,4-diamino-2-butene from 1,4-dibromo-2-butene and catalytic hydrogenation of the product with tritium gas. Rats of both sexes were treated with the labelled pyrrolizidine alkaloids (PAs) (75-215 microCi SPH or 40-485 microCi SON/kg body wt.) and killed after 6 h or 4-5 days. SON-treated females excreted 83.4 +/- 0.2% of applied radioactivity in faeces and urine within 4 days whereas equally treated males excreted 90.9 +/- 3.2% in the same time. Excretion of 3H-activity from SPH-treated females was completed within 5 days (104.7 +/- 6.4%). Corresponding with these results, tissue levels were highest in SON-treated females. DNA and proteins were isolated from liver, lungs and kidneys and covalent binding of the alkaloids to DNA was determined. A Covalent Binding Index (CBI, mumol alkaloid bound per mol nucleotides/mmol alkaloid administered per kg body wt.) of 210 +/- 12 was found for the liver from SON-treated females whereas binding to liver DNA of males was lower by a factor of 4. The DNA damage determined six hours after treatment persisted during the following 4 days. Administration of [3H]SPH to female and male rats resulted in a CBI of 69 +/- 7 and 73/92, respectively, for the liver DNA. Furthermore we found binding of both alkaloids to DNA of lungs and kidneys in male and female rats. The in vivo formation of [3H]SON derived DNA adducts could be proved by HPLC analysis of hydrolyzed DNA.

Covalent binding of ethinylestradiol and estrone to rat liver DNA in vivo.

The covalent binding of [6,7-3H] ethinylestradiol (EE) and [6,7-3H] estrone (E) to liver DNA of 200 g female rats was measured 8 h after the administration of 80 microgram (9.2 mCi) estrogen by gavage. The binding is 1.5 for EE and 1.1 for E, expressed as binding to DNA/dose, in units of mumol hormone/mol DNA phosphate/mmole hormone/kg body wt. It is the same order of magnitude as for benzene and about 10 000 times below the binding of typical liver carcinogens, such as aflatoxin B1 or N,N-dimethylnitrosamine.

In vivo covalent binding of aflatoxin B1 and aflatoxin M1 to liver DNA of rat, mouse and pig.

[14C]Aflatoxin B1 (AFB1) was isolated from cultures of Aspergillus parasiticus grown on [1-14C]sodium acetate. Covalent binding of AFB1 to liver DNA of rat and mouse was determined 6-8 h after oral administration. The effectiveness of covalent binding, expressed as DNA binding per dose in the units of a 'Covalent Binding Index' (CBI), (micromol aflatoxin/mol DNA nucleotides)/(mmol aflatoxin/kg animal), was found to be 10 400 for rats and 240 for mice. These CBI partly explain the different susceptibility of the two species for the incidence of hepatic tumors. The corresponding values for pig liver DNA, 24 and 48 h after oral administration, were found to be as high as 19 100 and 13 300. DNA-binding has not so far been reported for this species although it could represent an appropriate animal model for studies where a human-like gastrointestinal tract physiology is desirable. Aflatoxin M1 (AFM1) is a metabolite found in the milk of cows that have been fed AFB1-contaminated diet. [14C]AFM1 was also found to be produced by cultures of A. parasiticus giving a yield of about 0.3% of the total aflatoxins. A test for covalent binding to rat liver DNA revealed a CBI of 2100 showing that AFM1 must also be regarded as a strong hepatocarcinogen. It is concluded that AFB1 contaminations should be avoided in dairy feed.

Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster.

Sixteen pyrrolizidine alkaloids (PAs) were examined for their genotoxic potency in the wing spot test of Drosophila melanogaster following oral application. This in vivo assay tests for the induction of somatic mutation and mitotic recombination in cells of the developing wing primordia. All PAs tested except the C9-monoester supinine were clearly genotoxic. Depending on their chemical structure, however, genotoxicity of the PAs varied widely in a range encompassing about three orders of magnitude. In general, macrocyclic diester-type PAs were the most and 7-hydroxy C9-monoester types the least genotoxic representatives studied, while open diesters were intermediate in this respect. Stereoisomeric PAs mostly showed similar, but sometimes also clearly unequal genotoxicity. An increasing number of hydroxy groups in the PA molecule seemed to reduce its genotoxic potency. With respect to the structure/activity relationships, there appears to be a good correlation between hepatotoxicity of PAs in experimental rodents and genotoxicity in the wing spot test of Drosophila. This suggests that PAs are bioactivated along similar pathways in the mammalian liver and in the somatic cells of Drosophila. The genotoxic potential of PAs in the Drosophila wing spot test and their carcinogenic potential in mammals also seem correlated, although the information in the literature on carcinogenicity of the non-macrocyclic PAs with moderate to low genotoxic potency is concededly limited. Comparisons with other genotoxicity tests suggest that the wing spot test is particularly suitable for genotoxins like PAs, on the one hand because of the versatile metabolic bioactivation system of Drosophila and on the other hand also because of its excellent sensitivity to the crosslinking agents among the genotoxins.

Dose dependence of the excretion of maneb metabolites in urine of rats.

14C-activity in the urine of female rats appeared to be independent of dose when single oral doses of [14C]maneb, a fungicide, manganese ethylene-bis(dithiocarbamate), were given. This was found with [14C]maneb synthesized from [U-14C]ethylenediamine. In the dose range, 23 microgram/kg to 1.4 g/kg of maneb, the excreted activity in urine of 39 female rats was 48.8 +/- 12.6% (n=39).

Transfer of [3H]pyrrolizidine alkaloids from Senecio vulgaris L. and metabolites into rat milk and tissues.

[3H]Retronecine and [3H]necic acid-labelled senecionine and seneciphylline were prepared biosynthetically with seedlings of Senecio vulgaris L. using [2,3-3H]putrescine and [4,5-3H]isoleucine, respectively, as precursors. Lactating rats dosed with these differently labelled pyrrolizidine alkaloids (PAs) excreted within 3 h approx. 0.08% of the applied radioactivity in the milk mainly as yet not identified water-soluble retronecine-derived metabolites and with approx. 0.02% as unchanged PAs. Highest tissue levels of PAs and metabolites, 6 h after administration, were found in liver and lungs.

Lack of covalent binding to rat liver DNA of the hypolipidemic drugs clofibrate and fenofibrate.

14C-labelled clofibric acid and fenofibric acid were administered p.o. to 200 g male and female rats. After 10 h, liver nuclear DNA and protein were isolated and the radioactivity was determined. Binding to protein was clearly measurable whereas no binding to DNA could be detected from any drug. A comparison of the limit of detection of such DNA binding with well-known chemical carcinogens revealed that the known hepatocarcinogenicity of clofibrate cannot be based upon an initiating, DNA damaging, mode of action but must be due to other, nongenotoxic, mechanisms such as peroxisome proliferation, hepatomegaly, or cytotoxicity due to protein binding. The risk assessment in man and the interpretation of the carcinogenicity data for rodents are discussed.

The lacI transgenic mouse mutagenicity assay: quantitative evaluation in comparison to tests for carcinogenicity and cytogenetic damage in vivo.

The detection limit of the lacI transgenic mouse mutagenicity assay lies, in practice, at approximately a 50-100% increase in mutant frequency in treated animals over controls. The sensitivity of this assay in detecting genotoxins can be markedly improved by subchronic rather than acute application of the test compound. The lacI transgenic mouse mutagenicity assay was compared quantitatively to rodent carcinogenicity tests and to presently used in vivo mutagenicity assays. With the genotoxic carcinogens tested thus far, a rough correlation between mutagenic potency and carcinogenic potency was observed: on average, to obtain a doubling in lacI mutant frequency the mice had to be treated with a total dose equal to 50 times the TD50 daily dose level. This total dose could be administered either at a high dose rate within a few days or, preferably, at a low dose rate over several weeks. This analysis also indicated that a lacI experiment using a 250-day exposure period would give a detection limit approximately equal to that of a long-term carcinogenicity study. In comparison to the micronucleus test or the chromosome aberration assay, acute studies with the presently available lacI system offered no increase in sensitivity. However, subchronic lacI studies (3-4-month exposure) resulted in an increase in sensitivity over the established tests by 1-2 orders of magnitude (shown with 2-acetylaminofluorene, N-nitrosomethylamine, N-nitrosomethylurea and urethane). It is concluded that a positive result in the lacI test can be highly predictive of carcinogenicity but that a negative result does not provide a large margin of safety.

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay.

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.

Mutations in liver DNA of lacI transgenic mice (Big Blue) following subchronic exposure to 2-acetylaminofluorene.

2-Acetylaminofluorene (2-AAF) was administered at levels of 0, 300 and 600 ppm in the diet for 28 days to female transgenic mice bearing the lacI gene in a lambda vector (Big Blue mice). The lambda vector was excised from liver DNA and packaged in vitro into bacteriophage particles which were allowed to infect E. coli bacteria, forming plaques on agar plates. Approximately 10(5) plaques were screened per animal for the appearance of a blue colour, indicative of mutations in the lacI gene which had resulted in an inactive gene product. Background mutation rate was 2.7 x 10(-5) (pooled results of two animals, 8 mutant plaques/289,530 plaques). At 300 ppm in the diet, the rate of 3.5 x 10(-5) (8/236,300) was not significantly increased over background. At 600 ppm in the diet, the rate increased approximately 3 fold to 7.7 x 10(-5) (17/221,240). In comparison to the usual single or 5-day carcinogen exposure regimes, the 4-week exposure protocol allowed the use of much lower dose levels (10-1000 fold lower). Overt toxicity could thus be avoided. The daily doses used were somewhat higher than those required in 2-year carcinogenicity studies with 2-AAF.

Investigations on DNA binding in rat liver and in Salmonella and on mutagenicity in the Ames test by emodin, a natural anthraquinone.

Emodin (1,6,8-trihydroxy-3-methylanthraquinone), an important aglycone found in natural anthraquinone glycosides frequently used in laxative drugs, was mutagenic in the Salmonella/mammalian microsome assay (Ames test) with a specificity for strain TA1537. The mutagenic activity was activation-dependent with an optimal amount of S9 from Aroclor 1254-treated male Sprague-Dawley rats of 20% in the S9 mix (v/v) for 10 micrograms emodin per plate. Heat inactivation of the S9 for 30 min at 60 degrees C prevented mutagenicity. The addition of the cytochrome P-448 inhibitor 7,8-benzoflavone (18.5 nmoles per plate) reduced the mutagenic activity of 5.0 micrograms emodin per plate to about one third, whereas the P-450 inhibitor metyrapone (up to 1850 nmoles per plate) was without effect. To test whether a metabolite binds covalently to Salmonella DNA, [10-(14)C]emodin was radiosynthesized, large batches of bacteria were incubated with [10-(14)C]emodin and DNA was isolated. [G-3H]Aflatoxin B1 (AFB1) was used as a positive control mutagen known to act via DNA binding. DNA obtained after aflatoxin treatment could be purified to constant specific activity. With emodin, the specific activity of DNA did not remain constant after repeated precipitations so that it is unlikely that the mutagenicity of emodin is due to covalent interaction of a metabolite with DNA. The antioxidants vitamin C and E or glutathione did not reduce the mutagenicity. Emodin was also negative with strain TA102. Thus, oxygen radicals are probably not involved. When emodin was incubated with S9 alone for up to 50 h before heat-inactivation of the enzymes and addition of bacteria, the mutagenic activity did not decrease. It is concluded that the mutagenicity of emodin is due to a chemically stable, oxidized metabolite forming physico-chemical associations with DNA, possibly of the intercalative type. In order to check whether an intact mammalian organism might be able to activate emodin to a DNA-binding metabolite, radiolabelled emodin was administered by oral gavage to male SD rats and liver DNA was isolated after 72 h. Very little radioactivity was associated with the DNA. Considering that DNA radioactivity could also be due to sources other than covalent interactions, an upper limit for the covalent binding index, CBI = (mumoles chemical bound per moles DNA nucleotides)/(mmoles chemical administered per kg body weight) of 0.5 is deduced. This is 10(4) times below the CBI of AFB1.(ABSTRACT TRUNCATED AT 400 WORDS)

Genotoxicity of aniline derivatives in various short-term tests.

Various substituted aniline derivatives were tested for genotoxicity in several short-term tests in order to examine the hypothesis that a substitution at both ortho positions (2,6-disubstitution) could prevent genotoxicity due to steric hindrance of an enzymatic activation to electrophilic intermediates. In the Salmonella/microsome assay, 2,6-dialkylsubstituted anilines and 2,4,6-trimethylaniline (2,4,6-TMA) were weakly mutagenic in strain TA100 when 20% S9 mix was used, although effects were small compared to those of 2,4-dimethylaniline and 2,4,5-trimethylaniline (2,4,5-TMA). In Drosophila melanogaster, however, 2,4,6-TMA and 2,4,6-trichloroaniline (TCA) were mutagenic in the wing spot test at 2-3 times lower doses than 2,4,5-TMA. In the 6-thioguanine resistance test in cultured fibroblasts, 2,4,6-TMA was again mutagenic at lower doses than 2,4,5-TMA. Two methylene-bis-aniline derivatives were also tested with the above methods: 4,4'-methylene-bis-(2-chloroaniline) (MOCA) was moderately genotoxic in all 3 test systems whereas 4,4'-methylene-bis-(2-ethyl-6-methylaniline) (MMEA) showed no genotoxicity at all. DNA binding studies in rats, however, revealed that both MOCA and MMEA produced DNA adducts in the liver at levels typically found for moderately strong genotoxic carcinogens. These results indicate that the predictive value of the in vitro test systems and particularly the Salmonella/microsome assay is inadequate to detect genotoxicity in aromatic amines. Genotoxicity seems to be a general property of aniline derivatives and does not seem to be greatly influenced by substitution at both ortho positions.

Past and future in mycotoxin toxicology research.

Before 1960 the toxicology of mycotoxins was mainly of concern to veterinarians, because outbreaks of mycotoxicoses resulted occasionally in considerable loss of livestock. By a wider use of biotests, preferably in mammals, a further decline of such intoxications probably will occur. Following the discovery of the carcinogenicity of some aflatoxins, the focus turned to human health. Screening tests for carcinogenicity are still in development. The test used most frequently is the Ames test on microorganisms. Unfortunately, many problems still must be resolved before an extrapolation of results from these tests to man is possible. Examination of the carcinogenic activity of mycotoxins in long-term animal experiments is often difficult due to lack of resources, lack of test material, and the toxicity of the compounds, which precludes administration of sufficiently high dose levels. The available data regarding a possible carcinogenic activity of several important mycotoxins, such as the trichothecenes or patulin, do not fulfill currently used criteria. Therefore further studies are needed. A new approach is determination of the binding capacity to DNA of suspected carcinogens, which seems to correlate well with carcinogenic potency. By this method, a high carcinogenicity of aflatoxin M1 can be deduced. However, the macromolecular-bound residues of aflatoxin B1, which may occur in tissues of domestic animals, most probably do not show carcinogenic activity. Although many questions are still unanswered, it seems that the numerous mycotoxins identified since 1960 are less toxic or carcinogenic and occur less frequently in food than do aflatoxins.

Toxicity and mutagenicity of anthraquinones from Aspergillus chevalieri.

More than 100 strains of the Aspergillus glaucus group were cultivated on synthetic media for 11 days at 28 degrees C. Organic extracts of fungal material were screened by thin-layer chromatography (TLC) for the mycotoxins aflatoxins B1,2 and G1,2, sterigmatocystin, ochratoxin A, gliotoxin, patulin, and xanthocillin X. None of these toxins were produced in detectable amounts under experimental conditions. Nevertheless, organic extracts exhibited high toxicity after intraperitoneal (i.p.) administration in mice. Aspergillus chevalieri strain ZT 8268 was selected for further investigation of its toxic metabolites. The main toxic action was attributed to the four anthraquinone derivatives, physicion, physcionanthrone B, physciondianthrone, and erythroglaucin, which were isolated and identified. No toxic effects were found after oral administration. Using the Salmonella/mammalian microsome test, mutagenic activity (frame-shift) was detected in strain TA 1537 in the presence of S-9 liver microsome preparation.

Acute irritation tests in risk assessment.

The two most important elements in assessing the risk of topical injury from a chemical are its biological properties, in the context of skin and mucous membrane damage, and the likelihood and likely nature of topical contact with the chemical. Appropriate biological tests in model systems should be based on the probable circumstances of exposure. Topical contact takes place under two distinct sets of circumstances--intentional and accidental. Chemicals that are intended to come into contact with skin and mucous membranes include cosmetics and dermatological preparations. For such compounds the frequency and extent of skin contact is predictable and any irritant effects are unacceptable. The absence of irritant effects is established by testing in human volunteers or experimental animals. Since animal skin and mucous membranes are more susceptible to irritants than those of man, the amounts or concentrations tested need not be greater than those intended for human use. It is hoped that validated alternatives to animal models will soon be available. For household or industrial chemicals where skin and/or mucous-membrane contact occurs accidentally, topical contact should generally be avoided. In such cases the objective of irritancy testing should be to establish which compounds are particularly irritant and therefore need extra care in handling. We are convinced that this latter objective can be achieved by simpler and less cruel tests than the Draize eye-irritation test.