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Human volunteer studies of skin irritation have been carried out for decades, both for research and for safety evaluation purposes. For the majority of this time, and consequently for the majority of the studies, assessment of the skin reactions has been made visually. Typical endpoints include erythema, oedema, dryness and scaling, some or all of which would be rated on a simple scale, eg 0, +/-, +, ++, + + +. Such approaches can be criticized as subjective, of poor reproducibility, lacking in sensitivity and highly variable between observers and/or institutions. In consequence, instrumental methods of assessment have been strongly promoted and do indeed offer several advantages, not least their objectivity. However, it is possible to use the human eye, which is a very sensitive tool, to make detailed, accurate and reproducible descriptions of skin irritation reactions. To achieve this, it is necessary to give prolonged and thorough training to each observer. In this paper, 3 examples of human volunteer studies, in which different pairs of trained observers independently carried out double blinded scoring of the irritation reactions, are reported. The grading patterns produced were almost identical; statistical analysis showed that properly trained observers are in fact able to reliably measure a grade of erythema to within +/- 1 on a 10 point scale; 97.6% of scores were within 2 grade points on this scale. These results provide evidence that visual scoring can be sensitive, reliable and reproducible within a testing institution.

The human 4 hour patch test provides an opportunity to identify substances with significant skin irritation potential without recourse to the use of animals. The protocol is designed to avoid the production of more than mild irritant reactions and meets the highest ethical standards. This paper provides the background to the development of the method and comments on its performance in the light of recent intra- and inter-laboratory investigations. In particular, the value of the method in providing 'gold standard' data for the identification of those substances (or preparations) which should, or should not, be classified as irritant to skin in European legislation is discussed. On the basis of the published data and supplementary investigations, recommendations are made on both the conduct and interpretation of the human 4 hour patch test. Finally, the lack of any necessity for formal validation of this assay is addressed.

Current regulations require that the skin irritation/corrosion potential of new chemicals is assessed in the rabbit Draize test, although there are opportunities to use alternative methods to identify the most aggressive materials. Previously, we have proposed that it is possible to employ a strategy that avoids the use of animals and at the same time delivers a more relevant assessment of skin irritation/corrosion potential. The approach is to identify corrosive materials in vitro and then proceed to human volunteer testing for skin irritation. In this study, the human 4-h patch test, its interpretation, and results with 29 test materials are presented. Using 14 materials not classified as "Irritating to skin" by EU criteria, 13 classified as "Irritating to skin" by suppliers, and 2 as "Corrosive - causes burns", it is demonstrated that, by evaluating these on human skin in vitro and in vivo, a significant proportion are either over-classified or under-classified. In conclusion, we are convinced that by application of the approach described in detail here, it is possible to avoid the use of animals, whilst at the same time obtaining an assessment of skin irritation/corrosion potential that is more relevant to man, and which, if required, may be used directly for classification and labelling of substances and preparations within the European Union.

The corrosive potential of a series of fatty acids-propanoic acid (C3), butanoic acid (C4), hexanoic acid (C6), octanoic acid (C8), decanoic acid (C10) and dodecanoic acid (C12)-was investigated in the in vitro skin corrosivity test (IVSCT) using both rat skin and human skin. All the fatty acids with alkyl chain lengths up to and including C8 were found to be corrosive to rat skin. When human skin was used, the corrosive/non-corrosive threshold was shifted to around the C6 fatty acid. The results are discussed in the context of a QSAR for the corrosivity of organic acids, with the putative mechanism that corrosivity is a function of the ability of the chemical to permeate the skin together with its cytotoxicity, expressed in this case as acidity (pK(a)). This mechanistic interpretation is consistent with the known differences in barrier properties between rat and human skin.

The eukaryotic fatty acid desaturases are iron-containing enzymes that catalyze the NAD-(P)H- and O2-dependent introduction of double bonds into methylene-interrupted fatty acyl chains. Examination of deduced amino acid sequences for the membrane desaturases from mammals, fungi, insects, higher plants, and cyanobacteria has revealed three regions of conserved primary sequence containing HX(3 or 4)H,HX(2 or 3)HH, and HX(2 or 3)HH. This motif is also present in the bacterial membrane enzymes alkane hydroxylase (omega-hydroxylase) and xylene monooxygenase. Hydropathy analyses indicate that these enzymes contain up to three long hydrophobic domains which would be long enough to span the membrane bilayer twice. The conserved His-containing regions have a consistent positioning with respect to these potential membrane spanning domains. Taken together, these observations suggest that the membrane fatty acid desaturases and hydrocarbon hydroxylases have a related protein fold, possibly arising from a common ancestral origin. In order to examine the functional role of these conserved His residues, we have made use of the ability of the rat delta 9 desaturase gene to complement a yeast strain deficient in the delta 9 desaturase gene function (ole1). By site-directed mutagenesis, eight conserved His residues in the rat delta 9 desaturase were individually converted to Ala. Each His-->Ala mutation failed to complement the yeast ole1 mutant. In contrast, mutation of three nonconserved flanking His residues or a partially conserved Arg residue within the conserved motif to Ala allowed for complementation of the ole1 phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

There exist various regulatory instruments the purpose of which is to ensure that the intrinsic toxic hazards associated with substances and preparations are identified. In the context of identification of skin irritation potential, the method is normally the Draize test. Guidance notes provided by the OECD and the EEC expect that corrosive substances will have been screened out by a variety of methods. Substances or preparations which cause a sufficient degree of skin irritation will be classified as skin irritants. The primary motivation behind the present work was to introduce the concept that it is possible to assess the hazard potential of a substance or preparation to produce skin irritation in a human study. In the example presented here, 20% sodium lauryl sulfate (SLS) has been chosen as the positive control. With the protocol currently devised, occluded patch treatment with 20% SLS for up to 4 hr produces an irritant response in just over half of the panel. An irritant response is taken as a clinically evident and significant increase in erythema, oedema or dryness--a minimum of a+ reaction on the ICDRG scale. At such a level of response with the positive control (both in terms of intensity and in proportion of the panel), it is then possible to judge and/or to determine statistically, whether the test material has produced a level of skin irritation which is similar to, greater, or lower than the positive control. In this way a human patch test protocol can form a fundamental component of a strategy for the replacement of animals in determination of skin irritation and corrosion potential. By use of a careful and progressive protocol and by comparison of test data against a positive control it is both possible and practical to classify substances and preparations in terms of their skin irritation potential using that endpoint in the species of concern, man.

To date the use of in vitro/alternative tests to achieve formal classification in the EEC of the toxic properties of new substances is very limited. An opportunity exists in the area of skin irritation/corrosivity to adopt a strategic approach which will limit the need to use animals. The approach would involve use of human skin in vitro to identify corrosive materials followed by ethically approved human patch testing. In the patch test, the irritation potential of the substance or preparation would be judged against a suitable positive irritant control. In addition to the avoidance of the use of animals, a further benefit would be that use of human skin should lead to a more relevant classification of skin irritation/corrosion hazard for humans.

Regulations require that many substances and chemicals are tested for their cutaneous toxicity potential. At present this is assessed in an in vivo rabbit dermal test. A rapid, robust and reliable alternative for testing the corrosive potential of substances is the in vitro skin corrosivity test, which alleviates the need to assess corrosive substances in an in vivo test. The method is based on the observation that corrosive substances cause a significant reduction in the electrical resistance of skin. It was anticipated that the more resistant nature of human skin would lead to a number of substances being classified as non-corrosive, contrary to historical in vivo animal data. In the present study 15 surfactants and eight fatty acid substances were tested on human skin in the assay. Three surfactants and four fatty acids were classified as corrosive in vivo. In the assay, the three surfactants were identified as corrosive, but all the fatty acid substances were found to be non-corrosive. It is suggested that the fatty acid substances labelled as corrosive on the basis of animal data are over-labelled, that is, in humans the substances would not lead to a corrosive effect. It is envisaged that the in vitro human skin corrosivity test will be used in conjunction with human patch tests, currently being developed. This approach should allow a reduction in animal testing and a more relevant classification of skin irritation potential to humans.

To meet the EEC regulations for classification, packaging, labelling and transportation, many new substances have to be tested for their potential cutaneous toxicity. At present, this is assessed in an in vivo rabbit dermal test. The in vitro skin corrosivity test has been developed to identify those substances which would cause a corrosive response in an in vivo test, thereby alleviating the need to assess corrosive substances in an in vivo test. The method, which uses ex vivo rat skin, is based on the observation that corrosive substances cause a significant reduction in the electrical resistance of skin. The present study investigated the possibility of using human skin in the in vitro skin corrosivity test to provide a means of direct assessment of effect in humans. 12 substances were assessed in this model. The transcutaneous electrical resistance (TER) following treatment with distilled water is essentially the same as that for untreated skin. Of the six preparations that would be formally classified as corrosive by the EEC, on the basis of a 4-hr rabbit covered patch test, all caused a sharp fall in the TER for each specimen of human skin on which they were tested. On the basis of these data, the TER threshold for determination of potential corrosivity was judged to be 11.0 kohms/disc of skin. For the five preparations which would be classified as irritant or would not require a label, the reduction was usually much less marked. Continuing work in this laboratory is directed towards an assessment of a much wider range of chemicals using the human skin in vitro model, ultimately with a view to its use as a method of detecting those substances which may be corrosive to the skin of humans, leading to their classification and appropriate labelling.

The collaborative study reported here was performed to evaluate the reliability of the skin corrosivity test in vitro when performed in independent laboratories. Twenty substances were examined in each of three participating laboratories and the results were compared with existing data from standard assays in vivo. The skin corrosivity test is based on the assumption that corrosive substances destroy the skin's natural outer protective barrier, the stratum corneum. Corrosive action in vitro is measured by a fall in the transcutaneous electrical resistance (TER) below a predetermined threshold. A refined test using a MgSO(4) electrolyte solution for TER measurements has recently been shown to reduce the number of false positive results in the test, while maintaining excellent predictive value for skin corrosive substances. Although in the present study there was some variation between laboratories in terms of the absolute mean TER values obtained, all 6 substances corrosive in vivo were correctly predicted by the three laboratories. The other 14 substances ranged from being non-irritant to severe skin irritants in vivo, but the test was unable to discriminate between these different categories in any of the three laboratories. However, these inter-laboratory comparisons demonstrate that the refined skin corrosivity test is a robust and reliable method in vitro for identifying potential skin corrosive substances.

Administration of low levels of lead (0.001, 0.005 and 0.025 micrograms/g/day p.o.) to neonate rats from age three days to eight weeks failed to alter the activities of hepatic glucose-6-phosphatase, fructose-1,6-diphosphatase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase, the four key gluconeogenic enzymes. Administration of lead at a higher dose (0.1 micrograms/g/day p.o.) was also observed to produce no alterations in enzyme activity at eight weeks. However, the higher dose did enhance the activities of fructose-1,6-diphosphatase and phosphoenolpyruvate carboxykinase at age six weeks. Plasma insulin and glucagon were not significantly altered by up to 0.025 micrograms/g exposure to lead until eight weeks of age, although levels of these hormones appear to be slightly dose-responsive tending towards elevated glucagon and decreased insulin levels with increasing lead dosage. At 0.1 micrograms/g/day glucagon was significantly increased at eight weeks. Blood glucose and hepatic glycogen remained unaltered. Blood, hepatic and pancreatic lead levels were unchanged by treatment with lead up to 0.025 micrograms/g/day to eight weeks of age, but there was evidence of lead accumulation in pancreatic tissue whereas levels of the metal in the liver paralleled those in the blood. Significant increases were observed with 0.1 micrograms/g/day lead at six and eight weeks in blood and pancreas. Data are presented which suggest that six week old animals are more influenced by subacute lead exposure than are the eight week old animals, as reflected in some alteration of gluconeogenic enzyme activity in younger rats.

Maternal hypothyroidism, when induced by surgical thyroidectomy with parathyroid hormone replacement, results in fewer live fetuses and smaller fetuses at the 22nd day of gestation. The hypothyroid mother shows the ability to mobilize adequate amounts of glucose even at the expense of her own reserves but the supply of glucose to the fetus appears to be impaired. These fetuses have subnormal skeletal muscle and liver glycogen and are severely hypoglycemic. The impaired development of these fetuses may result from alterations of either transplacental carbohydrate transport or placentofetal carbohydrate metabolism.

The circadian spontaneous locomotor activity of rats exposed to 0.1 mg lead/kg, po from 3 days until 4 and 6 weeks of age was similar to that of controls. However, hyperactivity during initial hours of recording was observed in rats that were treated with lead (Pb) until 8 weeks of age. When treatment was discontinued for 2 weeks, previously Pb-exposed rats had a tendency to be hypoactive. The elevated locomotor activity in 8-week-old lead-treated rats was not accompanied by any significant changes of noradrenaline levels in the cerebral cortex or hippocampus. Alterations in noradrenaline content of the hippocampus were, however, observed in rats that had been treated with Pb for 4 and 6 weeks. The turnover rate of noradrenaline in the hippocampus was also found to be significantly reduced following treatment for 6 weeks. Regional distribution of Pb in the brains of lead-exposed rats showed a large accumulation of the metal in the hippocampus. The alterations of the noradrenergic function in the hippocampus may be associated with the preferential storage of lead in this region.

The present study examined the possibility that strabismic amblyopia may be explained in terms of binocular competition. This was done by measuring visual acuity in strabismic kittens whose deviating eye was placed at an advantage over the other. Four groups of kittens were studied: Normal/Normal (N/N); Lid-sutured/Normal (LS/N); Strabismic/Normal (S/N); Strabismic/Lid-sutured (S/LS). In conditions 2-4 kittens underwent lateral rectus section and/or unilateral lid suture around the time of natural eye opening. Visual acuity measurements were made using the jumping stand technique and the following results were obtained: all S/N kittens showed an acuity deficit in the strabismic eye; each S/LS kitten had a better acuity in the deviating eye than any S/N kitten; the acuity of the LS/S kittens was slightly below that of the N/N kittens or the normal eye of the LS/N kittens. These data suggest that binocular competition can account for essentially all of the strabismic amblyopia observed in kittens.

The effect of three different levels of 24-h hypobaric hypoxia (630 mm Hg, 520 mm Hg, and 340 mm Hg) on rat lung cAMP and cGMP was studied. Liver was also examined to evaluate comparatively with lung. Lung cAMP concentration averaged 33.6 +/- 2.2 (SE) pmol/mg protein, and cGMP averaged 2.4 +/- 0.21 (SE) pmol/mg protein. Twenty-four-hour hypoxia resulted in a significant 40% decrease in lung cAMP at 520 and 340 mm Hg. The magnitude of change was the same for both hypoxic levels suggesting threshold dependency. In contrast, liver cAMP was not affected by the hypoxic exposure. Lung and liver cGMP were also relatively refractory to hypoxia. The decreased cAMP concentration seen in the hypoxic lung returned to normal level within 24 hours. These data show that acute hypoxia has a selective action on lung cAMP.