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E Whittle

Publications and source records attributed to E Whittle.

At least 19 recordsLinked to original sources
PubMed2002

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products.

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Animal Testing Alternatives↗
PubMed2001

Engineering delta 9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor delta 9-18:0-ACP desaturase.

Six amino acid locations in the soluble castor Delta(9)-18:0-acyl carrier protein (ACP) desaturase were identified that can affect substrate specificity. Combinatorial saturation mutagenesis of these six amino acids, in conjunction with selection, using an unsaturated fatty acid auxotroph system, led to the isolation of variants with up to 15-fold increased specific activity toward 16-carbon substrates. The most improved mutant, com2, contained two substitutions (T117R/G188L) common to five of the 19 complementing variants subjected to further analysis. These changes, when engineered into otherwise wild-type 18:0-ACP desaturase to make mutant 5.2, produced a 35-fold increase in specific activity with respect to 16-carbon substrates. Kinetic analysis revealed changes in both k(cat) and K(m) that result in an 82-fold improvement in specificity factor for 16-carbon substrate compared with wild-type enzyme. Improved substrate orientation apparently compensated for loss of binding energy that results from the loss of desolvation energy for 16-carbon substrates. Mutant 5.2 had specific activity for 16-carbon substrates 2 orders of magnitude higher than those of known natural 16-carbon specific desaturases. These data support the hypothesis that it should be possible to reengineer archetypal enzymes to achieve substrate specificities characteristic of recently evolved enzymes while retaining the desired stability and/or turnover characteristics of a parental paralog.

Amino Acid Substitution↗
PubMed2001

Fate of ethanol topically applied to skin.

Ethanol is a major component of many aerosol sprays and consumer products that are designed to contact the skin. It is theoretically possible that small amounts of ethanol from alcohol-based sprays can be absorbed across the skin or inhaled during spraying. In order to assess the potential systemic dose, three parameters were measured: the evaporation of [14C]ethanol from the skin surface, the in vitro penetration of [14C]ethanol through excised pig skin and the ethanol concentration in the blood of human volunteers following simulated use of an alcohol based deodorant spray. The rate of evaporation from Benchkote and whole pig skin was similar (t(1/2)=13.6 sec and 11.7 sec, respectively) while that from glass was longer (t(1/2)=24.8 sec). Ethanol penetration through pig skin in vitro was greater in occluded cells than in non-occluded cells (2.19 mg/cm(2) and 0.10 mg/cm(2) in 24 hours, respectively). At the maximum flux seen in this experiment under occlusion, the amount of ethanol penetrating from a 1 m(2) area of skin would give a blood alcohol level of about 4 mg% in a 70-kg man. In the human use study, none of the blood samples taken from 16 human volunteers exhibited a detectable level of alcohol. These studies provide evidence that a systemic dose of ethanol is likely to be very low after the use of formulations delivering ethanol to the skin.

Administration, Topical↗
PubMed2001

Ethnic variation in the skin irritation response.

There is a widespread, but largely unsubstantiated, view that certain skin types may be more susceptible to the effect of skin irritants than others. One expression of this would be that certain ethnic groups may also be more likely to experience skin irritation. As a consequence, when evaluating the skin compatibility of substances/preparations, these differences may need to be taken into account. However, other evidence indicates that, within any particular group, inter-individual variation is likely to be much larger than the differences between means for distinct groups. In this study, we have investigated 2 carefully matched panels of Caucasian and Japanese women volunteers to determine their topical irritant reaction, both acute and cumulative, to a range of materials. The results indicated that the acute irritant response tended to be greater in the Japanese panel and this reached statistical significance with the stronger irritants. Cumulative irritation was investigated only with the weaker irritants and, although again the trend was to a higher response in Japanese compared to Caucasian panelists, this rarely reached significance. Nevertheless, where risk assessment for skin irritation was critical, then at the population level, these differences might be relevant, both for safety in use of substances and products and for skin acceptability.

Adult↗
PubMed1999

A two-center study of the development of acute irritation responses to fatty acids.

BACKGROUND: A human 4-hour patch test has been developed for evaluating the acute irritation potential of chemicals. This method was developed for comparative irritation assessments. Although skin irritation responses in human subjects can be quite variable, this test method has proven robust in both intra- and interlaboratory tests. However, the previous interlaboratory studies were not optimal in that slightly differing protocols were used and the studies were not controlled for time of year or source of test chemicals. As a result, some variation in acute irritation responses were seen that might have been reduced somewhat had these variables been controlled to a greater extent. OBJECTIVE: The purpose of the current study was to examine interlaboratory reproducibility of the 4-hour patch test when conducted under as identical as set of test conditions as possible. METHODS: Two laboratories conducted a direct comparison study of the acute irritation potential of three structurally related, undiluted fatty acids (octanoic acid, decanoic acid, and dodecanoic acid) in comparison to a benchmark positive control chemical (20% sodium dodecyl sulfate [SDS]). The studies were run within a 4-month period using the same commercial source of test chemicals. Test subjects were treated with each chemical under occluded patch conditions for gradually increasing exposure duration up to 4 hours. The results were then evaluated in terms of total cumulative incidence of positive responses and time response patterns. RESULTS: Using statistical comparisons of the proportion of the subjects with a positive irritant reaction to each substance, the rank order of irritation potential was decanoic acid >/= octanoic acid > SDS >> and dodecanoic acid. The statistical comparisons and the time-response patterns for each chemical were nearly identical at the two laboratories. There were also very similar, and intriguing, variations in the interchemical response patterns seen in the two studies. CONCLUSION: When conducted under as controlled a set of test conditions as was reasonably possible, this acute irritation protocol shows remarkably high consistency across independent test laboratories, further supporting its continued development and acceptance as a valid and more predictive tool for assessing skin irritation potential.

Adult↗
PubMed1998

Catalytic plasticity of fatty acid modification enzymes underlying chemical diversity of plant lipids.

Higher plants exhibit extensive diversity in the composition of seed storage fatty acids. This is largely due to the presence of various combinations of double or triple bonds and hydroxyl or epoxy groups, which are synthesized by a family of structurally similar enzymes. As few as four amino acid substitutions can convert an oleate 12-desaturase to a hydroxylase and as few as six result in conversion of a hydroxylase to a desaturase. These results illustrate how catalytic plasticity of these diiron enzymes has contributed to the evolution of the chemical diversity found in higher plants.

Amino Acid Substitution↗
PubMed1998

Susceptibility to skin stinging, non-immunologic contact urticaria and acute skin irritation; is there a relationship?

Adverse skin reactions cover many types of response: toxic, irritant, allergic, urticarial, sensory, etc. The relationships between an individual's tendency to develop different types of skin response are not well-described. We examined whether those who perceive stinging might be more likely to experience urticarial, sensory and irritation reactions in skin. A panel of 86 volunteers was tested with 10% lactic acid in the nasolabial fold to assess their ability to perceive stinging. At the same time, their capacity to develop non-immunologic contact urticaria was evaluated using chemicals of different structural type and urticant ability: methyl nicotinate, benzoic acid, cinnamic acid, cinnamaldehyde and dimethyl sulfoxide (DMSO). DMSO was also used to assess sensory effects and skin irritation. 44 were classes as "stingers" and 42 as "non-stingers". The pattern of urticant reactivity in the stingers and non-stingers was essentially the same, with neat DMSO generating the strongest reactions in both groups. Sensory reactions to DMSO (stinging, itching, tingling or burning) were similar in stingers and non-stingers; although the former may have reacted more quickly, a smaller proportion reacted (64% versus 76%). The skin irritation response to DMSO was also identical in stingers and non-stingers and the intensity of the urticant response in an individual did not correlate with the intensity of their subsequent irritant reaction. In conclusion, this study demonstrated that an individual's ability to perceive skin stinging does not give a general indication of their susceptibility to other types of non-immunologic skin response. Indeed, there appeared to be little evidence of correlations between any of the skin effects studied.

Acrolein↗
PubMed1998

Patch tests versus use tests in skin irritation risk assessment.

In the risk assessment of the ability of a substance or preparation to cause skin irritation, a patch test involving human volunteers is often seen as providing definitive information. However, the skin exposure conditions found in an occluded patch may be far removed from those in a particular use situation, not least for such reasons as the high dose per unit area, the duration of exposure and the maceration of the skin. Whilst these factors may enhance sensitivity in the identification of intrinsic hazard, they may do little to ensure accurate safety evaluation for real use. In this paper, we report data from a series of studies with an unmarketed facial skin cosmetic product. Whilst the product was unexpectedly highly irritating in a standard patch test in 30 volunteers, subsequent use tests, including a 6x daily open application to the elbow for 3 weeks and 2x daily application in a half-face test lasting 3 to 4 weeks and involving 52 volunteers failed to show any evidence of skin irritation. It is concluded that the most meaningful results for skin irritation risk assessment are likely to come from studies which involve relevant patterns of exposure.

Adult↗
PubMed1997

The classification of skin irritants by human patch test.

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.

Health Planning Guidelines↗
PubMed1997

Visual assessment of human skin irritation: a sensitive and reproducible tool.

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.

Dermatitis, Irritant↗
PubMed1996

Evaluation of a human patch test for the identification and classification of skin irritation potential.

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.

Acyclic Monoterpenes↗
PubMed1994

Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenase.

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)

Amino Acid Sequence↗
PubMed1994

Identification of irritation and corrosion hazards to skin: an alternative strategy to animal testing.

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.

Animal Testing Alternatives↗
PubMed1994

The identification and classification of skin irritation hazard by a human patch test.

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.

Adult↗
PubMed1984

The effects of low-level lead exposure in developing rats: changes in circadian locomotor activity and hippocampal noradrenaline turnover.

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.

Aging↗
PubMed1983

Effects of subacute low level lead exposure on glucose homeostasis.

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.

Animals↗