Refinement of the Dermal Sensitisation Threshold (DST) approach using a larger dataset and incorporating mechanistic chemistry domains.

An essential step in ensuring the toxicological safety of ingredients in consumer products is the evaluation of their skin sensitising potential. Where skin exposure is low, it is possible to conduct a risk assessment using the Dermal Sensitisation Threshold (DST), a process similar to that of the Threshold of Toxicological Concern. This paper describes work building on that previously published, whose aim was to produce a more robust tool for assessing the safety of consumer products. This consisted of expanding the Local Lymph Node Assay dataset used to define the original DST and classifying chemicals in the dataset according to their mechanistic chemistry domains. A DST of 900µg/cm(2) was derived for chemicals classified as non-reactive and non-proreactive. This value was benchmarked against human potency data for 58 fragrance allergens and was lower than the measured No Expected Sensitisation Levels for those classified as non-reactive. Use of this DST will help to eliminate the need for animal testing of non-reactive and non-proreactive chemicals where skin exposure is sufficiently low. For chemicals where a Quantitative Risk Assessment based on the DST does not give an adequate margin of safety, and those classified as reactive, a case-by-case risk assessment will be required.

An evaluation of the implementation of the Cramer classification scheme in the Toxtree software.

Risk assessment for most human health effects is based on the threshold of a toxicological effect, usually derived from animal experiments. The Threshold of Toxicological Concern (TTC) is a concept that refers to the establishment of a level of exposure for all chemicals below which there would be no appreciable risk to human health. When carefully applied, the TTC concept can provide a means of waiving testing based on knowledge of exposure limits. Two main approaches exist; the first of these is a General Threshold of Toxicological Concern; the second approach is a TTC in relation to structural information and/or toxicological data of chemicals. The structural scheme most routinely used is that of Cramer and co-workers from 1978. Recently this scheme was encoded into a software program called Toxtree, specifically commissioned by the European Chemicals Bureau (ECB). Here we evaluate two published datasets using Toxtree to demonstrate its concordance and highlight potential software modifications. The results were promising with an overall good concordance between the reported classifications and those generated by Toxtree. Further evaluation of these results highlighted a number of inconsistencies which were examined in turn and rationalised as far as possible. Improvements for Toxtree were proposed where appropriate. Notable of these is a necessity to update the lists of common food components and normal body constituents as these accounted for the majority of false classifications observed. Overall Toxtree was found to be a useful tool in facilitating the systematic evaluation of compounds through the Cramer scheme.

The Dermal Sensitisation Threshold- a TTC approach for allergic contact dermatitis.

The Threshold of Toxicological Concern (TTC) is a useful concept that is becoming of increasing interest as an addition to the arsenal of tools used for characterising the toxicological risk of human exposure to chemicals. Traditionally used for low level indirect additives, flavours and contaminants in foods, the TTC obviates the need for toxicological testing of chemicals where human exposure is low. Proposals have recently been made for the use of the TTC for low level ingredients in cosmetic and personal care products. However, use of the TTC is only protective for systemic toxicity endpoints, and cannot be used for local endpoints such as contact sensitisation. In this paper a probabilistic analysis of available sensitisation data, similar to that used in the development of the TTC, is presented. The incidence of sensitisers in the world of chemicals was estimated using the ELINCS (European List of Notified Chemical Substances) data set, and a distribution for sensitisation potency was established using a recently published compilation of Local Lymph Node Assay data. From the analysis of these data sets it is concluded that a Dermal Sensitisation Threshold (DST) can be established below which there is no appreciable risk of sensitisation, even for an untested ingredient. Use of a DST would preclude the need for sensitisation testing of ingredients where dermal exposure is sufficiently low.

Immediate contact reactions to chemicals in the fragrance mix and a study of the quenching action of eugenol.

In this study, the nature of non-immune immediate contact reactions (NIICR) produced by cinnamic aldehyde, benzoic acid and sorbic acid were investigated, with particular interest in the 'quenching' ability of eugenol. Three groups of human subjects were studied, and the guinea-pig ear was also used as a model of NIICR. Cinnamic aldehyde, benzoic acid and sorbic acid were all able to produce NIICR in the majority of subjects studied. There was a strong correlation between the susceptibility of each subject to each urticant, but no correlation between the susceptibility to NIICR and age, atopic status or tanning ability. Eugenol caused a reduction in NIICR induced by all three urticants. This 'quenching' effect was apparent even when the eugenol was applied up to 60 min prior to application of cinnamic aldehyde, and its effect was not eliminated by washing. In the guinea-pig-ear model, ear thickening was induced by all three urticants, and this response was inhibited by eugenol.

The effect and mode of action of zinc pyrithione on cell growth. I. In vitro studies.

The effects of zinc pyrithione (ZnPTO) were studied in a series of in vitro tests to determine whether its mode of action is primarily cytostatic or cytotoxic. Sodium pyrithione (NaPTO) was also studied, to check that pyrithione was the active moiety, and the known cytostatic chemical hydroxyurea was included for comparison. ZnPTO had a reversible inhibitory effect on the growth of BHK 21 cells at 0.1 microgram/ml, but had a rapid, irreversible inhibitory effect at 1 microgram/ml associated with cell rounding and detachment. NaPTO produced a similar effect but hydroxyurea produced an essentially reversible inhibition even at a dose well above that producing complete inhibition. ZnPTO and NaPTO both caused contraction, rounding and blebbing of BHK 21 cells in perfusion-chamber tests, at higher levels (1 and 10 micrograms/ml) than required for growth inhibition, but only 10 micrograms ZnPTO/ml caused lactate dehydrogenase (LDH) release. Hydroxyurea had no effects in these tests. ZnPTO and NaPTO also reduced the survival of Chinese hamster V79 cells sharply over a narrow dose range (0.01-0.03 microgram/ml), but the effect of hydroxyurea was not as sharp and occurred at much higher doses. All three showed elements of cytostasis and cytotoxicity as demonstrated by analysis of the relationship between survival and colony area. Of the three, only ZnPTO (at greater than or equal to 5 micrograms/ml) caused significant LDH release from the cells, though both ZnPTO and NaPTO (at 0.1-1 microgram/ml) inhibited cell growth as indicated by total LDH values. In studies with rat peritoneal mast cells, ZnPTO and NaPTO (at 10 ng/ml) both suppressed histamine release induced by 48/80 or Ca ionophore A23187, though neither caused histamine release directly. The combined results of these tests show that ZnPTO is primarily cytotoxic, rather than cytostatic.

Immunological studies on tartrazine and its metabolites. I. Animal studies.

Tartrazine is occasionally associated with some clinical changes which have been attributed to allergy. In tests on laboratory animals with tartrazine and its metabolites by methods which should have detected potential to induce antibody formation, no antibodies were detected except by methods which are artificial in terms of human exposure. Similarly, laboratory methods have shown that the metabolites of tartrazine, and in some cases tartrazine itself, can induce contact sensitization in guinea pigs, although there is little evidence that tartrazine can induce similar changes in man.

The effect of tartrazine on histamine release from rat peritoneal mast cells.

The release of histamine from purified rat peritoneal mast cells induced by specific antigen (egg albumin), compound 48/80 and calcium ionophore A23187 was modified by tartrazine. Histamine release induced by 48/80 and antigen was inhibited by the presence of 10(-5) to 10(-2)M tartrazine. The inhibitory effect on egg albumin induced histamine release was maximal when the tartrazine was added simultaneously with egg albumin, and was reduced by increased preincubation of the cells with tartrazine. Tartrazine had a small inhibitory effect on ionophore induced release at high concentrations, but augmented histamine release at tartrazine concentrations of 10(-3) and 10(-4)M. Augmentation of ionophore induced release was maximal at between 0-5 min preincubation of the cells with tartrazine.

In vitro cell perfusion. I. System for continuous observation of cells and analysis of perfusion fluid suitable for isolated mast cell and macrophage studies.

A system is described in which cells are perfused continuously with appropriate medium while simultaneously recording any change in cell morphology together with assay of released products (enzymes and mediators) following immunologic or non-immunologic perturbation. The released products in the perfusate are monitored continuously by automated fluorimetric assay procedures. The system is particularly applicable to investigate or toxicological studies of isolated mast cells or cultured macrophages.

An automated fluorimetric assay for the simultaneous determinations of histamine and 5-hydroxytryptamine released from rat mast cell preparations.

An automated assay suitable for the extraction and simultaneous fluorimetric determination of histamine and 5-hydroxytryptamine (5-HT) released from rat isolated peritoneal mast cells is described. The method gave good reproducibility and accuracy in use and required a sample volume of 0.5 ml for assay at a sampling rate of 30/h. The system was evaluated by assessment of its performance in the determination of histamine and 5-HT released from rat mast cells in response to compound 48/80. The detection limit for the procedure was 2 ng 5-HT base/ml and 20 ng histamine base/ml.