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This is the final report of the Management Team for a European Commission/British Home Office (EC/HO) validation study on alternatives to the Draize eye irritation test. The principal goal of the study was to establish whether one or more of nine non-animal tests could be used to replace the Draize test for all severely irritating materials (or those belonging to specific classes) or the animal test completely for chemicals with or without regard to chemical class. Sixty chemicals were independently selected, coded and supplied, then the data obtained in 37 laboratories were analysed independently. The results of comparisons between 27 alternative test index scores and the Modified Maximum Average Scores (MMASs) obtained in the Draize eye test were compared. Tables of results showing Pearson's product moment correlation coefficients and Spearman's rank coefficients for each laboratory are provided, and correlation matrices of alternative test index scores among the different groups of laboratories are shown for each endpoint. Scatterplots are provided, in which the alternative test scores obtained by the lead laboratories for the nine tests are plotted against the MMAS for the full set of chemicals and 12 surfactants. It is concluded that, with the possible exception of predicting the irritancy of surfactants, none of the nine tests met any of the four performance targets. Possible reasons for this outcome are discussed.

A retrospective analysis of the association between skin irritancy and the potential to cause contact sensitization has been performed employing a historical database for 50 chemicals and formulations. Correlations between the results of Draize skin irritation tests and skin sensitizing activity measured with the occluded patch test of Buehler have been examined. Weak, but nevertheless statistically significant, associations between contact sensitization and skin irritancy have been demonstrated. It is proposed that such correlations are consistent with the irritant properties of a material exerting an important influence on the extent to which contact sensitization is induced.

The induction of respiratory sensitization in guinea pigs to diphenylmethane-4,4'-diisocyanate (MDI), a known human respiratory allergen, has been investigated and different routes of exposure compared. Guinea pigs were exposed to MDI by i.d. injection, by topical application or by inhalation. Pulmonary hypersensitivity was measured subsequently as a function of changes in respiratory rate following challenge with atmospheres containing MDI. In addition, contact hypersensitivity was measured by topical challenge and antibody responses evaluated by enzyme-linked immunosorbent assay (ELISA) and passive cutaneous anaphylaxis (PCA). Attempts to sensitize guinea pigs by inhalation exposure to MDI were unsuccessful. Antibody responses and contact sensitization were both infrequent and low grade, and no animals exhibited pulmonary responses following challenge with atmospheric MDI. In contrast, sensitization by either i.d. injection or topical application of MDI induced antibody responses in the majority of animals. Moreover, a proportion of animals in each case exhibited pulmonary responses following subsequent inhalation challenge. These data indicate that the route of exposure influences markedly the effectiveness of sensitization to respiratory allergens such as MDI and that skin contact may be an important cause of occupational respiratory allergy.

The current OECD guideline for the assessment of eye irritation recommends, in initial considerations, the use of data from skin irritation tests as a prescreen to detect the most severely irritating materials, assuming that materials that are severely irritating to the skin are also significantly irritating to the eyes. However, analysis of data for 179 materials tested in this laboratory for both dermal and ocular irritancy, revealed that, at most, only 36% of severe eye irritants were also severe skin irritants. This resulted in a significant number of rabbits developing severe ocular effects that had not been predicted from the dermal responses. This study reports the results of an alternative approach for predicting severe eye irritants. The approach was a two-stage test battery in vitro: the first stage was a cytotoxicity assay utilizing the K562 cell line; the second was the isolated rabbit eye test. In contrast to the use of skin irritation tests, the in vitro battery was significantly more predictive (83% of severe eye irritants were detected). Although the incidence of false positive responses in each of the assays precludes their routine use as a replacement from the in vivo rabbit eye test they provide a powerful aid to reducing animal use and guiding in vivo studies to minimize the severity of effects. The need for an interlaboratory assessment to confirm and extend these findings is discussed.

The BTS/UKEMS Working Group was established to consider dose selection for in-vivo mutagenicity assays, and the feasibility of establishing criteria for identifying maximum dose levels that did not involve relating these to a high fraction (50-80%) of the estimated LD50 value. In view of the importance attached by regulatory authorities to negative results from in-vivo tests, namely reassurance that mutagenic potential seen in vitro could not be expressed in the whole animal, the need for the use of some form of MTD was accepted. The crucial question facing the group was whether the use of 'evident toxicity' rather than some index of lethality would result in any meaningful loss of sensitivity of the assays. The group endorsed the concept of a limit dose for relatively non-toxic compounds and supported the use of a value of 2 g kg-1 for single oral dosing and 1 g kg-1 for repeated dosing, in line with the general values used by the OECD and EEC. In order to assess the question of sensitivity of the assays the group considered the available data from the published literature, and from 'in-house' studies, on dose-response for mutagenicity and for toxicity, using the same dosing regime. It rapidly became apparent that few data were available, and that these were limited essentially to the micronucleus test; there were inadequate data to consider any other methods. In addition, there was the added complication that most of the available data related to protocols which were less rigorous than those currently recommended. The group thus concentrated on the micronucleus test because of its relatively wide use and since it had given rise to specific concerns due to a recent recommendation from the relevant EPA Gen-Tox group namely that the top dose should be 50-80% of the estimated LD50 value. It was noted that the EPA appear to be considering this approach as one alternative when dose setting, with the possibility of the use of a dose producing overt toxicity as another. Available data indicate that around 90% of tested mutagens would have been identified using an MTD based on non-lethal criteria. Moreover the percentage would be expected to be higher if all tests had been carried out to current protocol standards. However, the possibility of missing compounds could not be completely eliminated. Furthermore, it was important to put the bone marrow study in context. In the UK, Regulatory Authorities would not accept negative data from one tissue as providing adequate reassurance regarding the absence of in-vivo activity. Data from at least one other assay using a different tissue would be needed. It would not therefore be necessary to use 'heroic' and unrealistically high doses in the bone marrow assay in a misguided attempt to obtain absolute assurance from the one study. It is believed that Regulatory Authorities in most other countries would seek data from more than one in-vivo assay before discounting positive data from in-vitro studies. The group also considered in quantitative terms, the actual difference in MTD in the mouse if based on 'evident toxicity' or on lethality (an estimate of a dose equivalent to 50-80% the LD50 value). There was relatively little difference between the two levels, due to the steep dose response for toxicity seen in the mouse with most compounds.

The current OECD guideline for the assessment of eye irritation recommends, within its initial considerations, the use of data from skin irritation tests as a pre-screen to detect the most severely irritating materials, its being assumed that materials that are severely irritating to the skin are also significantly irritating to the eyes. However, analysis of data for 223 materials, tested in this laboratory for both dermal and ocular irritancy, revealed that only 23% of severe eye irritants were also severe skin irritants. This resulted in a significant number of rabbits developing severe ocular effects that had not been predicted from the dermal responses. This study reports the results of an alternative approach for predicting severe eye irritants. The approach was a two-stage in vitro test battery; the first stage was a cytoxicity assay using the K562 cell line; the second was the isolated rabbit eye test. In contrast to the use of skin irritation tests, the in vitro battery was significantly more predictive (83% of severe eye irritants were detected). Although the indicence of false positive responses in the assay precludes its routine use as a replacement for the in vivo rabbit eye test, the test battery provides a powerful aid to reducing animal use and guiding in vivo studies to minimize the severity of effects.

In vivo rabbit eye tests have attracted criticism on both scientific and ethical grounds. Consequently, there is a need to develop new approaches that still provide the necessary information on eye irritation hazard but that minimize or even avoid the use of whole laboratory animals. Cytotoxicity models have been used to predict the ocular irritancy of surfactants, since this class of chemicals has an essentially common action on cell membranes which involves membrane disruption. The aim of the present studies was to compare the predictive ability of two in vitro cytotoxicity tests, the K562 and the red blood cell lysis tests, in the assessment of the in vivo eye irritancy of surfactants. The results of these studies on 14 selected surfactant materials showed that the K562 assay was only modestly predictive of the in vivo response, with a specificity of 86% but a sensitivity of only 57%. In contrast, the red blood cell lysis test was more predictive, correctly identifying all irritants tested. In addition, all non-irritant surfactants examined were predicted and a high (89%) ability to rank irritant effect was demonstrated. The red blood cell lysis test could be a powerful addition to a testing strategy or pre-screen for the evaluation of surfactant chemicals.

Seventeen raw materials and chemical formulations were evaluated in the Eytex System to determine the ability of this assay in vitro to predict eye irritation potential in vivo. All the test samples, which represented a wide range of chemical types and eye irritancy potential in vivo, were provided by one of the participating laboratories. Historical data from tests in vivo were available for each of the test samples, so testing in vivo specifically for this study was not necessary. Samples were evaluated by both the membrane partition assay (MPA) and the rapid membrane assay (RMA). The sensitivity, specificity, predictivity and equivalence of the Eytex assay were determined by comparison with the rabbit eye irritation data, using each of the different Eytex Draize Equivalent (EDE) classification schemes. Regardless of the classification scheme used, the correlation between the scores in vivo and in vitro was poor. The Eytex System consistently overclassified materials of low irritancy in vivo and underclassified those test materials of moderate irritancy or above. On the basis of the results from the 17 materials tested in this study, the Eytex System appears unsuitable as a replacement in vitro for ocular irritancy testing of all types of chemical. However, Eytex may have a place as a pre-screening method used as part of a test battery.

The enucleated eye test (EET) for screening eye irritants was first described in 1981. In order to validate this method, an interlaboratory trial was established. Substances of varying degrees of ocular irritation were tested using both a 10 and a 60 s exposure period. The 10 s exposure time allowed the most irritant of substances to be identified by the EET. For the majority of substances tested, the 60 s exposure generated greater responses than the 10 s exposure. By assessing the in vitro response, each substance could be graded into one of four irritancy ratings. Despite the three laboratories adopting different in vitro grading systems, consistency amongst the laboratories was good, with 22 of the 27 substances tested being rated within one in vitro category. In comparison with existing in vivo data, the in vitro responses from a 10 s exposure correlated better than the reactions from a 60 s exposure protocol. The effects of liquid test substances were more successfully predicted in vitro than the ef...

Summary: The murine local lymph node assay (LLNA) has been developed as an alternative method for the identification of skin sensitizing chemicals. Measurement is made of the proliferation of lymphocytes within lymph nodes draining the site of exposure to the test chemical. This report describes a collaborative study in which 25 test chemicals were evaluated in each of four participating laboratories and the results compared with existing data from guinea pig predictive tests. Nineteen chemicals were predicted to be sensitizers in the guinea pig. Of these, 14 were correctly identified in the LLNA (9 by all laboratories and 5 by two or three laboratories). Five chemicals predicted to be contact allergens by guinea pig tests failed to elicit positive LLNA responses. With adoption of a 5 day rather than a 4 day exposure period to the test chemical and administration of maximum soluble test concentrations, positive reactions could be obtained with each of the chemicals initially negative in the LLNA. ...

With the exception of the Draize Test, the guinea-pig test methods currently accepted by regulatory authorities worldwide are well able to predict the potential of a material to cause skin sensitization. Nevertheless, (a) some methods are more sensitive than others (e.g. adjuvant tests are generally more sensitive than non-adjuvant tests); (b) methods cannot be sufficiently standardized to give full reproducibility of results between laboratories; and (c) most methods are based on subjective visual grading of skin reactions--difficulties thus arise when testing coloured or irritant materials. Laboratories must be able to show the sensitivity of the method(s) they use by demonstrating that positive reactions occur with mild/moderate contact allergens rather than the strong/extreme sensitizers currently recommended in certain guidelines, specifically in the EEC Test Method. The sensitivity of the adjuvant tests is such that it is possible to halve the minimum number of animals required by present regulatory guidelines without compromising the capacity of the tests to detect weak/mild sensitizers. A similar review has not yet been made for non-adjuvant tests. Alternative test methods, including some recently developed mouse models, offer several advantages, including more objective endpoints. These tests have not been extensively validated and this precludes their use at present for regulatory purposes other than to confirm the sensitization potential of a material. Two new test methods using mice, the Mouse Ear-swelling Test and the Local Lymph Node Assay, appear promising. They should undergo rigorous interlaboratory testing to determine their sensitivity and specificity. In vitro methods do not represent a viable alternative in the foreseeable future. An approach using quantitative structure-activity relationships is the most likely route to a non-animal model, but this will require considerable research, development and validation. Human sensitization tests have generally not been used for the classification of substances as non-sensitizers. This is because of an absence of internationally agreed test protocols, the lack of positive controls and because the methods for establishing the sensitivity of human tests are less developed than for animal tests. Nevertheless, for products for which direct human contact is intended, predictive tests in human volunteers can be considered. The EEC Directive for the Classification, Packaging and Labelling of Dangerous Substances provides a reasonable approach to the evaluation of skin sensitizers.(ABSTRACT TRUNCATED AT 400 WORDS)

A murine local lymph node assay has been developed for the identification of contact sensitizing chemicals. In the present study, the performance of the local lymph node assay has been evaluated with twenty-four coded chemicals of previously unknown skin sensitizing potential and the results compared with predictions made from concurrent occluded patch tests (Buehler tests) in guinea pigs. The data presented demonstrate that the local lymph node assay successfully identified those chemicals that were classified as moderate or strong skin sensitizers in the Buehler test. In the present series of experiments, chemicals predicted to be mild sensitizers in the Buehler test were classified as 'not strong sensitizers' in the local lymph node assay. In the majority of instances, the Buehler test and local lymph node assay were in agreement with regard to the identification of non-sensitizing chemicals. However, two chemicals that were classified as non-sensitizers in the guinea pig test exhibited positive responses in the local lymph node assay and were predicted to be sensitizers. Some coloured chemicals resulted in obscured Buehler readings and, here, assessment was based upon histological examination of the challenge site. These compounds were examined also in the local lymph node assay and similar predictions of sensitizing potential were made. Taken together, the data reveal close, but not absolute, concordance between the local lymph node assay and the Buehler test. The relative merits of these predictive test methods are discussed.

A lymphocyte transformation test (LTT) was able to distinguish between nickel-sensitive subjects and non-nickel-sensitive controls. Sixty-one out of 66 (92%) nickel-sensitive subjects had positive stimulation indices in 6- and/or 7-day assays using 5 micrograms/ml of nickel sulphate, whereas none of the 43 controls gave positive results. Stimulation indices were not enhanced by the patch testing of subjects to nickel before performing the LTT. A weak correlation was seen between the results of the LTT and the macroscopic degree of patch-test reactivity. The concentration of nickel sulphate used (5 micrograms/ml) did not have a significant non-specific mitogenic effect.

Inhalation exposure of the guinea pig to the respiratory sensitising chemicals trimellitic anhydride (TMA) or diphenylmethane di-isocyanate down-regulated the IgE antibody response to subsequent systemic challenge with alum-precipitated hapten-protein conjugate. The suppression was isotype and hapten specific, challenge with unrelated hapten-protein conjugate resulting in high-titre IgE and IgG1 antibody responses. Preliminary results indicate that the down-regulation of the IgE response to TMA markedly reduced the capacity of the animals to undergo a bronchoconstriction response following challenge with an aerosol of TMA, suggesting that the initial route of exposure may have profound effects upon the development of occupational respiratory allergy.

As a follow-up to a prevalidation study on in vitro tests for replacing the in vivo rabbit test for skin corrosivity, an international validation study was conducted during 1996 and 1997 under the auspices of ECVAM. The main objectives of the study were to: (a) identify tests capable of discriminating corrosives from non-corrosives for selected types of chemicals and/or all chemicals; and (b) determine whether these tests could identify correctly known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals. The tests evaluated were the rat skin transcutaneous electrical resistance (TER) assay, CORROSITEX(TM), the Skin(2TM) ZK1350 corrosivity test and EPISKIN(TM). Each test was conducted in three independent laboratories. 60 coded chemicals were tested. All of the tests evaluated showed acceptable intralaboratory and interlaboratory reproducibilities, and the TER, Skin(2) and EPISKIN tests proved applicable to testing a diverse group of chemicals of different physical forms, including organic acids, organic bases, neutral organics, inorganic acids, inorganic bases, inorganic salts, electrophiles, phenols and soaps/surfactants. Two of the four tests evaluated, the TER assay and EPISKIN, met the criteria agreed by the Management Team concerning acceptable underprediction and overprediction rates for them to be considered scientifically validated for use as replacements for the animal test for distinguishing between corrosive and non-corrosive chemicals for all of the chemical types studied [objective (a)]. EPISKIN was the only test able to distinguish between known R35 (UN packing group I) and R34 (UN packing groups II & III) chemicals, for all of the chemical types included, on an acceptable number of occasions [objective (b)]. The corrosive potentials of about 40% of the test chemicals could not be assessed with CORROSITEX, and the assay did not meet all of the criteria for it to be considered acceptable as a replacement test. However, CORROSITEX may be valid for testing specific classes of chemicals, such as organic bases and inorganic acids. The Skin(2) assay did not meet the criteria for it to be considered scientifically validated. Thus, the validities of (i) the TER and EPISKIN assays for discriminating corrosives from non-corrosives, and (ii) the EPISKIN assay for identifying correctly known R35/I and R34/II & III chemicals, have been demonstrated in this study. CORROSITEX appears to be valid when used only with certain types of chemicals.

This study evaluated a single intradermal injection model in the guinea pig with subsequent inhalation challenge and serological analysis as a method to predict the potential of chemicals to induce respiratory allergy. Four known respiratory allergens (trimellitic anhydride, diphenyl methane diisocyanate, phthalic anhydride and toluene diisocyanate (TDI)) were screened by two industrial research laboratories using this protocol. Dinitrochlorobenzene, a potent contact allergen, was included as a negative control material. In both laboratories, the respiratory allergens, but not the contact allergen, induced high titre antigen-specific antibodies in treated animals. The inhalation challenge results were similar in both laboratories but were less conclusive in that exposure to free TDI failed to induce pulmonary responses, probably because it fails to penetrate to the deep lung in sufficient concentration. Although the assay shows promise as a means of identifying chemical respiratory sensitisers, its use as a routine screen for the prediction of the ability of materials to induce respiratory allergy in man is probably questionable.

The guinea pig maximization test and the Buehler occluded patch test are used widely to identify the sensitization potential of new chemicals. This information enables toxicologists and/or regulatory authorities to determine whether a chemical should be classified formally as a skin sensitizer. Both to improve and to harmonize these assessments internationally, the OECD has recommended recently that moderate rather than strong contact sensitizers are used as positive control substances. The purpose is to ensure an adequate level of sensitivity in sensitization assays performed at specific testing establishments. Results from two laboratories reported here show that the minimum acceptable standard laid down by the OECD can be achieved and indeed commonly exceeded by a substantial margin. Furthermore, results with these positive controls in a new method, the local lymph node assay, also appear to satisfy similar criteria, suggesting results from this assay, including negative data, should be acceptable for classification purposes. However, a review of the way in which results with new chemicals will be interpreted for regulatory purposes, in the context of positive control data, reveals that considerable inadequacies still exist. It is recommended that ultimately, sensitization data can only be interpreted meaningfully (i.e. to protect humans from sensitization hazards) by considering the potency of the contact allergen in the context of the sensitivity of the assay performed at the particular testing institution.

A list of 176 chemicals, all of high or consistent purity and stable on storage, has been developed using available comprehensive in vivo rabbit skin irritation data. No new in vivo testing was conducted to qualify a chemical for inclusion in the list. The chemicals were tested undiluted in in vivo studies, apart from those chemicals where high concentrations could be expected to cause severe effects. The in vivo data were generated in studies carried out since 1981 according to OECD Test Guideline 404 and following the principles of Good Laboratory Practice. The data were obtained from tests normally using at least three rabbits evaluated at the same time, involving application of 0.5 g or 0.5ml to the flank under semi-occlusive patches for 4 hr, and in which observations were made at least 24, 48 and 72 hr after removal of the patch. The chemicals represent a range of chemical classes [acids, acrylates/methacrylates, alcohols, aldehydes, alkalis, amines, brominated derivatives, chlorinated solvents, esters, ethers, fatty acids and mixtures, fragrance oils, halogenated aromatics, hydrocarbons (unsaturated), inorganics, ketones, nitrites, phenolic derivatives, S-containing compounds, soaps/surfactants, triglycerides] and different degrees of irritancy. They are ranked for skin irritation potential on the basis of a 'primary irritation index'. These chemicals could be used in validation tests of promising alternatives to the in vivo rabbit skin irritation/corrosion test. This is an essential step in the progression to regulatory acceptance of alternative procedures.

In vivo rabbit eye tests have attracted criticism on both scientific and ethical grounds. Consequently there is a need to introduce methods that provide relevant information whilst avoiding the use of whole laboratory animals. Cytotoxicity models have been used to predict the ocular irritancy of surfactants since the action of this class of chemicals on cell membranes is essentially common and involves membrane disruption. The aim of the present studies was to compare the predictive ability of two in vitro cytotoxicity tests (the K562 and the red blood cell lysis tests) in the assessment of the in vivo eye irritancy of surfactants. The results of these studies on 14 selected surfactant materials showed that the K562 assay was less likely to over-estimate effect (when assessed over a wide range of surfactant concentration) than had previously been observed. This increased specificity (86%) was accompanied by a decrease in sensitivity (57%), the ability to correctly identify 'irritant' surfactants. In contrast, the red blood cell lysis test was more predictive, correctly identifying all irritants tested. In addition, all non-irritant surfactants examined were predicted by this test and a high (89%) ability to rank irritant effect was demonstrated. The red blood cell lysis test could be a powerful addition to a testing strategy or pre-screen for the evaluation of surfactant chemicals.

The expression of MHC class II (Ia) antigens on mouse keratinocytes was studied following both the induction and elicitation of contact sensitivity, and after primary irritant reactions. IA+ and IE+ keratinocytes were detected, using an indirect immunofluorescence assay on epidermal sheets, only after the induction and elicitation of contact sensitivity with the sensitizers oxazalone, picryl chloride and 2,4-dinitrochlorobenzene but not with formaldehyde. Ia+ keratinocytes were not detected after epicutaneous application of the non-sensitizing irritants croton oil, SDS and anthralin, or following attempted sensitization of nude mice, suggesting that the expression of Ia antigen on keratinocytes during contact sensitivity reactions is T-cell mediated. Because Ia antigen expression on keratinocytes could be detected only several days after induction or elicitation of contact sensitivity, and contact sensitization could also be demonstrated to occur independently of aberrant Ia expression, Ia+ keratinocytes cannot be involved in the initiation of these reactions. However, they might be important in exerting an immunomodulatory influence during the later stages of the responses to certain sensitizers.

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.

The purpose of this review is to identify the potential benefits and limitations of in vitro and ex vivo techniques in the identification of immunotoxicants and allergens, and in the investigation of the mechanisms through which chemicals perturb immune function.

SUMMARY The fate of 2,4-dinitrochlorobenzene, a potent contact sensitizing chemical, and 2,4-di-chloronitrobenzene, a non-sensitizcr, was compared following their application to the skin of BALB/c mice. Although both chemicals were able to bind to protein in vitro and were capable of being absorbed across mouse skin in vivo, only 2,4-dinitrochlorobenzene was able to bind to cells in the skin and to induce the movement of these cells from the epidermis into the dermis and ultimately into the draining lymph nodes. The sensitization potential of a chemical may therefore be dependent on its ability to associate with and stimulate the efflux of cutaneous antigen-presenting cells.

SUMMARY Serial biopsies during the first 24 hours after dinitrochlorobenzene (DNCB) challenge in fifteen sensitized patients have shown that DNCB associates with Langerhans cells within 1 hour of application, and has reached the dermis around the appendages by 6 hours.

Immunotoxicity is defined as the adverse effects of foreign substances (xenobiotics) on the immune system. Two types of effects are possible: immunosuppression (which may result in an increased susceptibility to infection or to the development of tumours) and immunopotentiation (which may manifest as an allergy or as autoimmunity). There is, as yet, little evidence that well controlled occupational exposure to industrial chemicals has led to clinically significant immunosuppression. In contrast, a number of industrial chemicals have been shown to cause immunopotentiation in exposed populations, producing occupational asthma and contact dermatitis and possibly autoimmunity. In experimental models, immunosuppression (usually assessed by in vivo or in vitro immune function tests) has been induced by a wide range of chemicals but there are a few reports of the immunosuppression leading directly to an increased susceptibility to infection or to the development of tumours. Predictive experimental models are available for type IV allergic reactions, but the identification of chemicals that have a potential to cause other types of allergy or autoimmune reactions requires further research and the development and validation of new animal models. It is considered that routine subacute and chronic toxicity studies should include a full gross and histopathological assessment of the lymphoid organs to more accurately detect the potential of a chemical to cause immunotoxicity. Should such studies indicate that a substance has affected the immune system directly, an assessment of overall immune competence and function tests may be necessary using dose levels below those which cause frank toxicity. However, precise interpretation of immune function tests in terms of their relevance to human health requires an improved understanding of the extent of the functional reserve of the immune system. A strategy for assessing immunotoxicity in exposed human populations demonstrates a need for reliable clinical assessment, accurate medical record-keeping, an environmental and biological monitoring for levels of contaminating chemicals and the judicious use of well-validated immune function tests.

An ELISA procedure has been developed to measure specific IgG antibodies against rat urine. Using this method antibody levels in individuals with differing exposure levels to rat have been measured. Mean antibody titres in groups of workers regularly exposed to rats were higher than those in groups currently or totally non-exposed. However, only in the group of workers exposed and sensitized to rats were IgG levels significantly elevated. Despite this significant difference in mean antibody titres there was considerable overlap between individuals in each group, and there was no direct relationship between exposure to rats and levels of IgG anti-rat urine antibody. Our results suggest a stronger correlation between sensitization to rats and the levels of antigen-specific IgG antibodies.

Guinea pigs could be immunologically sensitised (as shown by the development of antigen-specific homocytotropic antibodies) to toluene diisocyanate by exposing them for 3 h a day for 5 consecutive days to atmospheres containing free chemical. Pulmonary reactions could be elicited in many of the sensitised animals by challenging them with atmospheres containing protein conjugates of the chemical and then measuring changes in respiratory rate. Successful elicitation of pulmonary reactions appeared to depend upon a number of factors, including the quality of the protein conjugate used for the challenge, but possibly also the development of IgE as well as IgGl antibodies. Antigen-specific homocytotropic antibodies were detected in guinea pigs similarly exposed by inhalation to two non-isocyanate respiratory allergens, trimellitic anhydride and a reactive dye. Although the animals were immunologically sensitised to the chemicals, challenge with atmospheres containing appropriate chemical-protein conjugates failed to stimulate changes in respiratory rate.

Epicutaneous application of 2,4-dinitrothiocyanobenzene (DNTB) is said to result in specific immunological hyporesponsiveness and fails to induce contact sensitization. However, we demonstrate that topical exposure to DNTB causes activation of the draining lymph node in mice and the induction of contact sensitization in both rodents and a single human volunteer. In mice and rats, pre-exposure to DNTB failed to impair subsequent responsiveness to the cross-reactive allergen 2,4-dinitrochlorobenzene. These data provide evidence that DNTB cannot be regarded as an exclusive tolerogen when applied epicutaneously and indicate that attempts to define the characteristics of tolerising chemicals from analysis of this agent may be misleading.

Publisher Summary This chapter presents a study analyzing cellular events in human skin after application of dinitrochlorobenzene (DNCB). Fifteen subjects were studied—2 healthy volunteers and 13 patients with bronchogenic carcinoma. These patients entered the study at the time of diagnosis and all were ambulant, hematologically normal, and had a normal clinical sensitization reaction to DNCB. Sensitization was achieved with an occlusive patch test containing 100 μg DNCB, and the challenge procedure was similarly carried out at least 2 weeks later using 40 μg DNCB on forearm skin. Biopsies were taken from unchallenged skin of each subject (time 0) and from challenge sites at 1, 3, 6, 12, or 24 h after application of the patch. Serial sections have not revealed a close relationship between Langerhans cells and T lymphocytes during the challenge period. No apposition of these cell types was seen by electron microscopy before 12 h, and very few cells were apposed in either the epidermis or dermis at 12 or 24 h.

The injection of different doses of allogeneic adult blood leucocytes intraperitoneally into Xenopus laevis larvae at various stages of development failed to induce tolerance either in splenic mixed lymphocyte reactions or in skin graft rejection responses when the hosts were tested against their donors as 2–6 month old toadlets. The results are discussed in relation to the various methods used for the induction of transplantation tolerance.

Guinea-pigs injected intradermally with the known respiratory sensitiser trimellitic anhydride (TMA) developed high-titre antigen-specific homocytotropic (IgG1 and IgE) antibodies. Many of the sensitised animals responded to a challenge by inhalation with either free TMA or a TMA-protein conjugate with a change in respiratory rate, reflecting the onset of bronchoconstriction. Guinea-pigs were also injected intradermally with 2,4-dinitrochlorobenzene (DNCB), which is a potent skin sensitiser in man but which has not been reported to cause respiratory allergy. These animals developed only low-titre homocytotropic antibodies and were unresponsive to an inhalation challenge with either free or conjugated hapten. The animals were, however, contact-sensitised to the chemical.

Contact dermatitis, due to both allergenic (sensitising) and irritant (non-sensitising) agents is a major clinical problem in industry and the home. Although its prevalence in a population depends on the nature of employment and industry, and environmental factors, it has been shown that one type of contact dermatitis alone, hand eczema, affects 2% of men and 3% of women [1]. Industrial dermatitis in fact accounts for about 65% of all spells of absence from work in the United Kingdom recorded under prescribed diseases [9].

Mitogen- and antigen-driven T-cell proliferation is dependent upon the presence of MHC II+ accessory cells. The ability of plastic-adherent blood cells and epidermal cells to act as accessory cells was assessed using a modified 6-day lymphocyte transformation test (LTT). Accessory cells from both the blood and epidermis of patients contact-sensitized to nickel were able to mediate antigen- (nickel sulphate) and mitogen-(concanavalin A) induced proliferation of purified autologous T cells. The requirements for MHC II molecules in accessory cell function were assessed by the addition of monoclonal antibodies to the LTT Antibodies against HLA-DR markedly inhibited the antigen-specific response. In contrast, an antibody against HLA-DP only weakly inhibited cell proliferation and an antibody against HLA-DQ had no effect. These studies confirmed the importance of HLA-DR molecules in antigen-induced cell proliferation. Further work is needed to clarify the role of HLA-DP and HLA-DQ.