Criteria for the Research Institute for Fragrance Materials, Inc. (RIFM) safety evaluation process for fragrance ingredients.

The Research Institute for Fragrance Materials, Inc. (RIFM) has been engaged in the generation and evaluation of safety data for fragrance materials since its inception over 45 years ago. Over time, RIFM's approach to gathering data, estimating exposure and assessing safety has evolved as the tools for risk assessment evolved. This publication is designed to update the RIFM safety assessment process, which follows a series of decision trees, reflecting advances in approaches in risk assessment and new and classical toxicological methodologies employed by RIFM over the past ten years. These changes include incorporating 1) new scientific information including a framework for choosing structural analogs, 2) consideration of the Threshold of Toxicological Concern (TTC), 3) the Quantitative Risk Assessment (QRA) for dermal sensitization, 4) the respiratory route of exposure, 5) aggregate exposure assessment methodology, 6) the latest methodology and approaches to risk assessments, 7) the latest alternatives to animal testing methodology and 8) environmental risk assessment. The assessment begins with a thorough analysis of existing data followed by in silico analysis, identification of 'read across' analogs, generation of additional data through in vitro testing as well as consideration of the TTC approach. If necessary, risk management may be considered.

Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT).

The skin sensitization potential of eight unsaturated and one saturated lipid (bio)chemicals was tested in both the LLNA and the GPMT to address the hypothesis that chemicals with unsaturated carbon-carbon double bonds may result in a higher number of unspecific (false positive) results in the LLNA compared to the GPMT. Seven substances (oleic acid, linoleic acid, linolenic acid, undecylenic acid, maleic acid, squalene and octinol) gave clear positive results in the LLNA (stimulation index (SI)> or = 3) and thus would require labelling as skin sensitizer. Fumaric acid and succinic acid gave clearly negative results. In the GPMT, besides some sporadic skin reactions, reproducible skin reactions indicating an allergic response were found in a few animals for four test substances. Based on the GPMT results, only undecylenic acid would have to be classified and labelled as a skin sensitizer according to the European Dangerous Substance Directive (67/548/EEC) (results for linoleic acid were inconclusive), while the other seven test substances would not require labelling. Possible mechanisms for unspecific skin cell stimulation and lymph node responses are discussed. In conclusion, the suitability of the LLNA for unsaturated compounds bearing structural similarity to the tested substances should be carefully considered and the GPMT should remain available as an accepted test method for skin sensitization hazard identification.

Nickel allergy in mice: enhanced sensitization capacity of nickel at higher oxidation states.

Attempts to induce contact hypersensitivity to nickel in mice using, e.g., Ni(II)Cl2 often failed. Here, we report that sensitization was achieved by injecting Ni(II)Cl2 in combination with either CFA or an irritant, such as SDS and PMA, or IL-12, or by administering nickel at higher oxidation states, i.e., Ni(III) and Ni(IV). Although Ni(II), given alone, was ineffective in T cell priming, it sufficed for eliciting recall responses in vivo and in vitro, suggesting that Ni(II) is able to provide an effective signal 1 for T cell activation, but is unable to provide an adequate signal 2 for priming. Immunization of mice with nickel-binding proteins pretreated with Ni(IV), but not with Ni(II), allowed them to generate nickel-specific CD4+ T cell hybridomas. Ni(II) sufficed for restimulation of T cell hybridomas; in this and other aspects as well, the hybridomas resembled the nickel-specific human T cell clones reported in the literature. Interestingly, restimulation of hybridomas did not require the original Ni(IV)-protein complex used for priming, suggesting either that the nickel ions underwent ligand exchange toward unknown self proteins or peptides or that nickel recognition by the TCR is carrier-independent. In conclusion, we found that Ni(III) and Ni(IV), but not Ni(II) alone, were able to sensitize naive T cells. Since both Ni(III) and Ni(IV) can be generated from Ni(II) by reactive oxygen species, released during inflammation, our findings might explain why in humans nickel contact dermatitis develops much more readily in irritated than in normal skin.

T cell cross-reactivity to heavy metals: identical cryptic peptides may be presented from protein exposed to different metals.

The mechanisms by which metals induce activation of T cells and thus produce allergic and/ or autoimmune reactions are still obscure, and the same is true for the mechanisms that underly T cell cross-reactivity to different heavy metal ions. In the present study, we investigated induction by metals of T cell reactions to cryptic peptides of bovine RNase A. Murine CD4+ T cell hybridomas specific for cryptic RNase peptides presented from Au(III)-treated RNase were used as detection probes. We showed that in vitro treatment of RNase with Pd(II), Pd(IV), Ni(IV), and partially Pt(IV), but not Au(I), Ni(II), or Pt(II), induced presentation of the same cryptic peptides as those presented from Au(III)-treated RNase. That the former heavy metal ions, but not the latter, were able to alter the antigenicity of RNase was reflected by their ability to induce conformational changes of RNase, as detected by circular dichroism spectroscopy. Furthermore, upon immunization against RNase pretreated with these metals, CD4+ T cell hybridomas specific for unidentified cryptic peptides were obtained. In conclusion, "metal-specific" T cell reactions may be directed against cryptic peptides, and metal cross-reactivity in allergic individuals might be due to metal-induced presentation of overlapping, but not identical, panels of cryptic peptides.

Allergic and autoimmune reactions to xenobiotics: how do they arise?

Induction of allergic and autoimmune reactions by drugs and other chemicals constitutes a major public health problem. Elucidation of the underlying mechanisms might help improve diagnostic tools and therapeutic approaches. Here, Peter Griem and colleagues focus on several aspects of neoantigen formation by xenobiotics: metabolism of xenobiotics into reactive, haptenic metabolites; polymorphisms of metabolizing enzymes; induction of costimulatory signals; and sensitization of T cells.

Drug-induced inhibition of insulin recognition by T-cells: the antirheumatic drug aurothiomalate inhibits MHC binding of insulin peptide.

Previous work has shown that the Au(I) moiety of the antirheumatic drug disodium aurothiomalate (Au(I)TM) can selectively inhibit the response of murine CD4+ T-cell hybridomas to antigenic peptides containing two or more cysteine (Cys) residues. Here, we investigated the mechanism that underlies the inhibitory effect of Au(I)TM on T-cell recognition of bovine insulin (BI). We found that low concentrations of Au(I)TM (10 microM) inhibited the BI-induced proliferation of bulk T-cells from BI-immunized BALB/c mice as well as the IL-2 release of Ab- and Ad-restricted T-cell hybridoma clones. Au(I)TM was found to inhibit binding of the immunodominant BI peptide A1-14 to isolated MHC class II molecules. We suggest that Au(I) forms stable chelate complexes with thiol groups of two Cys residues in the BI A1-14 peptide. Conceivably, formation of these metal-peptide complexes keeps the peptide in a sterical conformation that cannot undergo binding to MHC class II molecules, resulting in an inhibition of T-cell activation due to insufficient peptide presentation.

Strain differences in tissue concentrations of mercury in inbred mice treated with mercuric chloride.

Tissue concentrations of mercury were determined by cold vapor atomic absorption spectrometry in different inbred mouse strains after continuous treatment with HgCl2 (3 weekly sc injections of 0.5 mg/kg bw) for up to 12 weeks. Except for the thymus, in which steadily increasing mercury concentrations were found, in steady state levels of mercury were reached in blood and liver after 4 weeks and in spleen and kidney after 8 weeks. In the closely related strains C57BL/6, B10.D2, and B10.S, which differ only or primarily at the major histocompatibility complex, mercury concentrations in blood and liver were about twofold lower and renal concentrations were about three- to fivefold lower than those detected in strains A.SW and DBA/2. Another strain difference was observed in the spleen: after 8 and 12 weeks of continuous HgCl2 treatment, mercury concentrations in the spleen of strains A.SW, C57BL/6, and B10.S were significantly higher than those in strains DBA/2 and B10.D2. The strain difference in the spleen, an organ of the immune system, correlates with the susceptibility to the HgCl2-induced systemic autoimmune syndrome in mice in that the strains showing a higher mercury accumulation in the spleen are susceptible to this form of chemically induced autoimmunity, whereas the strains with lower mercury concentrations in the spleen are resistant.

The popliteal lymph node assay in mice: screening of drugs and other chemicals for immunotoxic hazard.

The popliteal lymph node assay (PLNA) in mice represents a predictive test for assessing the sensitizing (allergenic and autoimmunogenic) potential of drugs and low molecular weight chemicals. Measuring activation of the draining lymph node of the hind paw, the PLNA facilitates the detection and analysis of immunotoxic effects in a rapid and reproducible manner. An attractive feature of the PLNA is that it can be performed in combination with the routine toxicity testing required for new drugs. Thus, it is possible to investigate whether animals exposed by the oral, intravenous, or inhalative route have been sensitized to the test compound or a reactive metabolite of the test compound generated in vivo. PLNAs may be appropriate supplements to routine toxicity screening of chemicals, thereby enhancing chemical safety.

[Gold antirheumatic drug: desired and adverse effects of Au(I) and Au(III) [corrected] on the immune system. / Das Antirheumatikum Gold: Erwünschte und unerwünschte Wirkungen von Au(I) und Au(II) auf das Immunsystem.

Three new findings are reviewed that help to understand the mechanisms of action of anti-rheumatic gold drugs, such as disodium aurothiomalate (Na2Au(I)TM): i) We found that Na2Au(I)TM selectively inhibits T-cell receptor-mediated antigen recognition by murine CD4+ T-cell hybridomas specific for antigenic peptides containing at least two cysteine residues. Presumably, Au(I) acts as a chelating agent forming linear complexes (Cys-Au(I)-Cys) which prevents correct antigen-processing and/or peptide recognition by the T-cell receptor, ii) We were able to show that Au(I) is oxidized to Au(III) in mononuclear phagocytes, such as macrophages. Because Au(III) rapidly oxidizes protein and itself is re-reduced to Au(I), this may introduce an Au(I)/Au(III) redox system into phagocytes which scavenges reactive oxygen species, such as hypochlorous acid (HOCl) and inactivates lysosomal enzymes, iii) Pretreatment with Au(III) of a model protein antigen, bovine ribonuclease A (RNase A), induced novel antigenic determinants recognized by CD4+ T lymphocytes. Analysis of the fine specificity of these "Au(III)-specific" T-cells revealed that they react to RNase peptides that are not presented to T-cells when the native protein, i.e., not treated with Au(III), is used as antigen. The T-cell recognition of these cryptic peptides did not require the presence of gold. This finding has important implications for understanding the pathogenesis of allergic and autoimmune responses induced by gold drugs. Taken together, our findings indicate that Au(I) and Au(III) each exert specific effects on several distinct functions of macrophages and the activation of T-cells. These effects may explain both the desired anti-inflammatory and the adverse effects of antirheumatic gold drugs.

Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides.

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by 'metal-specific' T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase)A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74-88; the latter also were obtained after immunization with native RNase. RNase/Au(III)-specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S-sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), A1(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7-21 and 94-108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.

Metal ion induced autoimmunity.

Metal-induced autoimmunity is a well established but poorly understood phenomenon. Recent work has begun to elucidate the molecular interactions of metal ions with immune cells and self-proteins. Metal-induced presentation of cryptic self-peptides emerges as a possible mechanism for activation of 'metal-specific' T cells, challenging the hypothesis of a random polyclonal activation of T and B cells by metals. A preferential T-helper cell type 2 response is involved in metal ion induced systemic autoimmune disease.

Mercuric-chloride-induced autoimmunity in mice involves up-regulated presentation by spleen cells of altered and unaltered nucleolar self antigen.

HgCl2 treatment of B10.S mice induces IgG autoantibodies to fibrillarin, a component of small nuclear ribonucleoprotein particles, and histone. Here, we demonstrate the activation by HgCl2 of autoreactive T cells specific for these nuclear proteins. Of nine CD4+ T cell hybridoma clones obtained from HgCl2-treated B10.S mice, one clone reacted to histone Hl and eight clones to fibrillarin. One of the fibrillarin-specific clones only recognized fibrillarin pretreated with HgCl2 (Hg2+ fibrillarin), suggesting that Hg2+ can induce the presentation of a novel fibrillarin epitope. Four fibrillarin-specific hybridoma clones studied for cytokine production were shown to produce interleukin (IL)-2, and three of them also produced IL-4. For stimulation of fibrillarin-specific T cell hybridomas, exogenous murine fibrillarin had to be added when antigen-presenting cells (APCs) came from untreated mice, but not when the APCs were obtained from HgCl2-treated animals. Apparently, HgCl2 treatment induces the presentation by APCs of a novel Hg2+ fibrillarin epitope and up-regulates the presentation of unaltered fibrillarin epitopes, thus activating 'Hg(2+)-specific' as well as autoreactive CD4+ T cells.

CD8 thymocytes derived from 3,3',4,4'-tetrachlorobiphenyl-exposed fetal thymi possess killing activity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and its congeners such as 3,3',4,4'-tetrachlorobiphenyl (TCB) cause immunosuppression in experimental animals and possibly in humans. In previous studies we found that exposure of murine fetal thymic organ cultures (FTOCs) to TCDD or TCB reduced the proliferative capacity of immature thymocytes. At the same time, the kinetics of thymocyte maturation was changed, and thymocyte differentiation was skewed toward CD4-CD8+ phenotypically mature cells. Here, we analyze the biological activities of thymocytes generated in TCB-exposed fetal thymus to determine whether these cells are also functionally mature. C57BL/6 fetal thymic lobes from Day 15 of gestation were explanted and grown for 8 days in FTOC in the presence or absence of 3.3 microM TCB. Then, the functions of total thymocytes or sorted subsets thereof were tested. We found that thymocytes from TCB-exposed lobes responded to stimulation by Con A or anti-CD3 and possessed cytotoxic activity upon cultivation in the presence of H-2 allogenic spleen cells. Further analysis showed that the overall cytotoxic activity of thymocytes was mainly due to the CD4-CD8+ cells. Our results suggest that the CD4-CD8+ cells, which are generated in substantially increased numbers in TCB-exposed fetal thymus, are functionally competent cells.

The antirheumatic drug disodium aurothiomalate inhibits CD4+ T cell recognition of peptides containing two or more cysteine residues.

The mechanism of action of antirheumatic gold drugs, such as disodium aurothiomalate (Au(I)TM), has not been clearly identified. Gold drugs inhibit T cell activation induced by mitogen and anti-CD3 mAb in vitro at relatively high concentrations. However, since gold drugs fail to induce immunosuppression in vivo, the pharmacologic relevance of this finding is doubtful. In this study, we asked whether Au(I)TM interferes with processing and presentation of defined Ags to T cells. Using a panel of murine CD4+ T cell hybridomas, we found that low concentrations of Au(I)TM (< or = 10 microM) led to a markedly reduced IL-2 release of T cell hybridoma clones that recognized peptides containing two or more cysteine (Cys) residues, such as bovine insulin A1-14. Since disodium thiomalate alone had no effect, the inhibition was due to Au(I). IL-2 production induced by anti-CD3 mAb stimulation was not affected by the low concentration of Au(I)TM used. Au(I)TM had no effect on the presentation of peptides containing no or only one Cys residue(s). In contrast to the unmodified insulin peptide A1-14, Au(I) could not inhibit recognition of an insulin peptide in which Cys residues in positions 6 and 11 were replaced by serine. Most likely, the observed inhibition is mediated by formation of chelate complexes between Au(I) and two Cys thiol groups of the affected antigenic peptides. The peptide-specific inhibitory effect of Au(I) on Ag presentation described here might contribute to the therapeutic effect of Au(I) compounds in rheumatoid arthritis.

The Antirheumatic Drug Gold, a Coin With Two Faces: AU(I) and AU(III). Desired and Undesired Effects on the Immune System.

Three new findings are reviewed that help to understand the mechanisms of action of antirheumatic Au(I) drugs, such as disodium aurothiomalate (Na(2)Au(I)TM): (i) We found that Na(2)Au(I)TM selectively inhibits T cell receptor (TCR)-mediated antigen recognition by murine CD(4+) T cell hybridomas specific for antigenic peptides containing at least two cysteine residues. Presumably, Au(I) acts as a chelating agent forming linear complexes (Cys-Au(I)-Cys) which prevent correct antigen-processing and/or peptide recognition by the TCR. (ii) We were able to show that Au(I) is oxidized to Au(III) in phagocytic cells, such as macrophages. Because Au(III) is re-reduced to Au(I) this may introduce an Au(I)/Au(III) redox system into phagocytes which scavenges reactive oxygen species, such as OCl(-) and inactivates lysosomal enzymes. (iii) Pretreatment with Au(III) of a model protein antigen, bovine ribonuclease A (RNase A), induced novel antigenic determinants recognized by CD(4+) T lymphocytes. Analysis of the fine specificity of these 'Au(III)-specific' T cells revealed that they react to RNase peptides that are not presented to T cells when the native protein, i.e., not treated with Au(III), is used as antigen. The T cell recognition of these cryptic peptides did not require the presence of gold. This finding has important implications for understanding the pathogenesis of allergic and autoimmune responses induced by Au(I) drugs. Taken together, our findings indicate that Au(I) and Au(III) each exert specific effects on several distinct components of macrophages and the subsequent activation of T cells; these effects may explain both the desired anti-inflammatory and the adverse effects of antirheumatic gold drugs.

Isolation of human minor histocompatibility peptides.

Incompatibility of human minor histocompatibility (hmH) antigens induces rejection of grafts in organ transplantation and graft versus host disease in bone marrow transplantation if donor and recipient are matched for human leukocyte antigen (HLA) genes. These antigens are recognized only by T cells. We describe here the isolation of hmH peptides recognized by a hmH antigen specific, HLA-B35 restricted CTL clone which was derived from a patient who rejected the kidneys from two HLA-identical sisters. Naturally occurring hmH peptides were isolated from a donor derived B cell line and an HLA-B35 transfected human B cell line by acid elution. Analysis of various HLA class I transfectant cells demonstrated that MHC class I molecules themselves determine the peptides which are naturally processed and presented to T cells.

Uneven tissue distribution of minor histocompatibility proteins versus peptides is caused by MHC expression.

Naturally processed minor histocompatibility (H) peptides corresponding to H-4b, H-Y, and an unmapped BALB.B minor H gene were quantified in a relative way in 15 different tissues of male BALB.B mice. For one of these minor H antigens, we could also determine the relative content of the respective protein. For each minor H peptide, an individual tissue distribution was found. Tissues expressing little or no MHC (major histocompatibility complex), like brain, contained only small amounts of minor H peptides or none at all, although the same tissues contained minor H protein in substantial quantities. By contrast, Kb-expressing brains from mice transgenic for Kb under control of the glial acidic protein promoter contained both minor H peptide and protein in high amounts. Thus, the expression of minor H peptides in a given tissue is dependent on coexpression of the restricting MHC class I molecules.