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The Risk Assessment Committee of the European Chemicals Agency issued an opinion on classifying titanium dioxide (TiO2) as a suspected human carcinogen upon inhalation. Recent animal studies indicate that TiO2 may be carcinogenic through the oral route. There is considerable uncertainty on the carcinogenicity of TiO2, which may be decreased if its mechanism of action becomes clearer. Here we consider adverse outcome pathways and present the available information on each of the key events (KEs). Inhalation exposure to TiO2 can induce lung tumors in rats via a mechanism that is also applicable to other poorly soluble, low-toxicity particles. To reduce uncertainties regarding human relevance, we recommend gathering information on earlier KEs such as oxidative stress in humans. For oral exposure, insufficient information is available to conclude whether TiO2 can induce intestinal tumors. An oral carcinogenicity study with well-characterized (food-grade) TiO2 is needed, including an assessment of toxicokinetics and early KEs. Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 61 is January 8, 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Significant advances have been made in the development of Adverse Outcome Pathways (AOPs) over the last decade, mainly focused on the toxicity mechanisms of chemicals. These AOPs, although relevant to manufactured nanomaterials (MNs), do not currently capture the reported roles of size-associated properties of MNs on toxicity. Moreover, some AOs of relevance to airborne exposures to MNs such as lung inflammation and fibrosis shown in animal studies may not be targeted in routine regulatory decision making. The primary objective of the present study was to establish an approach to advance the development of AOPs of relevance to MNs using existing, publicly available, nanotoxicology literature. A systematic methodology was created for curating, organizing and applying the available literature for identifying key events (KEs). Using a case study approach, the study applied the available literature to build the biological plausibility for ‘tissue injury’, a KE of regulatory relevance to MNs. The results of the analysis reveal the various endpoints, assays and specific biological markers used for assessing and reporting tissue injury. The study elaborates on the limitations and opportunities of the current nanotoxicology literature and provides recommendations for the future reporting of nanotoxicology results that will expedite not only the development of AOPs for MNs but also aid in application of existing data for decision making.

Background: Ensuring consistency of tetanus neurotoxin (TeNT) production by Clostridium tetani could help to ensure consistent product quality in tetanus vaccine manufacturing, ultimately contributing to reduced animal testing. The aim of this study was to identify RNA signatures related to consistent TeNT production using standard and non-standard culture conditions. Methods: We applied RNA sequencing (RNA-Seq) to study C. tetani gene expression in small-scale batches under several culture conditions. Results: We identified 1381 time-dependent differentially expressed genes (DEGs) reflecting, among others, changes in growth rate and metabolism. Comparing non-standard versus standard culture conditions identified 82 condition-dependent DEGs, most of which were specific for one condition. The tetanus neurotoxin gene (tetX) was highly expressed but showed expression changes over time and between culture conditions. The tetX gene showed significant down-regulation at higher pH levels (pH 7.8), which was confirmed by the quantification data obtained with the recently validated targeted LC-MS/MS approach. Conclusions: Non-standard culture conditions lead to different gene expression responses. The tetX gene appears to be the best transcriptional biomarker for monitoring TeNT production as part of batch-to-batch consistency testing during tetanus vaccine manufacturing.

Exposure of humans to metal oxide nanoparticles (NPs) occurs mainly via air, and inhaled metal oxide NPs may generate inflammation. The aim of this study was to investigate the proinflammatory potential of six metal oxide NPs (CeO2, Mn2O3, CuO, ZnO, Co3O4 and WO3; 27–108 μg ml−1) using human primary 3-dimensional airway epithelium (MucilAir™) and dendritic cell (DC) models. Metal oxide NPs were mainly aggregated/agglomerated in the cell media, as determined by dynamic light scattering, scanning electron microscopy and differential centrifugal sedimentation. WO3 and ZnO were highly soluble, both with and without respiratory mucus. Proinflammatory signalling by the epithelium was evaluated after a 24 hour exposure by increased interleukin-6 and -8 and monocyte chemoattractant protein 1 cytokine release, which occurred only for CuO. Moreover, maturation of immature human DCs, which play a key role in the lung immune system, were evaluated by expression of surface markers HLA-DR, CD80, CD83 and CD86 after a 48 hour exposure. Only Mn2O3 consistently upregulated DC maturation markers. Furthermore, by addition of medium from metal oxide NP-exposed 3-dimensional airway cultures to metal oxide NP-exposed DC cultures, the interplay between lung epithelium and DCs was studied. Such an interplay was again only observed for Mn2O3 and in one of five DC donors. Our results show that, even when using dosages that represent very high in vivo exposure levels, up to 27 hours of constant human airway exposure, metal oxide NPs cause minimal proinflammatory effects and that epithelial cells not necessarily interfere with DC maturation upon metal oxide NP exposure. The present approach exemplifies a relevant translation towards human safety assessment. Copyright © 2017 John Wiley & Sons, Ltd.

The discipline of immunotoxicology had its origins in the early 1970s, following the recognition of altered immune function and increased sensitivity to infections and cancers after exposure to environmental chemicals and therapeutic drugs. Reduced resistance to infectious disease was a well-documented consequence of primary and acquired immunodeficiencies, but a novel finding following xenobiotic exposure. The awareness of the consequences of altered immune function was likely heightened by the HIV epidemic, leading some to inappropriately characterize xenobiotic-induced immunosuppression as “chemical AIDS”, although it is now clear that mild to moderate suppression is the most likely outcome of inadvertent exposure. The human health implications of studies in which chemical exposure reduced resistance to infection, drove an early focus on immunosuppression within the toxicology community. Allergic hypersensitivity was well known to clinicians and symptoms were readily apparent, and therefore was not the initial focus of the developing toxicology subspecialty of immunotoxicology. The first review in the field of immunotoxicology was published by Vos in 1977, and, as research expanded during the years that followed, many of the assays, methodologies and approaches that are currently used to identify potential immunotoxicants were developed. Over the years, advances in our understanding of basic immunology have made it clear that allergy, immunosuppression and, in some cases, autoimmunity, are a matter of polarization of the immune response by immunotoxicants, rather than independent outcomes of chemical exposure.

Various nanomedicinal products (NMPs) have been reported to induce an adverse immune response, which may be related to their tendency to accumulate in or target cells of the immune system. Therefore, before their market authorization, NMPs should be thoroughly evaluated for their immunotoxic potential. Nonclinical regulatory immunotoxicity testing of nonbiological medicinal products, including NMPs, is currently performed by following the guideline S8 “Immunotoxicity Studies for Human Pharmaceuticals” of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). However, this guideline does not cover all the immunotoxicity endpoints reported for NMPs in the literature, such as complement activation related pseudo allergy, hypersensitivity and immunosuppression. In addition, ICH‐S8 does not provide any nanospecific testing considerations, which is important given their tendency to interfere with many commonly used toxicity assays. We therefore propose a nonclinical regulatory immunotoxicity assessment strategy, which considers the immunotoxicity endpoints currently missing in the ICH‐S8. We also list the known pitfalls related to the testing of NMPs and how to tackle them. Next to defining the relevant physicochemical and pharmacokinetic properties of the NMP and its intended use, the proposed strategy includes an in vitro assay battery addressing various relevant immunotoxicity endpoints. A weight of evidence evaluation of this information can be used to shape the type and design of further in vivo investigations. The final outcome of the immunotoxicity assessment can be included in the overall risk assessment of the NMP and provide alerts for relevant endpoints to address during clinical investigation. This article is categorized under:Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in NanomedicineToxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials, Proposed strategy for immunotoxicity assessment of nanomedicinal products.

The hazard assessment of skin sensitizers relies mainly on animal testing, but much progress is made in the development, validation and regulatory acceptance and implementation of non-animal predictive approaches. In this review, we provide an update on the available computational tools and animal-free test methods for the prediction of skin sensitization hazard. These individual test methods address mostly one mechanistic step of the process of skin sensitization induction. The adverse outcome pathway (AOP) for skin sensitization describes the key events (KEs) that lead to skin sensitization. In our review, we have clustered the available test methods according to the KE they inform: the molecular initiating event (MIE/KE1)-protein binding, KE2-keratinocyte activation, KE3-dendritic cell activation and KE4-T cell activation and proliferation. In recent years, most progress has been made in the development and validation of in vitro assays that address KE2 and KE3. No standardized in vitro assays for T cell activation are available; thus, KE4 cannot be measured in vitro. Three non-animal test methods, addressing either the MIE, KE2 or KE3, are accepted as OECD test guidelines, and this has accelerated the development of integrated or defined approaches for testing and assessment (e.g. testing strategies). The majority of these approaches are mechanism-based, since they combine results from multiple test methods and/or computational tools that address different KEs of the AOP to estimate skin sensitization potential and sometimes potency. Other approaches are based on statistical tools. Until now, eleven different testing strategies have been published, the majority using the same individual information sources. Our review shows that some of the defined approaches to testing and assessment are able to accurately predict skin sensitization hazard, sometimes even more accurate than the currently used animal test. A few defined approaches are developed to provide an estimate of the potency sub-category of a skin sensitizer as well, but these approaches need further independent evaluation with a new dataset of chemicals. To conclude, this update shows that the field of non-animal approaches for skin sensitization has evolved greatly in recent years and that it is possible to predict skin sensitization hazard without animal testing.

The use of engineered nanoparticles (NP) is widespread and still increasing. There is a great need to assess their safety. Newly engineered NP enter the market in a large variety; therefore safety evaluation should preferably be in a high-throughput fashion. In vitro screening is suitable for this purpose. TiO 2 NP exist in a large variety (crystal structure, coating and size), but information on their relative toxicities is scarce. TiO 2 NP may be inhaled by workers in e.g. paint production and application. In mice, inhalation of TiO 2 NP increases allergic reactions. Dendritic cells (DC) form an important part of the lung immune system, and are essential in adjuvant activity. The present study aimed to establish the effect of a variety of TiO 2 NP on DC maturation in vitro. Two NP of different crystal structure but similar in size, uncoated and from the same supplier, were evaluated for their adjuvant activity in vivo. Immature DC were differentiated in vitro from human peripheral blood monocytes. Exposure effects of a series of fourteen TiO 2 NP on cell viability, CD83 and CD86 expression, and IL-12p40 and TNF-α production were measured. BALB/c mice were intranasally sensitized with ovalbumin (OVA) alone, OVA plus anatase TiO 2 NP, OVA plus rutile TiO 2 NP, and OVA plus Carbon Black (CB; positive control). The mice were intranasally challenged with OVA. OVA-specific IgE and IgG1 in serum, cellular inflammation in bronchoalveolar lavage fluid (BALF) and IL-4 and IL-5 production in draining bronchial lymph nodes were evaluated. All NP dispersions contained NP aggregates. The anatase NP and anatase/rutile mixture NP induced a higher CD83 and CD86 expression and a higher IL-12p40 production in vitro than the rutile NP (including coated rutile NP and a rutile NP of a 10-fold larger primary diameter). OVA-specific serum IgE and IgG1 were increased by anatase NP, rutile NP, and CB, in the order rutile

[Verstraelen, Sandra; Jacobs, An; Wathiong, Bart; Deville, Sarah; Nelissen, Inge] Flemish Inst Techn Res, Dept Hlth, Mol, Belgium. [Wathiong, Bart; Deville, Sarah] Hasselt Univ, Biomed Res Inst, Diepenbeek, Belgium. [Pelaz, Beatriz; Moliman, Mahmoud Gamal Mohamed; Parak, Wolfgang] Philipps Univ Marburg, Fachbereich Phys, Marburg, Germany. [Gutleb, Arno; Serchi, Tommaso] Luxembourg Inst Sci & Technol, Environm Res & Innovat Dept, Luxembourg, Luxembourg. [Vandebriel, Rob] Natl Inst Publ Hlth & Environm, Bilthoven, Netherlands.

Respiratory sensitization provides a case study for a new approach to chemical safety evaluation, as the prevalence of respiratory sensitization has increased considerably over the last decades, but animal and/or human experimental/predictive models are not currently available. Therefore, the goal of a working group was to design a road map to develop an ASAT approach for respiratory sensitisers. This approach should aim at (i) creating a database on respiratory functional biology and toxicology, (ii) applying data analyses to understand the multi-dimensional sensitization response, and how this predisposes to respiratory inflammation and irritation, and (iii) building a systems model out of these analyses, adding pharmacokinetic-pharmacodynamic modeling to predict respiratory responses to low levels of sensitisers. To this end, the best way forward would be to follow an integrated testing approach. Experimental research should be targeted to (i) QSAR-type approaches to relate potential as a respiratory sensitizer to its chemical structure, (ii) in vitro models and (iii) in vitro-in vivo extrapolation/validation. © 2011 Elsevier Ltd.

The biocide and environmental pollutant bis(tri-n-butyltin)oxide (TBTO) causes thymus atrophy in rodents. Whether the depletion of thymic lymphocytes by tributyltin compounds may be the result of inhibition of cell proliferation or induction of apoptosis is subject of debate. We examined gene expression profiles in primary rat thymocytes exposed to TBTO in vitro at dose levels of 0, 0.1, 0.3, 0.5, and 1.0muM. By measuring cell viability and apoptosis, exposure conditions were selected that would provide information on changes in gene expression preceding or accompanying functional effects of TBTO. Several processes related to TBTO-induced toxicity were detected at the transcriptome level. Effects on lipid metabolisms appeared to be the first indication of disruption of cellular function. Many transcriptional effects of TBTO at higher dose levels were related to apoptotic processes, which corresponded to present or subsequent thymocyte apoptosis observed phenotypically. The gene expression profile was, however, not unambiguous since expression of apoptosis-related genes was both increased and decreased. Stimulation of glucocorticoid receptor signaling appeared to be a relevant underlying mechanism of action. These findings suggest that TBTO exerts its toxic effects on the thymus primarily by affecting apoptotic processes, but the possibility is discussed that this may in fact represent an early effect that precedes inhibition of cell proliferation. At the highest dose level tested, TBTO additionally repressed mitochondrial function and immune cell activation. Our in vitro toxicogenomics approach thus identified several cellular and molecular targets of TBTO that may mediate the toxicity towards thymocytes and thereby its immunosuppressive effects. AD - Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Health Risk Analysis and Toxicology (GRAT), Maastricht University, Maastricht, The Netherlands; National Institute of Public Health and the Environment (RIVM), Laboratory for Toxicology, Pathology and Genetics (TOX), Bilthoven, The Netherlands.

The original local lymph node assay (LLNA) is based on the use of radioactive labelling to measure cell proliferation. Other endpoints for the assessment of proliferation are also authorized by the OECD Guideline 429 provided there is appropriate scientific support, including full citations and description of the methodology (OECD, 2002. OECD Guideline for the Testing of Chemicals; Skin Sensitization: Local Lymph Node Assay, Guideline 429. Paris, adopted 24th April 2002.). Here, we describe the outcome of the second round of an inter-laboratory validation of alternative endpoints in the LLNA conducted in nine laboratories in Europe. The validation study was managed and supervised by the Swiss Agency for Therapeutic Products (Swissmedic) in Bern. Ear-draining lymph node (LN) weight and cell counts were used to assess LN cell proliferation instead of [3H]TdR incorporation. In addition, the acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears to identify skin irritation properties of the test items. The statistical analysis was performed in the department of statistics at the university of Bern. Similar to the EC(3) values defined for the radioactive method, threshold values were calculated for the endpoints measured in this modification of the LLNA. It was concluded that all parameters measured have to be taken into consideration for the categorisation of compounds due to their sensitising potencies. Therefore, an assessment scheme has been developed which turned out to be of great importance to consistently assess sensitisation versus irritancy based on the data of the different parameters. In contrast to the radioactive method, irritants have been picked up by all the laboratories applying this assessment scheme.

Predictive testing of immunotoxicity associated with chemical compounds is complicated and cannot be accomplished with a single test. As most of the existing tests for immunotoxicity employ experimental animals, there is an increasing need for alternative tests in vitro. We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. This system named "fluorescent cell chip" (FCC) is based on a number of genetically modified cell lines that regulate the expression of a transgene coding for fluorescent protein enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-1beta, IL-2, IL-4, IFN-gamma, IL-10, TNF-alpha, and beta-actin. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promotors were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells. Two chemicals with known immunotoxic activities, cyclosporine A and potassium tetrachloro-platinate(II), mediated compound-specific pattern of inhibition and activation of reporter gene expression. Thus, the "fluorescent cell chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well defined biological activity.

Predictive tests to identify the sensitizing properties of chemicals are carried out using animals. There is as yet, no accepted in vitro method for the identification of skin sensitizing chemicals. Such in vitro tests should encompass (parts of) the sensitization phase of contact hypersensitivity. Two cell types are predominantly involved in this process, keratinocytes (KC) and Langerhans cells, the latter being a specialized type of skin dendritic cells (DC). Low molecular weight chemicals act as haptens; KC respond to allergen contact by, among others, producing proinflammatory cytokines, while DC take up the haptenized protein, migrate, and present antigen to T-cells. During migration, DC mature, resulting in a loss of antigen uptake capacity, thereby increasing expression of certain surface molecules. Thus, both cytokine production by KC and surface marker expression by DC may be used as in vitro models for the identification of sensitizers. Several reports have shown that intracellular IL-1 alpha is a promising candidate to identify sensitizers using KC. We have recently shown that the potency of sensitizers may be determined by dose-response analysis of intracellular IL-1 alpha and IL-18 using a murine KC cell line. The ranking of potency using this in vitro method was similar to the ranking previously established using the local lymph node assay. Using DC, effects on the expression of various cell surface markers, cytokines, and molecules involved in antigen uptake have been shown to identify sensitizers. One study showed that also the potency of sensitizers may be determined using DC. Additional studies are required to establish whether KC or DC, or combinations thereof, are most suitable for in vitro identification and potency assessment of sensitizers.

Assessment of allergenic potency of low molecular weight compounds is generally performed using animal models, such as the guinea pig maximisation test and the murine local lymph node assay (LLNA). Progress in unravelling the mechanisms of skin sensitisation, including effects on the production of cytokines by the different cell types of the skin, provides us with the opportunity to develop in vitro tests as an alternative to in vivo sensitisation testing. The aim of the present study was to establish an in vitro method to assess the potency of allergens, on the basis of their induction of cytokine production by murine and human keratinocytes. In the present study we used test systems comprised of the murine epidermal keratinocyte cell line HEL-30 and the human keratinocyte cell line HaCaT. We exposed these cell lines to the allergens ethyl-p-aminobenzoate (benzocaine), diethylamine (DEA), 2,4-dinitrochlorobenzene (DNCB), and phthalic anhydride (PA). IL-1alpha and IL-18 dose-response data were evaluated by non-linear regression analysis and at a stimulation index of 3 of cytokine production of treatment versus control, the corresponding allergen concentration was calculated. For HEL-30, for both cytokines DNCB showed the strongest potency followed in this order by PA, benzocaine, and DEA. This classification was similar to our previous findings obtained in the LLNA. For HaCaT, unfortunately, such ranking proved to be much less feasible. In conclusion, to assess the potency of allergens the murine keratinocyte cell line HEL-30 may be a useful in vitro test system, alternative to in vivo models, although this requires further testing using a much wider range of compounds.

Safety assessment of chemicals and drugs is an important regulatory issue. The evaluation of potential adverse effects of compounds on the immune system depends today on animal experiments. An increasing demand, however, exists for in vitro alternatives. Cytokine measurement is a promising tool to evaluate chemical exposure effects on the immune system. Fortunately, this type of measurement can be performed in conjunction with in vitro exposure models. We have taken these considerations as the starting point to develop an in vitro method to efficiently screen compounds for potential immunotoxicity. The T-cell lymphoma cell line EL-4 was transfected with the regulatory sequences of interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma or actin fused to the gene for enhanced green fluorescent protein (EGFP) in either a stabile or a destabilised form. Consequently, changes in fluorescence intensity represent changes in cytokine expression with one cell line per cytokine. We used this prototype "Cell Chip" to test, by means of flow cytometry, the immunomodulatory potential of 13 substances and were able to detect changes in cytokine expression in 12 cases (successful for cyclosporine, rapamycin, pentamidine, thalidomide, bis(tri-n-butyltin)oxide, house dust mite allergen (Der p I), 1-chloro-2,4-dinitrobenzene, benzocaine, tolylene 2,4-diisocyanate, potassium tetrachloroplatinate, sodium dodecyl sulphate and mercuric chloride; unsuccessful for penicillin G). In conclusion, this approach seems promising for in vitro screening for potential immunotoxicity, especially when additional cell lines besides T-cells are included.

This is the report of a workshop organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods that are of importance to the biosciences and which replace, reduce or refine the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organization of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (Anonymous, 1994). The workshop on "The use of in vitro systems for evaluating Immunotoxicity" was held at ECVAM (Ispra), Italy, on 24th-26th November 2003. The participants represented academia, national organizations, international regulatory bodies and industry. The aim of the workshop was to review the state-of-the-art in the field of in vitro immunotoxicology, and to develop strategies towards the replacement of in vivo testing. At the end of this report are listed the recommendations that should be considered for prevalidation and validation of relevant and reliable procedures, that could replace the use of animals in chemical and cosmetics toxicity testing.

Impact of exposure duration by low molecular weight compounds on interferon-gamma and interleukin-4 mRNA expression and production in the draining lymph nodes of mice.Vandebriel RJ, De Jong WH, Hendriks JJ, Van Loveren H.Laboratory for Pathology and Immunobiology, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands. r.vandebriel@rivm.nlThe local lymph node assay (LLNA) is used to identify allergens by means of dermal exposure. For hazard identification, besides identification also the distinction between contact and respiratory allergens is of importance. We have previously shown that a modified LLNA can be used to identify respiratory allergens, on the basis of Con A induced IL-4 production. Here we show a good qualitative correlation between mRNA expression and production of IFN-gamma and IL-4. This suggests that distinction between contact and respiratory allergens may also be studied at the mRNA expression level. Secondly, another assay, similar to the modified LLNA but differing in the duration and the number of allergen applications as well as in the ex vivo culture conditions, here denoted as 'longer' assay, has been reported to be able to identify contact allergens, on the basis of (spontaneous) IFN-gamma production. In the present study we have compared these assays. Similar to our previous findings, in the modified LLNA exposure to the respiratory allergen trimellitic anhydride (TMA) resulted in a approximately 10-fold higher Con A induced IL-4 production compared with the contact allergen dinitrochlorobenzene (DNCB), while exposure to both allergens resulted in a similar Con A induced IFN-gamma production. In the 'longer' assay, TMA exposure resulted in Con A induced IL-4 production whereas DNCB exposure did not. Importantly, only a 2-fold higher spontaneous IFN-gamma production was induced by DNCB compared with TMA, the difference being not statistically significant. Thus, although the 'longer' assay indeed showed a somewhat higher IFN-gamma induction by DNCB compared with TMA, the magnitude and robustness of this effect question its applicability. These results favor the modified LLNA since it is shorter, and combines identification of allergens (by cell proliferation) with identification of respiratory allergens (by IL-4 production). Compounds that induce cell proliferation with a low concomitant IL-4 production may thus be identified as contact allergens, although the need to positively identity such allergens remain.

Determination of the sensitising activity of the rubber contact sensitisers TMTD, ZDMC, MBT and DEA in a modified local lymph node assay and the effect of sodium dodecyl sulfate pretreatment on local lymph node responses.De Jong WH, Tentij M, Spiekstra SW, Vandebriel RJ, Van Loveren H.Laboratory for Pathology and Immunobiology, National Institute of Public Health and the Environment, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands. w.de.jong@rivm.nlA modified local lymph node assay (LLNA) was used to determine the sensitising activity of four chemicals used for the production of natural rubber latex products. Tetramethylthiuramdisulfide (TMTD), 2-mercaptobenzothiazole (MBT) and zincdimethyldithiocarbamate (ZDMC), three moderate human sensitisers, and diethylamine (DEA) a known human sensitiser, were epicutaneously administered on the ear and the proliferating activity in the draining (auricular) lymph node (LN) was determined by ex vivo (3)H-thymidine incorporation. Consistent results were obtained for TMTD and ZDMC with stimulation indices (SI) above 3, identifying these compounds as sensitiser, while for DEA and MBT inconsistent results were obtained. For all parameters determined such as LN weight, LN cell number, cell proliferation per 2 x 10(6) cells, and cell proliferation per LN statistical significant increases were observed. The SI, expressed as cellular proliferation per LN or per animal (left and right LN combined), was the most sensitive parameter with an optimum at day 5 after start of treatment.Furthermore, we investigated whether the use of sodium dodecyl sulfate (SDS) was able to enhance weak responses in the LLNA. SDS treatment with dosages of 10% and higher resulted in a SI above 3, while a dosage of 1% SDS showed no activity. Pretreatment with 1% SDS 1 h before application of the rubber chemicals enhanced the responses to these chemicals consistently, identifying also DEA and MBT as sensitisers. Our results indicate that SDS had synergistic activity on the LN responses of the administered rubber chemicals in the LLNA.For the moderately responding sensitisers TMTD and ZDMC both IFN-gamma and IL-4 production was observed. For the weakly responding sensitisers DEA and MBT both IFN-gamma and IL-4 cytokine production was only observed after pretreatment of the animals with 10% SDS. For 10% and 20% SDS, inducing approximately a SI of 20 in the LLNA, no induction of cytokines was observed.

Titanium dioxide white pigment consists of particles of various sizes, from which a fraction is in the nano range (

Background: Synthetic Amorphous Silica (SAS) is commonly used in food and drugs. Recently, a consumer intake of silica from food was estimated at 9.4 mg/kg bw/day, of which 1.8 mg/kg bw/day was estimated to be in the nano-size range. Food products containing SAS have been shown to contain silica in the nanometer size range (i.e. 5 - 200 nm) up to 43% of the total silica content. Concerns have been raised about the possible adverse effects of chronic exposure to nanostructured silica.Methods: Rats were orally exposed to 100, 1000 or 2500 mg/kg bw/day of SAS, or to 100, 500 or 1000 mg/kg bw/day of NM-202 (a representative nanostructured silica for OECD testing) for 28 days, or to the highest dose of SAS or NM-202 for 84 days.Results: SAS and NM-202 were extensively characterized as pristine materials, but also in the feed matrix and gut content of the animals, and after in vitro digestion. The latter indicated that the intestinal content of the mid/high-dose groups had stronger gel-like properties than the low-dose groups, implying low gelation and high bioaccessibility of silica in the human intestine at realistic consumer exposure levels. Exposure to SAS or NM-202 did not result in clearly elevated tissue silica levels after 28-days of exposure. However, after 84-days of exposure to SAS, but not to NM-202, silica accumulated in the spleen. Biochemical and immunological markers in blood and isolated cells did not indicate toxicity, but histopathological analysis, showed an increased incidence of liver fibrosis after 84-days of exposure, which only reached significance in the NM-202 treated animals. This observation was accompanied by a moderate, but significant increase in the expression of fibrosis-related genes in liver samples. Conclusions: although only few adverse effects were observed, additional studies are warranted to further evaluate the biological relevance of observed fibrosis in liver and possible accumulation of silica in the spleen in the NM-202 and SAS exposed animals respectively. In these studies, dose-effect relations should be studied at lower dosages, more representative of the current exposure of consumers, since only the highest dosages were used for the present 84-day exposure study. © 2014 van der Zande et al.; licensee BioMed Central Ltd.

The guinea pig maximization test (GPMT) has been used as a method for the prediction of skin sensitizing potential for over 30 years. Besides hazard identification, risk assessment of sensitizing chemicals requires the assessment of potency. For the determination of potency based on lowest effective dose levels, dose-response studies are required. In the standard GPMT a single concentration is used for intracutaneous and topical induction and the assay provides a qualitative assessment of allergenicity. This paper presents data derived from quantitative evaluation of the sensitizing potency of chemicals in the GPMT, based on multiple concentrations. We performed the GPMT in accordance with the original procedure of Magnusson and Kligman; and included in this procedure a range of intradermal and topical concentrations for induction. Three allergens with different sensitizing potencies, diethylamine (DEA), tetramethyl thiuram disulfide (TMTD) and zinc dimethyl dithiocarbamate (ZDMC) were tested. The data obtained with this test procedure were compared to data we previously obtained using the local lymph node assay (LLNA). Both the GPMT and the LLNA showed dose response relationships for the three chemicals tested. For the chemicals tested, both tests differed in the relative potencies based on benchmark concentrations. While both tests ranked DEA as the least potent allergen, the GPMT ranked ZDMC more potent than TMTD, the reverse being found in the LLNA. The nature of the data provided in the LLNA makes it likely that benchmarks as defined with this test are more reliable than that defined in the GPMT. However, further validation with human data is necessary. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Chemicals/CAS: Allergens; diethylamine, 109-89-7; Diethylamines; Thiram, 137-26-8; Ziram, 137-30-4

In general toxicity testing, maximal acceptable concentrations are derived from no-observed adverse effect levels (NOAEL) in rodents. Risk assessment then considers safety factors for the interspecies difference, and intraspecies variability. This approach can be used for assessing maximal acceptable concentrations for chemicals inducing direct immunotoxicity, resulting in e.g. reduced resistance to infections. As for predictive testing of chemicals in terms of sensitization, laboratory animal data are mostly used for risk assessment as well. Generally, the assessment of risk for chemicals that induce contact sensitivity is limited to hazard identification, and risk management is restricted to labeling. An alternative type of evaluation of the risk of adverse effects due to exposure to immunotoxic chemicals may be the so called parallellogram approach. In this parallellogram there are four cornerstones, one of which is the health effect of exposure to a chemical, assessed as an endpoint (e.g. infection model) in experimental animals, and another the quantitative prediction of this endpoint in humans. The other cornerstones are assays of parameters that are relevant to the mechanism of the adverse effect in experimental animals and humans, and are used for species comparison. Species comparisons between the animal species used for hazard identification and humans are crucial for extrapolation of animal data to the human situation. This approach can be used to provide relevant information on the dose-response relationship in humans. In concert with information on actual exposure, such data can then be used for the characterization of risk for adverse health effects in humans. Such approaches have been used for chemicals that exert direct immunotoxic activity (bis(tri-n-butyltin)oxide (TBTO)), and may hold promise for the risk evaluation of chemicals that exert skin sensitizing properties.

Molecular immunotoxicology is aimed at analysing exposure effects on the temporal expression of important immunoregulatory genes. Cytokines play key roles in the immune system and thus molecular immunotoxicology has focused on the analysis of cytokine (expression) levels. These targets offer important new avenues to explore both in terms of mechanistic understanding of immunotoxicity and in terms of developing new assays and tests for predicting the immunotoxic potential of novel compounds. Effects on cytokine levels can be analysed on two different levels, these being mRNA and protein. The choice essentially depends on the aim of the study. Proteins comprise the biological activity so they are a more direct measure than mRNA. mRNA on the other hand, measures at a specific point in time within a tissue or organ, whereas protein is measured in a body fluid, possibly as a spill-over from tissue, or in a supernatant as a summation over a culture period. mRNA levels are assayed using Northern or dot blotting that both comprise hybridisation and using reverse transcription-polymerase chain reaction (RT-PCR). Although the latter technique has both enormous sensitivity and relative ease of operation as important advantages, it requires much more effort in terms of quantitation. References to the nucleic acid sequences of human, murine, and rat cytokines and their receptors are presented (with accession numbers). Examples in which molecular techniques were successfully employed to assess immunotoxicity and (in some cases) understand mechanisms of action are also presented.

Analysis of cytokine (receptor) mRNA levels has been suggested to be a sensitive technique for predicting the immunomodulatory potential of drugs and chemicals. Furthermore, this type of analysis is thought to be important in unraveling mechanisms of immunotoxicity. To study these issues, male Wistar rats were exposed to the immunotoxic environmental contaminants bis(tri-n-butyltin) oxide (TBTO; 5, 20, or 80 mg/kg diet for 6 weeks), hexachlorobenzene (HCB; 50, 150, or 450 mg/kg diet for 6 weeks), or benzo(a)pyrene (B(a)P; 3, 10, 30, or 90 mg/kg body wt for 5 weeks by a daily (5 times a week) oral intubation). Spleen cells were cultured with Con A and analyzed by dot blot hybridization for IL-2, IFN-gamma, IL-2 receptor alpha-chain (IL-2R alpha; CD25), and IL-4 mRNA levels. In addition, spleen and thymus sections of TBTO-exposed animals were assayed immunohistochemically for CD25 expression. Exposure to TBTO resulted in a dose-dependent decrease in IL-2R alpha mRNA levels from 5 mg/kg, a dose-dependent increase in IFN-gamma mRNA levels from 20 mg/kg, and increased IL-2 mRNA levels at 80 mg/kg diet. Exposure to HCB resulted in a dose-dependent increase in IL-2 and IFN-gamma mRNA levels from 150 mg/kg and increased IL-2R gamma mRNA levels at 450 mg/kg diet. Exposure to B(a)P resulted in a dose-dependent increase in IL-2 and IFN-gamma mRNA levels from 10 mg/kg and increased IL-2R alpha mRNA levels at 90 mg/kg body wt. No effects were seen on IL-4 mRNA levels. Spleen and thymus sections of TBTO-exposed animals showed reduced CD25 expression from 5 mg/kg diet. These results show that (1) the correlation between altered cytokine (receptor) mRNA levels and functional endpoints is variable, depending on the type of functional endpoint tested and the compound studied, (2) these assays are among the most sensitive ones for TBTO and HCB immunotoxicity, and among the more sensitive ones for B(a)P immunotoxicity, and (3) for TBTO, these assays provide a possible clue to a mechanism for thymus atrophy, resulting from exposure to this compound: reduced IL-2R expression may impede thymocyte maturation, resulting in thymus atrophy.

There are a large number of chemicals in current use for which adequate toxicity data are not available. Whilst there are clear ethical and legal obligations to obtain data from sources other than in vivo experiments wherever possible, in certain cases in vivo assays may be deemed necessary. In such circumstances, it is essential to ensure that the maximum amount of high quality data is obtained from the minimum number of animals, using the most humane procedures, in accordance with the philosophy of reduction, refinement and replacement (3Rs). The aim of this report is to provide a strategy for anyone involved in animal experimentation, for either toxicological or pharmacological purposes, as to how in vivo experiments may be optimised. The impact of generic and endpoint specific sources of variability has been highlighted in a proof-of-principle analysis considering the variation in protocols for assays for four human health endpoints (skin sensitisation, reproductive/developmental toxicity, mutagenicity and carcinogenicity). Other factors such as operator training, experimental/statistical design, use of lower species and use of combined assays are also discussed. Recommendations for optimisation of in vivo assays, in terms of the 3Rs philosophy, applied to performing tests, harvesting data and appropriate reporting are summarised as a checklist of issues to be addressed prior to undertaking such assays.

Many chemicals can induce allergic contact dermatitis. Because evaluation of skin sensitizing potential by animal testing is prohibited for cosmetics, and screening of many chemicals is required within Registration, Evaluation, Authorisation and Restriction of Chemicals, urgent need exists for predictive in vitro assays to identify contact allergens. Keratinocytes (KC) are the first cells encountered when chemicals land on the skin. Therefore, KC form an important site of haptenization and their metabolism is likely to be important. Moreover, KC secrete mediators that affect processing and presentation of haptenized proteins by dendritic cells. To develop a KC-based in vitro assay to predict sensitizing potential of chemicals, in vitro exposure effects of eight contact sensitizers and six irritants on the KC cell line HaCaT were examined by gene profiling. Classifiers predictive of the class sensitizers or irritants were calculated, based on support vector machine (SVM) and random forest (RF) algorithms. Classifiers using high-ranking genes were 70% (SVM) and 62% (RF) accurate, based on three (SVM) and two to five (RF) features. Classifiers using oxidative stress pathway gene sets were 68-73% (SVM) and 69-71% (RF) accurate. Crossvalidation showed that the top-3 of most discriminating genes added up to 13 genes and included oxidative stress gene HMOX1 irrespective of the chemical left out. Moreover, HMOX1 was the most significantly regulated gene. Gene Set Enrichment Analysis showed upregulation of "Keap1 dependent" and "oxidative stress" gene lists. In conclusion, KC expression profiling can identify contact sensitizers, providing opportunities for nonanimal testing for sensitizing potential. Moreover, our data suggest that contact sensitizers induce the oxidative stress pathway in KC. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

Predictive tests to identify the sensitizing properties of chemicals are carried out using animals. In the European Union timelines for phasing out many standard animal tests were established for cosmetics. Following this policy, the new European Chemicals Legislation (REACH) favors alternative methods, if validated and appropriate. In this review the authors aim to provide a state-of-the art overview of alternative methods (in silico, in chemico, and in vitro) to identify contact and respiratory sensitizing capacity and in some occasions give a measure of potency. The past few years have seen major advances in QSAR (quantitative structure-activity relationship) models where especially mechanism-based models have great potential, peptide reactivity assays where multiple parameters can be measured simultaneously, providing a more complete reactivity profile, and cell-based assays. Several cell-based assays are in development, not only using different cell types, but also several specifically developed assays such as three-dimenionally (3D)-reconstituted skin models, an antioxidant response reporter assay, determination of signaling pathways, and gene profiling. Some of these assays show relatively high sensitivity and specificity for a large number of sensitizers and should enter validation (or are indeed entering this process). Integrating multiple assays in a decision tree or integrated testing system is a next step, but has yet to be developed. Adequate risk assessment, however, is likely to require significantly more time and efforts.

Effect of prolonged exposure to low antigen concentration for sensitization.van Och FM, Vandebriel RJ, De Jong WH, van Loveren H.Laboratory for Pathology and Immunobiology, National Institute of Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven, The Netherlands.The local lymph node assay (LLNA) is an assay in mice to identify potential allergens. Compounds that do not induce a stimulation index (SI)>or=3 are not considered sensitizers. Of the chemicals that do, the SI of 3 is used as a benchmark, and indicates the sensitizing potency of a chemical. Compared to the exposure duration of the LLNA (3 days), real life exposure often lasts for months or years. We therefore investigated whether prolonged exposure to sensitizers at concentrations that do not induce a SI>or=3 in the LLNA, were able to surpass this threshold. Mice were treated for 2 months at 7-day intervals with a range of concentrations of the known allergens ethyl-p-aminobenzoate (benzocaine, BENZ), 2,4-dinitrochlorobenzene (DNCB), and tetramethyl thiuram disulfide (TMTD). Both proliferative activity and cytokine production were established at day 60. Neither BENZ nor TMTD showed a significant increase in the proliferation rate compared to vehicle controls. Only DNCB at concentrations originally above the EC(3) a significant increase in proliferation was seen after prolonged exposure. No significant effect on IFN-gamma and IL-4 production was observed for all three compounds compared. These findings indicate that for classification of sensitizers the shorter exposure period employed in the standard LLNA is sufficient, and longer periods of exposure have no bearing on this classification.

Ranking of allergenic potency of rubber chemicals in a modified local lymph node assay.De Jong WH, Van Och FM, Den Hartog Jager CF, Spiekstra SW, Slob W, Vandebriel RJ, Van Loveren H.Laboratory for Pathology and Immunobiology, National Institute for Public Health and the Environment, RIVM, P.O.Box 1, 3720 BA Bilthoven, The Netherlands. w.de.jong@rivm.nlA modified local lymph node assay (LLNA) with ex vivo tritium thymidine (3H-TdR) labeling of the proliferating lymph node cells was used for determination of the allergenic potency of chemicals used in the production of rubber for latex medical gloves. Fifteen chemicals known to induce contact hypersensitivity reactions in man, including various thiuram, carbamate, and benzothiazole compounds, and one amine were tested. The EC3 (effective concentration inducing a 3-fold increase in proliferation of lymph node cells [Stimulation Index, SI = 3]) was calculated with nonlinear regression analysis, including a bootstrap method for determination of the 5-95% confidence interval of the EC3 value. This procedure identified 14 out of the 15 chemicals tested as sensitizers, while for one chemical, ZDBC, no EC3 could be calculated due to low responses and a lack of a dose-response relationship in the data obtained. The ranking order of the chemicals with increasing EC3 values (and thus decreasing allergenic potency) was found to be in the following order: ZDEC < TMTD < TETD < ZPC < ZDMC < MBTS < PTD < TMTM < MBT < MBI < PTT < ZMBT < TBTD < DEA < ZDBC. Our results indicate that the chemicals of choice for use in the production of natural rubber latex products would be for the thiuram compounds, TBTD; for the carbamates, ZDBC; and for the benzothiazoles, ZMBT. However, one has to be aware that besides potency, the total amount of residual chemical present in the final product is also important for allergy induction

In laboratory animals, an adequate way to assess effects of environmental exposures on the immune system is to study effects on antigen-specific immune responses, such as after sensitization to T-cell-dependent antigens. This probably also applies to testing effects in the human population. It has thus been suggested that antibody responses to vaccination might be useful in this context. Vaccination responses may be influenced by a variety of factors other than environmental ones. One factor is the vaccine itself; a second is the vaccination procedure used. In addition, the intrinsic capacity of the recipient to respond to a vaccine, which is determined by sex, genetic factors, and age, is important. Psychological stress, nutrition, and (infectious) diseases are also likely to have an impact. We reviewed the literature on vaccine response. With regard to exogenous factors, there is good evidence that smoking, diet, psychological stress, and certain infectious diseases affect vaccination titers, although it is difficult to determine to what extent. Genetic factors render certain individuals nonresponsive to vaccination. In general, in epidemiologic studies of adverse effects of exposure to agents in the environment in which vaccination titers are used, these additional factors need to be taken into consideration. Provided that these factors are corrected for, a study that shows an association of exposure to a given agent with diminished vaccination responses may indicate suboptimal function of the immune system and clinically relevant diminished immune response. It is quite unlikely that environmental exposures that affect responses to vaccination may in fact abrogate protection to the specific pathogen for which vaccination was performed. Only in those cases where individuals have a poor response to the vaccine may exogenous factors perhaps have a clinically significant influence on resistance to the specific pathogen. An exposure-associated inhibition of a vaccination response may, however, signify a decreased host resistance to pathogens against which no vaccination had been performed.

The local lymph node assay (LLNA) is a new and promising test in mice used to identify contact allergens by means of dermal exposure. Experimentally this assay, which comprises a sensitizing phase only, is also used to identify respiratory allergens. Another, experimentally used test in mice to identify allergens is also based on dermal exposure, but comprises both a sensitizing and effector phase. In this latter test, it has been shown that contact allergens preferentially induce a T-helper 1 (TH1) response, whereas respiratory allergens preferentially induce a T-helper 2 (TH2) response. These responses can be discriminated on the basis of cytokine production, such as IFN-gamma, which is produced by TH1 cells, and IL-4, which is produced by TH2 cells. The aim of the study was to establish whether the LLNA was sufficient to not only identify allergens but also mark them as either a contact or a respiratory allergen. To this end, LLNA responses to the contact allergen dinitrochlorobenzene (DNCB) and the respiratory allergen trimellitic anhydride (TMA) were determined using IFN-gamma and IL-4 mRNA expression and production as parameters. Topical application of TMA resulted in a threefold higher lymphocyte proliferation compared to DNCB 3 and 5 days after the first application, while a similar proliferation was found from Day 7 and onward. RT-PCR showed a similar induction of IFN-gamma and IL-4 mRNA expression. While both DNCB and TMA induced IFN-gamma production, TMA but not DNCB induced IL-4 production. Thus, only IL-4 production seemed a suitable parameter to discriminate between the two compounds. In a second study, the respiratory allergens toluene-2,4-diisocyanate (TDI) and phthalic anhydride (PA) were also assayed 7 days after the first application. Topical application of DNCB and PA resulted in a similar lymphocyte proliferation, while application of TMA and TDI resulted in a 1.8-fold higher proliferation. IFN-gamma production was similar for DNCB, TMA, and TDI, and fourfold lower for PA, while IL-4 production was similar for TMA, TDI, and PA, and 24-fold lower for DNCB. In summary, both studies showed induction of IL-4 production by respiratory allergens, with little or no induction by the contact allergen, holding promise for the possibility of identifying respiratory allergens within the LLNA by measuring IL-4 production 7 days after the first application.

Rat thymocytes and splenocytes were exposed in vitro to the model compounds Cyclosporin A (CsA), an immunosuppressive drug, and bis(tri-n-butyltin)oxide (TBTO), an immunotoxic environmental contaminant. The lymphocyte transformation test (LTT), cytokine (receptor) mRNA expression (RT-PCR and dot blot hybridisation), and flow cytometry were evaluated as assays for in vitro immunotoxicity, at dose levels that did not show effects on viability, this being the aim of the study. LTT and RT-PCR proved useful assays. Lymphocyte transformation was suppressed by both compounds, while IL-2 mRNA expression was suppressed by CsA but not by TBTO, and both compounds suppressed IL-2R mRNA expression in splenocytes but not in thymocytes. Furthermore, the data obtained suggest that antiproliferative effects may be more relevant than apoptosis induction for TBTO induced thymus atrophy.

Bis(tri-n-butyltin)oxide (TBTO) has been shown to be immunotoxic in rodents, resulting in decreased resistance to infections. The no-effect level assessed by estimating effects on host resistance in rats has been found to lie between 0.5 and 5.0 mg TBTO/kg food (0.025 and 0.25 mg/kg body weight). For risk assessment such animal data need to be extrapolated to the human situation. In risk assessment procedures uncertainty factors are used to account for interspecies variation (extrapolation from animal to man) and for variation within the human species. For both factors a value of to is often used, based on international guidelines. Hence, exposures below 0.00025 mg/kg body weight should not pose a risk for the human population.

Erratum to ‘‘Comparison of dose–responses of contact allergens using the guinea pig maximization test and the local lymph node assay’’ [Toxicology 167 (2001) 207–215] Francois M.M. van Och , Rob J. Vandebriel , Menk K. Prinsen , Wim H. De Jong , Wout Slob , Henk van Loveren * a National Institute of Public Health and the En irontnent (RIVM), P.O. Box 1, 3720 BA, Biltho en, The Netherlands b TNO Nutrition and Food Research Institute, P.O. Box 360, 3700 AJ, Zeist, The Netherlands