Further experience with the local lymph node assay using standard radioactive and nonradioactive cell count measurements.

In a previous study, the predictive capacity of a modified local lymph node assay (LLNA) based on cell counts, the LNCC, was demonstrated to be closely similar to that of the original assay. In addition, a range of substances, including some technical/commercial materials and a range of agrochemical formulations (n = 180) have also been assessed in both methods in parallel. The results in the LNCC and LLNA were generally consistent, with 86% yielding an identical classification outcome. Discordant results were associated with borderline data and were evenly distributed between the two methods. Potency information derived from each method also demonstrated good consistency (n = 101), with 93% of predictions being close. Skin irritation was observed only infrequently and was most commonly associated with positive results; it was not associated with the discordant results. Where different vehicles were used with the same test material, the effect on sensitizing activity was modest, consistent with historical data. Analysis of positive control data indicated that the LNCC and LLNA displayed similar levels of biological variation. When taken in combination with the previously published results on LLNA Performance Standard chemicals, it is concluded that the LNCC provides a viable non-radioactive alternative to the LLNA for the assessment of substances, including potency predictions, as well as for the evaluation of preparations.

Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements.

The local lymph node assay (LLNA) is the preferred test for identification of skin-sensitizing substances by measuring radioactive thymidine incorporation into the lymph node. To facilitate acceptance of nonradioactive variants, validation authorities have published harmonized minimum performance standards (PS) that the alternative endpoint assay must meet. In the present work, these standards were applied to a variant of the LLNA based on lymph node cell counts (LNCC) run in parallel as a control with the standard LLNA with radioactivity measurements, with threshold concentrations (EC3) being determined for the sensitizers. Of the 22 PS chemicals tested in this study, 21 yielded the same results from standard radioactivity and cell count measurements; only 2-mercaptobenzothiazole was positive by LLNA but negative by LNCC. Of the 16 PS positives, 15 were positive by LLNA and 14 by LNCC; methylmethacrylate was not identified as sensitizer by either of the measurements. Two of the six PS negatives tested negative in our study by both LLNA and LNCC. Of the four PS negatives which were positive in our study, chlorobenzene and methyl salicylate were tested at higher concentrations than the published PS, whereas the corresponding concentrations resulted in consistent negative results. Methylmethacrylate and nickel chloride tested positive within the concentration range used for the published PS. The results indicate cell counts and radioactive measurements are in good accordance within the same LLNA using the 22 PS test substances. Comparisons with the published PS results may, however, require balanced analysis rather than a simple checklist approach.

Experience with the HET-CAM method in the routine testing of a broad variety of chemicals and formulations.

Data on eye irritation are generally needed for the hazard identification of chemicals. For the routine testing of a broad variety of chemicals and formulations, we have used the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method. In the course of a tiered-testing strategy, and due to the lack of global regulatory acceptance of the HET-CAM method, we have also performed the Rabbit Eye Irritation test according to OECD Test Guideline 405. Of the 145 substances tested, 76% were classified as non-irritant/mild irritant and 13% were identified as irritant in vivo, according to the EU classification system (61% and 28%, respectively, with the GHS classification). The remaining 11% were severe irritants in vivo, based on the irreversibility of the effects and not due to sufficiently high irritation scores in the three days following application. The retrospective analysis revealed that the overall accuracy of the HET-CAM assay was 65% and the overall rates of false-negatives (FN) and false-positives (FP) were 50% and 33%, respectively. The HET-CAM assay was sufficiently specific (few FP) for water-soluble substances, but failed to identify nearly all the severe irritants within this group. In contrast, it was highly sensitive (no FN) for non-soluble and oil-soluble substances, but the specificity for this group was rather low. Therefore, we conclude that the HET-CAM assay is not useful in our tiered-testing strategy for eye irritation testing. However, for water-insoluble substances, it might be applicable in combination with another in vitro method, provided that regulatory acceptance is gained.

Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months.

Carbon nanotubes (CNT) are of great commercial interest. Theoretically, during processing and handling of CNT and in abrasion processes on composites containing CNT, inhalable CNT particles might be set free. For hazard assessment, we performed a 90-day inhalation toxicity study with a multiwall CNT (MWCNT) material (Nanocyl NC 7000) according to Organisation for Economic Co-operation and Development test guideline 413. Wistar rats were head-nose exposed for 6 h/day, 5 days/week, 13 weeks, total 65 exposures, to MWCNT concentrations of 0 (control), 0.1, 0.5, or 2.5 mg/m(3). Highly respirable dust aerosols were produced with a proprietary brush generator which neither damaged the tube structure nor increased reactive oxygen species on the surface. Inhalation exposure to MWCNT produced no systemic toxicity. However, increased lung weights, pronounced multifocal granulomatous inflammation, diffuse histiocytic and neutrophilic inflammation, and intra-alveolar lipoproteinosis were observed in lung and lung-associated lymph nodes at 0.5 and 2.5 mg/m(3). These effects were accompanied by slight blood neutrophilia at 2.5 mg/m(3). Incidence and severity of the effects were concentration related. At 0.1 mg/m(3), there was still minimal granulomatous inflammation in the lung and in lung-associated lymph nodes; a no observed effect concentration was therefore not established in this study. The test substance has low dust-forming potential, as demonstrated by dustiness measurements, but nonetheless strict industrial hygiene measures must be taken during handling and processing. Toxicity and dustiness data such as these can be used to compare different MWCNT materials and to select the material with the lowest risk potential for a given application.

Development of a short-term inhalation test in the rat using nano-titanium dioxide as a model substance.

Evidence suggests that short-term inhalation studies may provide comparable prediction of respiratory tract toxicity to 90-day studies, presenting the opportunity to save time and resources in screening inhalation toxicity of test substances. The aim of this study was to develop a short-term inhalation test that could be employed to provide early evidence on respiratory tract effects which might occur from long-term exposure to aerosols of nano-materials. Male Wistar rats were exposed to aerosols of 0 (control), 2, 10 and 50 mg/m(3) nano-titanium dioxide (TiO2) by inhalation for 6 h/day for 5 days. Necropsies were performed either immediately after the last exposure or after 3 and 16 days post exposure (study days 5, 8 and 21, respectively). Treatment with nano-TiO2 resulted in morphological changes in the lung, with 50 mg/m(3) nano-TiO2 producing an increase in lung weight. Lung inflammation was associated with dose-dependent increases in bronchoalveolar lavage fluid (BALF) total cell and neutrophil counts, total protein content, enzyme activities and levels of a number of cell mediators. No indications of systemic effects could be found by measurement of appropriate clinical pathology parameters. Cell replication (determined by incorporation of 5-bromo-2'-deoxyuridine) was increased at all nano-TiO2 dose levels in large/medium bronchi and terminal bronchioles. The effects on the parameters measured were most prominent either on study day 5 or 8, with some endpoints returning to control levels by day 21. Overall, the pulmonary effects of nano-TiO2 observed in this short-term study were comparable to those previously reported in subchronic inhalation studies.

Local lymph node assay (LLNA): comparison of different protocols by testing skin-sensitizing epoxy resin system components.

Thirteen epoxy resin system components were tested in the LLNA with regard to their sensitizing potency. Lymph node stimulation was quantified not only by measuring the incorporation of [3H]-thymidine into the ear lymph nodes but also the counts of cells recovered from these organs. Equivalent figures were obtained with both endpoints used for the evaluation of lymph node cell proliferation if the reference stimulation indices were adjusted. When dissolved in acetone, all test substances showed skin-sensitizing potential, mainly on the boundary between "strong" and "moderate" according to common potency evaluation schemes. Replacing acetone with acetone/olive oil (4:1) as a vehicle for four selected test items, resulted in considerably lower estimated concentrations for sensitization induction. The challenges in comparing the results obtained by different LLNA variations are discussed.

Assessment of the human epidermis model SkinEthic RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431.

Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing of chemicals, the National Co-ordinators of OECD Test Guideline Programme endorsed in 2002 two new test guidelines: TG 430 'Transcutaneous Electrical Resistance assay' and TG 431 'Human Skin Model Test'. To allow all suitable in vitro human reconstructed (dermal or epidermal) models to be used for skin corrosion testing, the OECD TG 431 defines general and functional conditions that the model must meet before it will be routinely used for skin corrosion testing. In addition, the guideline requires correct prediction of 12 reference chemicals and assessment of intra- and inter-laboratory variability. To show that the OECD TG 431 concept works, in 2003 ZEBET tested several chemicals from the ECVAM validation trials on the SkinEthic reconstituted human epidermal (RHE) model. Based on knowledge that reconstructed human skin models perform similarly in toxicological studies, it was decided to adopt the validated EpiDerm skin corrosion test protocol and prediction model to the SkinEthic model. After minor technical changes, classifications were obtained in concordance with those reported for the validated human skin models EPISKIN and EpiDerm. To allow adequate determination of inter-laboratory reproducibility, a blind trial was conducted in three laboratories -- ZEBET (D), Safepharm (UK) and BASF (D), in which the 12 endorsed reference chemicals were tested. Results obtained with the SkinEthic epidermal model were reproducible, both within and between laboratories, and over time. Concordance between the in vitro predictions of skin corrosivity potential obtained with the SkinEthic model and the predictions obtained with the accepted tests of OECD TG 430 and TG 431 was very good. The new test was able to distinguish between corrosive and non-corrosive reference chemicals with an accuracy of 93%.

Effects of 5-day styrene inhalation on serum prolactin and dopamine levels and on hypothalamic and striatal catecholamine concentrations in male rats.

In several studies a hypersecretion of the pituitary hormone prolactin (PRL) in styrene-exposed workers has been described. This should cause reproductive problems like oligomenorrhea, secondary amenorrhea and reduced fertility [Arfini et al. (1987) J Occup Med 29:826-830, Bergamaschi et al. (1996) Neurotoxicology 17:753-760, Mutti and Smargiassi (1998) Toxicol Ind Health 14:311-323]. Secretion of PRL is tonically inhibited by the catecholamine dopamine (DA), which is released from hypothalamic neurons. It has been suggested that the activity of the enzyme dopamine-beta-hydroxylase (DBH) in the serum is a peripheral marker of central dopaminergic function. A slight reduction of such enzymatic activity was observed in styrene-exposed workers, which was associated with hypersecretion of PRL. To further investigate the putative effects of styrene on PRL release, male rats were exposed to styrene vapors (645, 2150 and 6450 mg/m(3)) for 6 h/day on 5 consecutive days. Animals were killed either directly following the last exposure (immediate group) or after a recovery period of 24 h (recovery group). Serum PRL and DA levels were measured by radioimmunoassay. Concentrations of catecholamines and their metabolites in the striatum and mediobasal hypothalamus (MBH) were determined by high performance liquid chromatography with electrochemical detection. Neither in the immediate nor in the recovery group were any statistically significant changes of serum PRL levels observed. Likewise, concentrations of catecholamines and their metabolites in the striatum and MBH remained unaffected. We conclude from these data that styrene, even at very high concentrations, has no adverse effects on the neuroendocrine mechanisms regulating PRL release and DA levels in the brain. With the limitations inherent in any animal model, we suggest that our data indicate that styrene also has no adverse neuroendocrine effects in humans.