Non-clinical safety evaluation of repeated intramuscular administration of the AS15 immunostimulant combined with various antigens in rabbits and cynomolgus monkeys.

Combination of tumor antigens with immunostimulants is a promising approach in cancer immunotherapy. We assessed animal model toxicity of AS15 combined with various tumor antigens: WT1 (rabbits), or p501, dHER2 and recPRAME (cynomolgus monkeys), administered in seven or 20 dose regimens versus a saline control. Clinical and ophthalmological examinations, followed by extensive post-mortem pathological examinations, were performed on all animals. Blood hematology and biochemistry parameters were also assessed. Antigen-specific antibody titers were determined by enzyme-linked immunosorbent assay. Additional assessments in monkeys included electrocardiography and immunohistochemical evaluations of the p501 expression pattern. Transient increases in body temperature were observed 4 h or 24 h after injections of recPRAME + AS15 and dHER2 + AS15. Edema and erythema were observed up to 1 week after most injections of recPRAME + AS15 and all injections of dHER2 + AS15. No treatment-related effects were observed for electrocardiography parameters. Mean fibrinogen levels were significantly higher in all treated groups compared to controls, but no differences could be observed at the end of the treatment-free period. Transient but significant differences in biochemistry parameters were observed post-injection: lower albumin/globulin ratios (p501 + AS15), and higher bilirubin, urea and creatinine (dHER2 + AS15). Pathology examinations revealed significant increases in axillary lymph node mean weights (recPRAME + AS15) compared to controls. A 100% seroconversion rate was observed in all treated groups, but not in controls. p501 protein expression was observed in prostates of all monkeys from studies assessing p501 + AS15. These results suggest a favorable safety profile of the AS15-containing candidate vaccines, supporting the use of AS15 for clinical development of potential anticancer vaccines.

Subchronic (13-week) oral toxicity study in rats with fungal chitin-glucan from Aspergillus niger.

Chitin-glucan is an insoluble biopolymer, composed of chitin and beta-(1,3)-D-glucan, that is a component of the fungal cell wall. This study was conducted to assess the safety of chitin-glucan from the mycelium of Aspergillus niger (Artinia brand) for use as dietary supplement and food ingredient. Chitin-glucan was fed to Wistar rats (20/sex/group) at dietary levels of 0, 1, 5 and 10% for 13 weeks. Clinical and neurobehavioural observations, growth, feed and water consumption, ophthalmoscopy, haematology, clinical chemistry, urinalysis, organ weights, necropsy and histopathological examination revealed no adverse effects of chitin-glucan. Rats fed chitin-glucan at 10% consumed more feed than controls, probably due to lower energy density of their diet. Water intake was increased slightly at all dose levels. These changes were not toxicologically significant. Full and empty caecum weights were increased in mid-dose males and high-dose males and females. This caecal enlargement was a physiological response to the consumption of a high amount of poorly digestible carbohydrate and considered of no toxicological concern. In conclusion, feeding chitin-glucan at dietary levels up to 10% for 13 weeks was tolerated without any signs of toxicity. This level corresponded to 6.6 and 7.0 g chitin-glucan/kg body weight/day in male and female rats, respectively.

The accuracy of extended histopathology to detect immunotoxic chemicals.

The accuracy of extended histopathology to detect immunotoxic chemicals in female B6C3F1 mice was evaluated under the auspices of the National Toxicology Program (NTP). A workgroup was formed consisting of four pathologists who conducted extended histopathological evaluation of lymphoid tissues obtained from a subset of NTP toxicology studies, in which previously detailed immunotoxicity assessment was performed. In addition, a positive control data set of three known immunosuppressive agents, one negative control data set, and an additional negative control group composed of the vehicle only treated groups were included. Data obtained from extended histopathology evaluations were compared to more traditional immune test results (both functional and nonfunctional) from previously conducted immunotoxicity assessments. Analyses of the data indicated that the ability to identify immunotoxic chemicals using histological endpoints decreased linearly as the level of stringency used to determine significant histopathological changes increased. A relatively high (80%) accuracy level was achieved when histological changes were considered in toto (i.e., any histological abnormality in the three tissues examined), using minimal or mild criteria for scoring. When minimal or mild histological changes were considered significant for a specific tissue, a 60% level of accuracy in identifying immunotoxic chemicals was obtained as compared to a 90% accuracy level that was achieved with this data set using the antibody plaque forming cell response, considered to represent the most predictive functional test. A minimal classification was obtained in the analyses of the negative control groups, suggesting that use of the minimal classification for hazard identification is inappropriate as it will likely result in a high incidence of false positives. This was not the case when mild classifications were used as an indicator of significance, which in most instances allowed the successful identification of negatives. When moderate to marked histopathological changes were used to identify immunotoxic chemicals, the level of accuracy that could be achieved was poor. A considerably higher level of accuracy was obtained for the positive control data set than the test chemical data set suggesting that the ability to detect an immunotoxic agent histologically is proportional to the potency of the immunotoxic agent. Comparison of immune function test results and histopathological results obtained from the high-dose treatment groups and the lower-dose treatment group did not reveal any significant differences between the two endpoints to predict immunotoxicity as a function of dose. Of the three lymphoid organs examined, (i.e., lymph node, thymus, and spleen), the most consistent and discernible histological lesions were observed in the thymus cortical region. These lesions correlated with thymus: body weight ratios and to a slightly lesser extent, the antibody plaque forming cell response. Addition of general toxicological endpoints such as body weight and leukocyte counts did not significantly improve the sensitivity of extended histopathology for this data set. Taken together, these data suggest that, while not as sensitive as functional analyses, extended histopathology may provide a reasonable level of accuracy as a screening test to identify immunotoxic chemicals, provided the level of stringency used to score histological lesions is carefully considered to allow for detection of immunotoxic agents while limiting false positives.

Extended histopathology in immunotoxicity testing: interlaboratory validation studies.

There has been considerable interest in the use of expanded histopathology as a primary screen for immunotoxicity assessment. To determine the utility of a semiquantitative histopathology approach for examining specific structural and architectural changes in lymphoid tissues, a validation effort was initiated. This study addresses the interlaboratory reproducibility of extended histopathology, using tissues from studies of ten test chemicals and both negative and positive controls from the National Toxicology Program's immunotoxicology testing program. We examined the consistency between experienced toxicologic pathologists, who had varied expertise in immunohistopathology in identifying lesions in immune tissues, and in the sensitivity of the individual and combined histopathological endpoints to detect chemical effects and dose response. Factor analysis was used to estimate the association of each pathologist with a so-called "common factor" and analysis-of-variance methods were used to evaluate biases. Agreement between pathologists was highest in the thymus, in particular, when evaluating cortical cellularity of the thymus; good in spleen follicular cellularity and in spleen and lymph node-germinal center development; and poorest in spleen red-pulp changes. In addition, the ability to identify histopathological change in lymphoid tissues was dependent upon the experience/training that the individual pathologist possessed in examining lymphoid tissue and the apparent severity of the specific lesion.

Ninety-day oral toxicity study of lycopene from Blakeslea trispora in rats.

Lycopene, as a suspension in sunflower oil (20% w/w), was tested for subchronic toxicity by administration at dietary concentrations of 0, 0.25, 0.50, and 1.0% to groups of 20 male and 20 female Wistar rats for a period of 90 days. The lycopene examined in this study was derived from a fungal biomass (Blakeslea trispora). Lycopene intake was calculated to be 0, 145, 291, and 586mg/kg body weight/day in control through high-dose males and 0, 156, 312, and 616mg/kg body weight/day in control through high-dose females. The results from this study do not provide any evidence of toxicity of lycopene at dietary levels up to 1.0% as demonstrated by the findings of clinical observations, neurobehavioral observations, motor activity assessment, body weight and food consumption measurements, ophthalmoscopic examinations, hematology, clinical chemistry, urinalysis, organ weights, gross pathology, or histopathology. The No-Observed-Effect Level (NOEL) was 1.0% in the diet, the highest dietary concentration tested.

Respiratory irritation by trimellitic anhydride in Brown Norway and Wistar rats.

Several acid anhydrides are known for their sensitizing and irritative properties. Since both irritation and respiratory allergy can cause changes of lung function, proper testing of allergen-dependent effects on the respiratory tract requires knowledge of the respiratory irritant effects. To study the latter effects, groups of female Brown Norway (BN) and Wistar rats were exposed for 30 min to a range of concentrations (10 to 300 mg/m(3)) of the well-known respiratory allergen trimellitic anhydride (TMA). Breathing pattern and frequency were monitored before, during, and after exposure. Animals were necropsied and lung weights were determined 1 day after exposure. In BN rats, changes in breathing pattern were seen at levels of 29 mg/m(3) and higher and decreases in frequency at 60 mg/m(3) and higher, whereas in Wistar rats changes in both pattern and frequency (increases followed by decreases) were seen at levels of 34 mg/m(3) and higher. Changes in breathing pattern consisted of a spiked form instead of a wave form of the respiratory cycle, with a pause between breaths at the end of expiration. The length of the pause increased with increasing concentrations of TMA while the duration of the respiratory cycle decreased slightly, implying that breathing frequency was mainly determined by the magnitude of the increase in pause. These reversible changes in breathing pattern and frequency were considered to be suggestive of lower airway irritation, rather than upper airway irritation. No concentration-related changes in lung weights were observed. The highest level at which no acute airway irritation as based on both breathing pattern and frequency was observed in both rat strains was 14 mg/m(3).

Health risks associated with inhaled nasal toxicants.

Health risks of inhaled nasal toxicants were reviewed with emphasis on chemically induced nasal lesions in humans, sensory irritation, olfactory and trigeminal nerve toxicity, nasal immunopathology and carcinogenesis, nasal responses to chemical mixtures, in vitro models, and nasal dosimetry- and metabolism-based extrapolation of nasal data in animals to humans. Conspicuous findings in humans are the effects of outdoor air pollution on the nasal mucosa, and tobacco smoking as a risk factor for sinonasal squamous cell carcinoma. Objective methods in humans to discriminate between sensory irritation and olfactory stimulation and between adaptation and habituation have been introduced successfully, providing more relevant information than sensory irritation studies in animals. Against the background of chemoperception as a dominant window of the brain on the outside world, nasal neurotoxicology is rapidly developing, focusing on olfactory and trigeminal nerve toxicity. Better insight in the processes underlying neurogenic inflammation may increase our knowledge of the causes of the various chemical sensitivity syndromes. Nasal immunotoxicology is extremely complex, which is mainly due to the pivotal role of nasal lymphoid tissue in the defense of the middle ear, eye, and oral cavity against antigenic substances, and the important function of the nasal passages in brain drainage in rats. The crucial role of tissue damage and reactive epithelial hyperproliferation in nasal carcinogenesis has become overwhelmingly clear as demonstrated by the recently developed biologically based model for predicting formaldehyde nasal cancer risk in humans. The evidence of carcinogenicity of inhaled complex mixtures in experimental animals is very limited, while there is ample evidence that occupational exposure to mixtures such as wood, leather, or textile dust or chromium- and nickel-containing materials is associated with increased risk of nasal cancer. It is remarkable that these mixtures are aerosols, suggesting that their "particulate nature" may be a major factor in their potential to induce nasal cancer. Studies in rats have been conducted with defined mixtures of nasal irritants such as aldehydes, using a model for competitive agonism to predict the outcome of such mixed exposures. When exposure levels in a mixture of nasal cytotoxicants were equal to or below the "No-Observed-Adverse-Effect-Levels" (NOAELs) of the individual chemicals, neither additivity nor potentiation was found, indicating that the NOAEL of the "most risky chemical" in the mixture would also be the NOAEL of the mixture. In vitro models are increasingly being used to study mechanisms of nasal toxicity. However, considering the complexity of the nasal cavity and the many factors that contribute to nasal toxicity, it is unlikely that in vitro experiments ever will be substitutes for in vivo inhalation studies. It is widely recognized that a strategic approach should be available for the interpretation of nasal effects in experimental animals with regard to potential human health risk. Mapping of nasal lesions combined with airflow-driven dosimetry and knowledge about local metabolism is a solid basis for extrapolation of animal data to humans. However, more research is needed to better understand factors that determine the susceptibility of human and animal tissues to nasal toxicants, in particular nasal carcinogens.

Chronic pulmonary effects of respirable methylene diphenyl diisocyanate (MDI) aerosol in rats: combination of findings from two bioassays.

Two independent bioassays are available which have examined the potential carcinogenicity of monomeric and polymeric methylene diphenyl diisocyanate (MDI) following long-term inhalation exposure in rats. These studies are not directly comparable, however, due to differences in design and conduct of the in-life phase, and differences in nomenclature used for some of the histopathological findings. This paper presents a definitive overview ofthe pulmonary toxicity of MDI developed following a thorough review of both investigations. As part of this process, the test materials and the designs of the studies were compared, and an in-depth review of lung lesions was conducted by an independent reviewing pathologist. This included the re-examination of the original lung slides, supported by an analysis of the exposure regimens, the results of which were used to develop an accurate profile of the doses received by the animals in the two studies. Histopathological findings were then combined with this information to give an overall dose-response curve for both studies as a whole. The range of total inhalation exposures to MDI was calculated as 559, 1972, 2881, 6001, 17,575 and 17,728 mgh/m3. Major pulmonary effects included increased lung weights together with bronchiolo-alveolar adenomas and hyperplasia, and interstitial fibrosis which occurred consistently in both studies, indicating a very similar qualitative response of the lungs to polymeric and monomeric MDI. The quantitative response of the lung was clearly dose-related in each study, and when the studies were considered as a whole a reasonable overall dose-response relationship was apparent for major lung lesions. Lung tumours (in low incidences) only occurred at the highest dose level in both studies (17,575 and 17,728 mgh/m3). For inflammatory and other non-neoplastic pulmonary changes, the lowest dose examined (559 mgh/m3) was regarded as a no-observed-adverse-effect-level for both polymeric and monomeric MDI. It was concluded that the results of the two studies could be combined to serve as a basis for human risk assessment of MDI.

Disposition, accumulation and toxicity of iron fed as iron (II) sulfate or as sodium iron EDTA in rats.

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate. It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO(4). Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO(4) and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects.

Histopathologic approaches to detect changes indicative of immunotoxicity.

Toxicologic pathology is crucial in the identification and characterization of health effects following exposure to xenobiotics, mainly in toxicity experiments in rodents. Regarding regulatory toxicology, histopathology of lymphoid organs and tissues is a cornerstone in the identification of immunotoxic compounds. A 2-tier testing system is usually employed in which the first tier is a general screen for (immuno)toxicity and the second tier consists of specific immune function studies, including host resistance tests or mechanistic studies. The role attributed to histopathology of lymphoid organs in the updated Organisation for Economic Cooperation and Development and Food and Drug Administration guidelines requires improvement and standardization of the histopathology procedures. Optimalization and standardization was started in an international collaborative immunotoxicity study (ICICIS). However, several problems were left unaddressed, mostly because of the few compounds tested in this study. Based on the results of the ICICIS study and the morphologic changes induced by immunotoxic/immunomodulatory compounds observed in other investigations, suggestions are given to further improve the identification and (semi)quantification of histopathologic changes in lymphoid organs and tissues.

Predictive testing for pathogenic autoimmunity: the morphological approach.

The term autoimmunity refers to physiologically normal immune processes against self-antigens. In rare cases, the regulatory mechanisms become deflective and the uncontrolled production of autoantibodies or activation of autoreactive T-cells can subsequently cause disease. Substances may be capable of evoking autoimmune disease, and it is a challenge in routine toxicology to recognize such substances. In in vivo toxicity studies, uncommon inflammation in exposed animals should be discussed in terms of non-immune toxicity (e.g. irritation), infection, allergy and autoimmunity, taking into account that a response in even a few animals may be significant. Moreover, early morphological indicators of inflammation and lymphoid organ alterations can direct further investigation.

Intestinal T lymphocytes of different rat strains in immunotoxicity.

In order to study the intestinal mucosal immune cells, with emphasis on single T lymphocytes, an inventory was made of single and organized lymphocytes in the epithelium and lamina propria of the small intestines of untreated Wistar, Fischer 344, and Lewis rats. The single and organized lymphocytes were examined microscopically. In addition, the single lymphocytes in the epithelium (IEL) and lamina propria (LPL) were analyzed by flow cytometry. Next, the use of flow cytometry analysis was explored to detect changes in the IEL T-lymphocyte population in subacute oral studies with the immunomodulating agents azathioprine and hexachlorobenzene. Untreated random-bred Wistar rats exhibited a large interindividual variability in IEL composition, while the variability was small in inbred Fischer 344 and Lewis rats. The explorative study with the 2 model immunomodulating compounds demonstrated that hexachlorobenzene increased the number of intraepithelial T lymphocytes with CD8+ phenotype at the cost of T cells with CD4+ phenotype in Lewis rats. Azathioprine did not induce distinct effects on the percentages of IEL. The data indicate that the intraepithelial lymphocytes in the intestines are a potential target for orally administered immunomodulating compounds and should therefore receive more attention in toxicologic pathology studies.

Airway morphology and function of rats following dermal sensitization and respiratory challenge with low molecular weight chemicals.

Local lymph node activation and increased total serum IgE levels are suggested to be predictive parameters of airway hypersensitivity caused by low molecular weight (LMW) chemicals. Whether increases of total serum IgE are indicative of actual induction of specific airway reactions (morphological and functional) after inhalation challenge was examined in the present study. In Brown Norway (BN) and Wistar rats, serum IgE concentrations were examined following topical exposure of chemicals with known diverse sensitization potential in humans: trimellitic anhydride (TMA), a dermal and respiratory sensitizer; dinitrochlorobenzene (DNCB), a dermal sensitizer with no known potential to cause respiratory allergy; and methyl salicylate, a skin irritant devoid of sensitizing properties. Functional and histopathological changes in the respiratory tract were examined after subsequent inhalatory challenge with these chemicals. Of the three tested chemicals, only topical exposure to TMA resulted in a significant increase in total serum IgE concentrations in the high-IgE-responding BN rat. Upon subsequent inhalatory challenge of these rats, TMA induced specific airway reactions which included a sharp decrease in respiratory rate during challenge, followed by an increase in breathing rate with a concomitant decrease in tidal volume 24 and 48 h after inhalatory challenge, and histopathological changes in the larynx and lungs of animals necropsied 48 h after challenge. Interestingly, despite low IgE levels, TMA induced histopathological changes in the larynx and lungs of Wistar rats too. Laryngeal changes were also observed in Wistar rats upon sensitization and challenge with DNCB. These data suggest that increased total serum IgE after topical sensitization is associated with immediate-type specific airway reactivity after inhalation challenge in BN rats and thus may be a valuable parameter in testing for respiratory sensitization potential of LMW compounds. Histopathological examination upon subsequent inhalation challenge of sensitized low-IgE-responders may provide information on other allergic inflammatory airway reactions.

Carcinogenic response of the nasal cavity to inhaled chemical mixtures.

Nasal cancers occur in experimental animals following chronic exposure to a wide range of inhaled chemicals. Although exposure to several of these chemicals is common in industrial as well as domestic environments, epidemiological studies have not provided convincing evidence that exposure to these chemicals individually is associated with nasal cancer in humans. The reverse seems to be true for inhalation of chemical mixtures. The evidence for nasal carcinogenicity of inhaled chemical mixtures in experimental animals is very limited, while there is ample evidence in humans that occupational exposure to certain chemical mixtures is associated with increased risk of nasal cancer. Examples of such (complex) chemical mixtures are wood dust, textile dust, chromium- and nickel-containing materials and leather dust. It is remarkable that these mixtures are aerosols, suggesting that a 'dusty working environment' may increase nasal cancer risk.

Thymic microenvironment at the light microscopic level.

The thymus is a primary lymphoid organ that serves the immune system by providing an optimal microenvironment for developing T cells to rearrange the genes encoding the T-cell receptor and to undergo positive and negative selection in shaping the peripheral T-cell repertoire. The microenvironment of the organ is peculiar among lymphoid organs, as the supporting stroma consists of reticular epithelial cells. Bone marrow-derived interdigitating cells and macrophages are the main accessory cell populations. The epithelium, interdigitating cells, and macrophages each contribute to the T-cell selection process. During the last decade knowledge has been gathered that these cell populations show a considerable heterogeneity, as documented for subcellular features and immunologic phenotype. This heterogeneity may reflect various stages in differentiation, but may otherwise be linked to the functional activity of the cells. The authors survey the major cell populations, i.e., epithelial cells and lymphocytes. Macrophages and interdigitating cells are briefly discussed. Emphasis is given to functional aspects of histologic/ cytologic features.

Nitrite-induced adrenal effects in rats and the consequences for the no-observed-effect level.

In a previous subchronic oral toxicity study with potassium nitrite, hypertrophy of the adrenal zona glomerulosa was observed for all nitrite levels examined including the lowest level of 100 mg/litre. This present study was carried out, therefore, to establish a no-observed-effect level (NOEL) for nitrite. Groups of 10 male and 10 female 6-wk-old Wistar rats received KNO2 at levels of 12.5, 25, 50, 100 or 3000 mg/litre or NaNO2 at levels of 81 or 2432 mg/litre in the drinking water for 13 wk. The nitrite content of the drinking water in the latter two groups was equal to that of the 100 and 3000 mg KNO2/litre groups, respectively. Potassium and sodium concentrations were equalized in the corresponding test groups with KCl and NaCl, respectively. General health, behaviour and survival were not affected by the ingestion of nitrite. Body weight and food and liquid intake were slightly decreased in the 3000 mg KNO2/litre and 2432 mg NaNo2/litre groups for both sexes. Methaemoglobin concentration was significantly elevated in rats of both high-dose nitrite groups in wk 4 and 12, while slight increases in a number of red blood cell variables occurred with 3000 mg KNO2/litre in females in wk 12. Relative kidney weights were increased in both high-dose nitrite groups. In wk 4, plasma aldosterone and corticosterone levels were slightly decreased in males with 2432 mg NaNO2/litre and plasma corticosterone in females with 3000 mg KNO2/litre but not in wk 13. Systolic blood pressure was not affected by nitrite. Microscopic examination revealed slight hypertrophy of the adrenal zona glomerulosa in animals of the 100 and 3000 mg KNO2/litre and of the 81 and 2432 mg NaNO2/litre groups, the incidence and degree being dose related. The results obtained with 100 and 3000 mg KNO2/litre in the drinking water were comparable with those found at the same levels in the previous 90-day study. The effects with sodium nitrite were similar to those observed with potassium nitrite. The biological significance of the adrenal zona glomerulosa hypertrophy is discussed. It is concluded that the NOEL of KNO2 is 50 mg/litre in the drinking water, equivalent to about 5 mg/kg body weight/day.

Subacute toxicity of a mixture of nine chemicals in rats: detecting interactive effects with a fractionated two-level factorial design.

The present study was intended (1) to find out whether simultaneous administration of nine chemicals at a concentration equal to the "no-observed-adverse-effect level" (NOAEL) for each of them would result in a NOAEL for the combination and (2) to test the usefulness of fractionated factorial models to detect possible interactions between chemicals in the mixture. A 4-week oral/inhalatory study in male Wistar rats was performed in which the toxicity (clinical chemistry, hematology, biochemistry, and pathology) of combinations of the nine compounds was examined. The study comprised 20 groups, 4 groups in the main part (n = 8) and 16 groups in the satellite part (n = 5). In the main study, the rats were simultaneously exposed to mixtures of all nine chemicals [dichloromethane, formaldehyde, aspirin, di(2-ethylhexyl)phthalate, cadmium chloride, stannous chloride, butyl hydroxyanisol, loperamide, and spermine] at concentrations equal to the "minimum-observed-adverse-effect level" (MOAEL), NOAEL, or 1/3NOAEL. In the satellite study the rats were simultaneously exposed to combinations of maximally five compounds at their MOAEL. These combinations jointly comprise a two-level factorial design with nine factors (=9 chemicals) in 16 experimental groups (1/32 fraction of a complete study). In the main part many effects on hematology and clinical chemistry were encountered at the MOAEL. In addition, rats of the MOAEL group showed hyperplasia of the transitional epithelium and/or squamous metaplasia of the respiratory epithelium in the nose. Only very few adverse effects were encountered in the NOAEL group. For most of the end points chosen, the factorial analysis revealed main effects of the individual compounds and interactions (cases of nonadditivity) between the compounds. Despite all restrictions and pitfalls that are associated with the use of fractionated factorial designs, the present study shows the usefulness of this type of factorial design to study the joint adverse effects of defined chemical mixtures at effect levels. It was concluded that simultaneous exposure to these nine chemicals does not constitute an evidently increased hazard compared to exposure to each of the chemicals separately, provided the exposure level of each chemical in the mixture is at most similar to or lower than its own NOAEL.

Local lymph node activation and IgE responses in brown Norway and Wistar rats after dermal application of sensitizing and non-sensitizing chemicals.

The local lymph node assay (LLNA) and the IgE test in the mouse are proposed models for predictive recognition of low molecular weight chemicals causing IgE-mediated allergic airway reactions in man. Since rats are commonly used in routine toxicity studies and a previous study (Arts et al. (1996) Food Chem. Toxicol. 34, 55-62) has shown that several rat strains were found appropriate for the LLNA, the suitability of the rat for the IgE test was examined in the present study. Serum IgE concentrations were examined following topical exposure of Brown Norway (BN) and Wistar rats to each of four chemicals with known diverse sensitization potential in humans: trimellitic anhydride (TMA), a dermal and respiratory sensitizer, dinitrochlorobenzene (DNCB), a dermal sensitizer with no or limited potential to cause respiratory allergy; formaldehyde (FA), a skin irritant and dermal sensitizer with equivocal evidence for respiratory sensitizing potential; methyl salicylate (MS), a skin irritant devoid of sensitizing properties. Of the four tested chemicals, only exposure to TMA resulted in a significant increase in serum IgE concentration and this response was only evoked in the high-IgE-responding BN rat. The latter two chemicals were also tested for lymph node activation, in casu the ear-draining lymph nodes. FA caused a dose-dependent activation of the draining lymph nodes whereas MS was inactive. The results as obtained with TMA, DNCB and MS in the rat are in agreement with human data. The results with FA though, indicate the need for further studies of chemicals that have both irritant and sensitizing properties at about similar concentrations or may act through non-IgE-mediated immune mechanisms.

Subchronic oral toxicity studies with erythritol in mice and rats.

Erythritol is a sugar alcohol (polyol) with potential applications as a low-calorie, bulk sweetener. Ingested erythritol is efficiently absorbed and excreted unchanged via the urine since it is not metabolized systemically by the animal or human body. Erythritol was administered to four groups of 10 male and 10 female Swiss CD-1 mice and four groups of 15 male Wistar Crl:(WI) WU BR rats at dietary levels of 0, 5, 10, or 20% for 90 days. A fifth group of rats received a diet containing 20% erythritol on a time-restricted basis (6 hr/day), and a sixth group received a diet containing 20% mannitol for comparison. There were no treatment-related mortalities in either mice or rats. Soft stools and occasional diarrhea were observed in rats fed diets with 20% erythritol or mannitol but not in mice. Body weights were slightly yet significantly reduced in rats fed 20% erythritol or mannitol and in male mice of the 20% dose group. Erythritol intake in the high-dose group was approximately 12 g/kg body wt in rats and 44 and 45 g/kg body wt in male and female mice, respectively. Hematological and clinicochemical examinations of blood and plasma did not reveal any treatment-related effects. Urine output increased with increasing erythritol dose. In male and female mice of the 20% erythritol group, the creatinine-normalized urinary excretion of protein, K-glutamyltransferase (GGT), and electrolytes (Na+, K+, Ca2+, Pi, citrate) was significantly increased while urinary N-acetylglucosaminidase (NAG) remained unchanged. At the 10% level, significantly increased urinary protein (both sexes) and GGT (males only) excretion were seen. In rats, the creatinine-normalized urinary excretion of GGT, NAG, and some electrolytes (Na+, K+, and Ca2+) was increased in some erythritol groups but a clear dose-response relationship was evident only for calcium. On termination of the study, cecal enlargement was seen in rats of the 10 and 20% dose groups and in mice of the 20% dose group. Increased relative and absolute kidney weights were observed in both sexes of mice in the 20% erythritol group, in male mice of the 5 and 10% groups, and in rats of the 10 and 20% erythritol groups. Histopathological examination did not reveal any treatment-related abnormalities in either mice or rats. In conclusion, the ingestion of erythritol for 90 days at dietary levels of up to 20% did not produce signs of toxicity in mice or rats. In particular, the morphological integrity of the kidneys was not adversely affected by the treatment in either species. The increases in urinary excretion of protein, GGT, NAG, and electrolytes were considered to result from extensive osmotic diuresis and a potential overload of the renal excretory system at the high dose levels employed.

Local lymph node activation in rats after dermal application of the sensitizers 2,4-dinitrochlorobenzene and trimellitic anhydride.

Five rat strains were compared for their performance in the local lymph node assay (LLNA), a promising test system for the identification of the skin-sensitizing potential of chemicals in the mouse. The contract sensitizer 2,4-dinitrochlorobenzene (DNCB) and the contact and respiratory sensitizer trimellitic anhydride (TMA) were used as model chemicals and responses in rats were compared with those in BALB/c mice. The chemicals were applied to the dorsum of both ears, once daily for three consecutive days; 2 days (mice) or 3 days (rats) thereafter, proliferating cells were labelled by i.p. injection of BrdU 2 hr before the animals were killed. Systemic effects were subsequently assessed by determination of spleen, liver and kidney weights, skin effects by determination of swelling and inflammatory cell infiltration of the ears, and immune effects by determination of weight and proliferative activity of the local lymph nodes (LLN). Following application (x 3) of DNCB or TMA, minor systemic effects were observed, as indicated by slightly elevated spleen and liver weights in a few rat strains and the mice. Skin effects, consisting of increased ear thickness and presence of mononuclear inflammatory cell infiltrates, were observed in all rat strains treated with DNCB or TMA, LLN weights had increased, as had the proliferative activity in these nodes. It was concluded that effects induced by DNCB and TMA in all five rat strains were comparable with those in mice.