THE HEMOLYTIC PLAQUE-FORMING CELL ASSAY IN TILAPIA (OREOCHROMIS NILOTICUS) EXPOSED TO BENZO[a]PYRENE: ENHANCED OR DEPRESSED PLAQUE FORMATION DEPENDS ON DOSING SCHEDULE

The prospect of utilizing the cichlid teleost tilapia (Oreochromis niloticus) as an alternative experimental model to mammals for preliminary chemical immunotoxicity risk assessment is being evaluated by examining the National Toxicology Program's standard battery of rodent immunotoxicity assays in chemicaltreated tilapia. The present report examines the hemolytic plaque forming cell assay (PFC), a quantitative indicator of antibody production, in tilapia exposed to the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P). Reduced antibody production against sheep red blood cell (SRBC) antigen in response to B\[a]P was observed using the PFC assay, via reduction in plaque number. Under specific immunization circumstances, increased plaque formation was observed in chemical-exposed fish, an effect also reported in rodents. Although the normal teleost immune responsiveness was weaker than seen with mice under comparable conditions (presumably due to differences in antibody structure of teleosts), tilapia werefound toexhibit well-defined primary and secondary humoral responses to SRBC, and an immunotoxic response to B[a]P comparable to the rodent model.     

Suppression of Humoral Immune Responses by Dialkylnitrosamines: Structure-Activity Relationships

Suppression of Humoral Immune Responses by Dialkylnitrosamines:Structure-Activity Relationships. KAMINSKI, N. E., JORDAN, S.D., PAGE, D., KIM, B. S., AND HOLSAPPLE, M. P. (1989). Fundam.Appl Toxicol 12,321-332. Comparisons between chemical structureof N, N-dialkylnitrosamine congeners and their ability to alterthe Day 4 IgM antibody response to sRBC, body weights. and organweights of female B6C3F1 mice were investigated. Short-chainnitrosamine congeners were selected for these studies on thebasis of two criteria: (1) congeners wth symmetrical aliphaticchain length [N-nitrosodimethylamine (DMN), N-nitrosodiethylamine(DEN), N-nitrdipropylamine (DPN), N-nitrosodibutylamine (DBN)]and (2) congeners possessing an N-methyl group [N-nitrosomethylethylamine(MEN), N-nitrosomethylpropylamine (MPN), and N-nitrosomethylbutylamine(MBN)]. The immunotoxicity of each congener was evaluated basedon the compound's ability to suppress the in vivo sRBC antibodyresponse following 7 consecutive days of treatment. An ED50dose was calculated, using a linear regression analysis, foreach congener and represents the millimoles of congener perkilogram body weight required to cause a 50% suppression ofthe sRBC response. These studies demonstrated two general trends:(1) those dialkylnitrosamine congeners that possessed an N-methylgroup were most immunotoxic and exhibited comparable ED50 concentrations(42-183 µmol/kg); and (2) dialkylnitrosamine congenerspossessing symmetrical aliphatic chains demonstrated an inverserelationship between aliphatic chain length and immunotoxicpotency—DMN (62 µmol/kg) > DEN (276 µmol/kg)> DPN (467 µmol/kg) > DMN (1557 µmol/kg).Comparisons were also made between the immunotoxic potency ofvarious nitrosamine congeners in the whole animal and theirpotency in an in vitro hepatocyte-spleen cell coculture system.     

Immunotoxicogenomics: the potential of genomics technology in the immunotoxicity risk assessment process.

Evaluation of xenobiotic-induced changes in gene expression as a method to identify and classify potential toxicants is being pursued by industry and regulatory agencies worldwide. A workshop was held at the Research Triangle Park campus of the Environmental Protection Agency to discuss the current state-of-the-science of "immunotoxicogenomics" and to explore the potential role of genomics techniques for immunotoxicity testing. The genesis of the workshop was the current lack of widely accepted triggering criteria for Tier 1 immunotoxicity testing in the context of routine toxicity testing data, the realization that traditional screening methods would require an inordinate number of animals and are inadequate to handle the number of chemicals that may need to be screened (e.g., high production volume compounds) and the absence of an organized effort to address the state-of-the-science of toxicogenomics in the identification of immunotoxic compounds. The major focus of the meeting was on the theoretical and practical utility of genomics techniques to (1) replace or supplement current immunotoxicity screening procedures, (2) provide insight into potential modes or mechanisms of action, and (3) provide data suitable for immunotoxicity hazard identification or risk assessment. The latter goal is of considerable interest to a variety of stakeholders as a means to reduce animal use and to decrease the cost of conducting and interpreting standard toxicity tests. A number of data gaps were identified that included a lack of dose response and kinetic data for known immunotoxic compounds and a general lack of data correlating genomic alterations to functional changes observed in vivo. Participants concluded that a genomics approach to screen chemicals for immunotoxic potential or to generate data useful to risk assessors holds promise but that routine use of these methods is years in the future. However, recent progress in molecular immunology has made mode and mechanism of action studies much more practical. Furthermore, a variety of published immunotoxicity studies suggest that microarray analysis is already a practical means to explore pathway-level changes that lead to altered immune function. To help move the science of immunotoxicogenomics forward, a partnership of industry, academia, and government was suggested to address data gaps, validation, quality assurance, and protocol development.     

Sensitivity of a Tier I Screening Battery Compared to an in Utero Exposure for Detecting the Estrogen Receptor Agonist 17{beta}-Estradiol

A Tier I screening battery for detecting endocrine active compounds(EACs) has been evaluated for its ability to identify 17ß-estradiol,a pure estrogen receptor agonist. In addition, the responsesobtained with the Tier I battery were compared to the responsesobtained from F₁ generation rats from a 90-day/one-generationreproduction study with 17ß-estradiol in order tocharacterize the sensitivity of the Tier I battery against thesensitivity of an in utero exposure for detecting EACs. TheTier I battery incorporates two short-term in vivo tests (5-dayovariectomized female battery; 15-day Intact male battery) andan in vitro yeast transactivation system (YTS) for identifyingcompounds that alter endocrine homeostasis. The Tier I femalebattery consists of traditional uterotrophic endpoints coupledwith biochemical and hormonal endpoints. It is designed to identifycompounds that are estrogenic/antiestrogenic or modulate dopaminelevels. The Tier I male battery consists of organ weights coupledwith microscopic evaluations and a comprehensive hormonal assessmentIt is designed to identify compounds that have the potentialto act as agonists or antagonists to the estrogen, androgen,progesterone, or dopamine receptor; steroid biosynthesis inhibitors(aromatase, 5     

Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part VI: When Salt and Sugar and Vegetables Are Positive, How Can Genotoxicity Data Serve to Inform Risk Assessment?

This opinion piece examines the current approaches in the designand evaluation of genotoxicity data and recommends an alternativethat would provide information that could be more useful tohuman risk assessment. It is suggested that genotoxicity studies,both in vitro and in vivo, be designed similar to other traditionaltoxicology studies, such that a dose-response relationship ischaracterized, including identification of a "no-observed-adverse-effect-level"dose. It is further suggested that genotoxicity tests shouldno longer be designed or interpreted in isolation but shouldbe examined in the context of other available data includingtoxicokinetics, mechanism of genotoxicity, and relevant exposureinformation. The answer to improving genetic toxicology testingdoes not lie in coming up with better, "more sensitive" genotoxicitytest systems but rather in the incorporation of contextual improvementsin both the experimental design and the interpretation of datacollected using the current models. Such a strategy will betterposition the toxicology and risk assessment communities to copewith the current intellectually uncomfortable dichotomy thatdirects disproportionate scientific resource to addressing genetictoxicity findings of anthropogenic substances, regardless ofdose-exposure context, while at the same time ignoring the plethoraand comparatively large amounts of genotoxic and toxic substancesthat are inescapably present in what are otherwise regardedas healthy foods (salt, sugar, and vegetables).     

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.     

Acute Respiratory Exposure of Human Volunteers to Octamethylcyclotetrasiloxane (D4): Absence of Immunological Effects

Humans are exposed to silicones in a number of commercial andconsumer products. Some of these silicones, including octamethycy-clotetrasiloxane(D₄), are volatile. Therefore, there is a potential for respiratoryexposure. A pharmacokinetic analysis of respiratory exposureto D₄ is presented in the accompanying paper (M. J. Utell etal., 1998, Toxicol. Sci. 44, 206–213). Possible immuneeffects of respiratory exposure to D₄ are investigated in thispaper. Normal volunteers were exposed to 10 ppm D₄ or air for1 h via a mouthpiece using a double-blind, crossover study design.Assays were chosen to screen for immunotoxicity or a systemicinflammatory response. Assessment of immunotoxicity includedenumeration of peripheral lymphocyte subsets and functionalassays using peripheral blood mono-nuclear cells. Because inhumans there is no direct test for adjuvant effect of respiratoryexposure, we analyzed proinflammatory cyto-kines and acute-phasereactants in peripheral blood, markers for a systemic inflammatoryresponse, as surrogate markers for adjuvancy. These tests wererepeated when the volunteers were reexposed to D₄ approximately3 months after this initial exposure. Blood was obtained priorto exposure, immediately postexposure, and 6 and 24 h postexposure.In these short-term, controlled human exposures, no immunotoxicor proinflammatory effects of respiratory exposure to D₄ Werefound.     

Risk Assessment in Immunotoxicology: II. Relationships between Immune and Host Resistance Tests

We have reported on the design and content of a screening batteryusing a "tier" approach for detecting potential immunotoxiccompounds in mice (Luster et al, Fundam. Appl. Toxicol., 10,2–19, 1988). The data base generated from these studies,which consists of over 50 selected compounds, has been collectedand analyzed in an attempt to improve future testing strategiesand provide information to aid in developing future quantitativerisk assessment for immunotoxicity. In a recent study it wasshown that as few as two or three immune parameters were neededto predict immunotoxicants in mice (Luster et al., Fundam. Appl.Toxicol., 18, 200–210, 1992). In particular, enumerationof lymphocyte populations and quantitation of the T-dependentantibody response were particularly beneficial. Furthermore,commonly employed apical measures (e.g., leukocyte counts, lymphoidorgan weights) were fairly insensitive. The present analysesfocus on the use of this data base to develop statistical modelsthat examine the qualitative and quantitative relationship(s)between the immune function and host resistance tests. The conclusionderived from these analyses are: (1) A good correlation existsbetween changes in the immune tests and altered host resistancein that there were no instances where host resistance was alteredwithout affecting an immune test(s). However, in some instancesimmune changes occurred without corresponding changes in hostresistance. (2) No single immune test could be identified whichwas fully predictive for altered host resistance, although mostassays were relatively good indicators (i.e., >70%). Severalothers, such as proliferative response to lipopolysaccharideand leukocyte counts, were found to be relatively poor indicatorsfor host resistance changes. (3) The ability to resist infectiousagent challenge is dependent upon the degrees of immunosuppressionand the quantity of infectious agent administered. (4) Logisticand standard regression modeling using one extensive chemicaldata set from the immunosuppressive agent, cyclophosphamide,indicated that most immune function-host resistance relationshipsfollowed linear rather than linear-quadratic (threshold-like)models. For most of the relationships this could not be confirmedusing a large chemical data set and, thus, a more mechanisticallybased approach for modeling will need to be developed. (5) Usingthis limited data set, methods were developed for modeling theprecise quantitative relationships between changes in selectedimmune tests and host resistance tests.     

Respiratory immunotoxicity: An in vitro assessment

As yet, in vitro assessment of the immunotoxic potency of respiratory agents is not possible. The complexity of the endpoint and the respiratory tract, and the limited availability of well-documented respiratory agents are the main reasons.

The evidence that epithelial cells (ECs) are triggered by compounds to express in vitro surface proteins and soluble mediators, has stimulated their use for developing tests for respiratory immunotoxicity. A variety of airway ECs and EC-lines have been assessed, but the available information seems to point at human alveolar cells (e.g., A549) as the most convenient cell type. EC-based test formats with various degrees of complexity have been assessed. Sofar, promising results were obtained using a 3D model using the human A549 lung cell line. Dendritic cells (DCs) have been subjected to intensive research. However, currently available tests are not well suited to discern among the potency of sensitizers. Potential explanations include the lack of standardised protocols for the generation of DCs, no good standards for estimating the quality of in vitro derived DC-cultures, and limited dynamics of the currently used end-points. Alveolar macrophages (AMs) have sofar received less attention. This may proof unjustified as macrophages may link innate responses to adaptive immunity.

The observation that ECs, DCs and AMs affect each other, suggests that test formats are required combining at least two of these cell types if ranking of compounds according to their sensitising potency is the aim. In addition, the capacity of compounds to cross a cellular membrane is an important property of an immunotoxic compound, which can be assessed only in 3D reconstituted human tissue models. While promising data have been reported for the skin, immunocompetent 3D reconstituted human lung remains to be evaluated for respiratory immunotoxicity. Obviously, the success of any of these simplified test (as compared to the complexity of the immune response) is highly dependent on the availability of early stage biomarkers (expressed at mucosal barrier level) that are predictive for relevant immunotoxicity mechanisms occurring down-stream of the immune response. As yet, such biomarkers are not yet available.     

Surface Marker-Defined Head Kidney Granulocytes and B Lymphocytes of Rainbow Trout Express Benzo[a]pyrene-Inducible Cytochrome P4501A Protein

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene(BaP) are immunotoxic to fish. Metabolism of PAHs in immunecells has been implicated in PAH immunotoxicity in mammals,but for fish the presence of metabolic enzymes in immune cellsis less clear. The objective of this study was to examine localizationand induction of the BaP-metabolizing biotransformation enzyme,cytochrome P4501A (CYP1A), in head kidney immune cells of rainbowtrout (Oncorhynchus mykiss). In the first step, we measuredinduction of CYP1A-dependent 7-ethoxyresorufin-O-deethylase(EROD) activity and CYP1A protein in head kidney of rainbowtrout treated with a single intraperitoneal (ip) injection of25 mg BaP/kg body weight. From days 3 to 10 postinjection, theBaP treatment led to a significant elevation of EROD and CYP1Aprotein in head kidney and liver, with CYP1A expression levelsin the head kidney being much lower than in the liver. Next,we examined the cellular localization of CYP1A protein in thehead kidney cell types: vascular endothelial, endocrine andlymphoid cells. CYP1A immunoreactivity was detectable only inBaP-treated trout, where it was localized in endothelial andlymphoid cells. Finally, we aimed to clarify which of the hematopoieticcell types possess CYP1A protein. Using double immunostainingfor CYP1A and surface markers of rainbow trout immune cells,we identified B lymphocytes and granulocytes expressing inducibleCYP1A protein and being the likely sites of BaP metabolism inthe head kidney.     

Immunotoxicity and disease resistance in Japanese quail (Corturnix coturnix japonica) exposed to malathion

The purpose of this study was to evaluate the impact of malathion on the immune system of wild birds, using Japanese quail (Coturnix coturnix japonica) as a model. Quail were exposed to malathion in drinking water at environmentally realistic concentrations (0 ppm, 1 ppm, and 10 ppm). In the fifth week, several arms of the immune response were tested using the T-cell based phytohemagglutinin (PHA) skin test, the B-cell mediated antibody response, and the chemiluminescence assay measuring innate immunity. After the sixth week of malathion exposure, quail were challenged with E. coli O2. The bursa of Fabricius and the spleen were assessed for histopathology. No clinical signs of malathion toxicity were observed. Morbidity or mortality subsequent to E. coli exposure tended (P = 0.08) to be higher in the high exposure group (50.0%) compared to the control (22.2%) group. There was no difference in the innate immune response in the malathion exposed birds, however, humoral immunity was suppressed (P = 0.03) with the higher malathion exposure. Histopathological evaluation revealed an immunosuppressive effect of malathion on the bursa of Fabricius; bursal atrophy, decreased B-cell density and increased apoptosis in the medulla, and increased connective tissue thickness of the follicular epithelium. Antibody suppression was correlated with bursal changes and peripheral blood lymphocyte count, the organ and cells involved in antibody production. Following the same pattern as other immunotoxicity tests, the PHA T-cell proliferative response also tended to be suppressed in the high exposure group. This study provides evidence that subchronic, moderate malathion exposure is immunotoxic to quail and that testing integrated, functional immunity using an infectious challenge is a better predictor of immunotoxicity than individual responses to immunotoxicity tests. The secondary antibody response, circulating lymphocyte populations, and bursal histopathology were the most sensitive indicators of immune status, as these predicted decreased disease resistance with malathion exposure.     

Immunotoxicologic Assessment of Subacute Exposure of Rats to Carbon Tetrachloride with Comparison to Hepatotoxicity and Nephrotoxicity

Immunotoxicologic Assessment of Subacute Exposure of Rats toCarbon Tetrachloride with Comparison to Hepatotoxicity and Nephrotoxicity.SMIALOWICZ, R.J., SIMMONS, J. E., LUEBKE, R. W., AND ALLIS,J. W. (1991). Fundam. Appl. Toxicol 17, 186-196. The immunotoxicity,hepatotoxicity, and nephrotoxicity of subacute exposure to carbontetrachloride (CCI4) were evaluated in young adult (8-9 weeksold) male Fischer 344 rats dosed by gavage with CCI4 for 10consecutive days at 0, 5, 10, 20 or 40 mg/kg/day. Two days followingthe last treatment rats were evaluated for alterations in immunefunction by monitoring the following; body and lymphoid organweights; mitogen and mixed leukocyte reaction lymphoproliferativeresponses; natural killer cell activity; and cytotoxic T lymphocyteresponses. A separate group of similarly dosed rats was immunizedwith sheep red blood cells (SRBQ on Day 9 of dosing, and theprimary antibody response was assessed 4 days later. Hepaticand renal toxicity were assessed 2 days after the last treatmentby monitoring organ weights, serum indicators of hepatic andrenal damage, and hepatic cytochrome P450 levels, as well asby histological evaluation. Significant increases in relativeliver weights were observed in rats dosed at 40 mg/kg/day. Histologically,these livers displayed mild to moderate vacuolar degenerationand minimal to mild hepatocellular necrosis. In addition, serumlevels of aspartate aminotransferase and alanine aminotransferasewere elevated at this dosage, as well as at 20 mg/kg/day. Therewere no renal effects observed at these dosages of CCU. In addition,no consistent alterations were observed in the immune parametersexamined in these same animals nor in the rats immunized withSRBC. Furthermore, there was no difference in the antibody responseto SRBC in another set of rats dosed at 40, 80, or 160 mg/kg/dayCCI⁴. These results indicate that CCU is not immunotoxic inthe rat at dosages that produce overt hepatotoxicity.     

Gene toxicity studies on titanium dioxide and zinc oxide nanomaterials used for UV-protection in cosmetic formulations

Abstract

Titanium dioxide and zinc oxide nanomaterials, used as UV protecting agents in sunscreens, were investigated for their potential genotoxicity in in vitro and in vivo test systems. Since standard OECD test methods are designed for soluble materials and genotoxicity testing for nanomaterials is still under revision, a battery of standard tests was used, covering different endpoints. Additionally, a procedure to disperse the nanomaterials in the test media and careful characterization of the dispersed test item was added to the testing methods. No genotoxicity was observed in vitro (Ames' Salmonella gene mutation test and V79 micronucleus chromosome mutation test) or in vivo (mouse bone marrow micronucleus test and Comet DNA damage assay in lung cells from rats exposed by inhalation). These results add to the still limited data base on genotoxicity test results with nanomaterials and provide congruent results of a battery of standard OECD test methods applied to nanomaterials.     

The Comet Assay with Multiple Mouse Organs: Comparison of Comet Assay Results and Carcinogenicity with 208 Chemicals Selected from the IARC Monographs and U.S. NTP Carcinogenicity Database

ABSTRACT

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology.

Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organspecific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally.

Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data.

Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Approaches to the identification and recording of findings in the lymphoreticular organs indicative for immunotoxicity in regulatory type toxicity studies

Recent validation studies showed that histopathological examination of the hematopoietic and lymphoreticular system is one of the most sensitive tools in the evaluation of non-specific immune stimulatory or immune suppressive effects and hazard identification of potential immunotoxicity in routine safety tests. Most immunotoxic effects have a classical dose-response relationship and an immediate effect (i.e. do not need an induction phase). The findings associated with a specific immunomodulatory response are generally not detected morphologically in routine sections of the immune system in safety studies, but may be detected, because of their effects on other organs such as skin (contact dermatitis) or joints and kidneys (immune complex deposits). Careful detailed examination of the immune system may give valuable clues for the possible mechanism of action of the test material, as was also demonstrated in these validation studies.     

Preclinical Toxicology Studies with Acyclovir: Genetic Toxicity Tests

Preclinical Toxicology Studies with Acyclovir: Genetic ToxicityTests. Clive, D., Turner, N.T., Hozier, J., Batson, A.G. andTucker, W.E., Jr. (1983). Fundam. Appl. Toxicol 3: 587–602.Acyclovir (ACV), an antiviral drug active in the treatment oforal and genital Herpes infections, has been evaluated for mutagenicand carcinogenic potential in a battery of in vitro and in vivoshort-termassays. Negative results were obtained in the following in vitrotests: Ames Salmonella, plate incorporation and preincubationmodification assays; E. coli polA⁺/polA^– DNA repair; yeast(S. cerevisiae D4) gene conversion; Chinese hamster ovary cells(HGPRT, APRT loci and ouabain-resistance marker); L5178 Y mouselymphoma cells (HGPRT locus and ouabain-resistance marker);and C3H/10Tmouse fibroblast neo-plastic transformation assay.All except the last assay were performed in the presence andabsence of an exogenous metabolic activation system. ACV waspositive at high concentrations x exposure times in the absenceof exogenous metabolic activation in the following in vitrosystems and at the indicated concentrations: BALB/c-3T3 neoplastictransformation (50 /µg/mL, 72 h exposure); human lymphocytecytogenetics (250–500 µg/mL, 48 h exposure); andL5178Y mouse lymphoma cells (TK locus, 400–2400 µg/mL,4 h exposure; predominantly small colony mutants of chromosomalorigin produced). No effects were seen in vivo (mouse dominantlethal assay; rat and Chinese hamster bone marrow cytogenetics)at up to maximum tolerated doses (MTD). An unusual clastogeniceffect was seen in Chinese hamsters at 5 times the MTD. Overall,positive effects were seen only at either high concentrations(250 µg/mL in vitro or plasma levels) or prolonged exposure(72 hr in the BALB/ c-3T3 neoplastic transformation assay).These studies support the view that ACV is a chromosomal mutagen,i.e., one which causes multi-locus damage but not single geneeffects. The significance of these results for the genetic riskof ACV to man is discussed.     

Comparison of ELISA and Plaque-Forming Cell Assays for Measuring the Humoral Immune Response to SRBC in Rats and Mice Treated with Benzo[a]pyrene or Cyclophosphamide

Comparison of ELISA and Plaque-Forming Cell Assays for Measuringthe Humoral Immune Response to SRBC in Rats and Mice Treatedwith Benzo[a]pyrene or Cyclophosphamide. TEMPLE, L., KAWABATA,T. T., MUNSON, A. E., AND WHITE, K. L., JR. (1993). Fundam.Appl. Toxicol. 21, 412–419. The humoral immune response against sheep red blood cells (SRBC)is one of the most sensitive and frequently used endpoints inevaluating the immunotoxicity of drugs and chemicals in experimentalanimals. Currently, most immunotoxicology studies measure theSRBC IgM antibody response by quantitating the number of SRBC-specificIgM antibody-forming cells using the hemolytic plaque assay.On the other hand, measurement of serum SRBC-specific IgM couldbe an easier, more cost effective endpoint in evaluating theSRBC antibody response in rodents. A validated method to measureSRBC-specific IgM, however, has not been developed. Thus, theobjectives of the studies presented were to develop and validatean enzyme-linked immunosorbent assay (ELISA) for SRBC-specificIgM. Hemoglobin-free, detergent-solubilized membrane preparationswere chosen as antigen for the ELISA. Various sources of SRBCwere found to be equally useful, and as little as 0.1 µgof protein per well was optimal. Kinetic studies of the IgMresponse showed the peak day to be on Day 5 (mice) and Day 6(rats). To validate the usefulness of the method for immunotoxicologicstudies, serum SRBC-specific IgM levels and number of SRBC-specificplaque-forming cells were compared in rats and mice treatedwith two well-characterized immunosuppressive agents: benzo[a]pyreneand cyclophosphamide. Administration of these chemicals wasfound to produce very similar dose-dependent decreases in serumSRBC IgM and IgM-specific plaque-forming cells. These two endpointswere equally sensitive to the effects of the immunosuppressivedrugs. Based on the present findings and the sensitivity, simplicity,and potential for automation of the ELISA for SRBC-specificIgM, the ELISA could potentially replace the plaque assay asthe first step in testing for potential immunotoxic chemicals.