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This paper describes a new screening method for assessing groundwater vulnerability to pollution from hydrocarbon exploitation in the subsurface. The method can be used for various hydrocarbon energy sources, including conventional oil and gas, shale gas and oil, coal bed methane and underground coal gasification. Intrinsic vulnerability of potential receptors is assessed at any particular location by identifying possible geological pathways for contaminant transport. This is followed by an assessment of specific vulnerability which takes into account the nature of the subsurface hydrocarbon activity and driving heads. A confidence rating is attached to each parameter in the assessment to provide an indication of the confidence in the screening. Risk categories and associated confidence ratings are designed to aid in environmental decision making, regulation and management, highlighting where additional information is required. The method is demonstrated for conventional gas and proposed shale gas operations in northern England but can be adapted for use in any geological or hydrogeological setting and for other subsurface activities., (Copyright © 2019. Published by Elsevier Ltd.)

Shale gas is considered by many to have the potential to provide the UK with greater energy security, economic growth and jobs. However, development of a shale gas industry is highly contentious due to environmental concerns including the risk of groundwater pollution. Evidence suggests that the vertical separation between exploited shale units and aquifers is an important factor in the risk to groundwater from shale gas exploitation. A methodology is presented to assess the vertical separation between different pairs of aquifers and shales that are present across England and Wales. The application of the method is then demonstrated for two of these pairs-the Cretaceous Chalk Group aquifer and the Upper Jurassic Kimmeridge Clay Formation, and the Triassic sandstone aquifer and the Carboniferous Bowland Shale Formation. Challenges in defining what might be considered criteria for 'safe separation' between a shale gas formation and an overlying aquifer are discussed, in particular with respect to uncertainties in geological properties, aquifer extents and determination of socially acceptable risk levels. Modelled vertical separations suggest that the risk of aquifer contamination from shale exploration will vary greatly between shale-aquifer pairs and between regions and this will need to be considered carefully as part of the risk assessment and management for any shale gas development.

6:2 fluorotelomer alcohol (6:2 FTOH; CF3[CF2]5[CH2]2OH, CAS# 647-42-7) was evaluated for acute, genetic, and subchronic toxicity using in vitro and in vivo methods. In rats, 6:2 FTOH was considered to be slightly toxic by the oral (LD50=1,750 mg/kg), and dermal (LD50 > 5,000 mg/kg) routes. In rabbits, 6:2 FTOH was not a primary skin or eye irritant, and it did not produce a dermal sensitization response in mice. In a 90-day subchronic study, 6:2 FTOH was administered to rats by oral gavage (0, 5, 25, 125, 250 mg/kg/day). Mortality was observed at 125 and 250 mg/kg/day; deaths occurred after approximately three weeks of dosing and continued sporadically. The NOAEL in the subchronic study was 5mg/kg/day based on hematology and liver effects. 6:2 FTOH was not mutagenic in the bacterial reverse mutation test or in the mouse lymphoma assay and was not clastogenic in a chromosome aberration assay in human lymphocytes. The hazard classification for human health endpoints of 6:2 FTOH according to the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) is Category 4 for acute oral toxicity based on an LD50 of 1,750 mg/kg. Other acute health endpoints including eye and skin irritation, skin sensitization, as well as genotoxicity, did not meet the criteria for hazard classification. Benchmark Dose Analysis was performed on the most sensitive endpoints from the 90-day oral gavage study and these levels were all above the study NOAEL of 5mg/kg/day. For risk assessment purposes, the recommended point of departure is the more conservative study NOAEL of 5mg/kg/day., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Neuroscience affords knowledge that can be leveraged in the ontological valuation of individuals, groups, and species. Sociocultural sentiments, norms, and mores may impede embracing such knowledge to revise moral attitudes, ethics, and policies. We argue that the practices of neuroethics will be valuable in that they ground ethico-legal discourse in (1) naturalistic philosophy; (2) the current epistemological capital of neuroscience; (3) the issues, problems, and solutions arising in and from neuroscientific research and its applications; and 4) the use of neurocentric criteria-such as painience-to define and resolve ethical decisions regarding attitudes toward and treatment of nonhuman animals.

As experience is gained with toxicology testing and as new assays and technologies are developed, it is critical for stakeholders to discuss opportunities to advance our overall testing strategies. To facilitate these discussions, a workshop on practices for assessing immunotoxicity for environmental chemicals was held with the goal of sharing perspectives on immunotoxicity testing strategies and experiences, developmental immunotoxicity (DIT), and integrated and alternative approaches to immunotoxicity testing. Experiences across the chemical and pharmaceutical industries suggested that standard toxicity studies, combined with triggered-based testing approaches, represent an effective and efficient approach to evaluate immunotoxic potential. Additionally, discussions on study design, critical windows, and new guideline approaches and experiences identified important factors to consider before initiating DIT evaluations including assay choice and timing and the impact of existing adult data. Participants agreed that integrating endpoints into standard repeat-dose studies should be considered for fulfilling any immunotoxicity testing requirements, while also maximizing information and reducing animal use. Participants also acknowledged that in vitro evaluation of immunosuppression is complex and may require the use of multiple assays that are still being developed. These workshop discussions should contribute to developing an effective but more resource and animal efficient approach for evaluating chemical immunotoxicity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Guidance for determining the sensitizing potential of chemicals is available in EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances; REACH guidance from the European Chemicals Agency; and the United Nations Globally Harmonized System (GHS). We created decision trees for evaluating potential skin and respiratory sensitizers. Our approach (1) brings all the regulatory information into one brief document, providing a step-by-step method to evaluate evidence that individual chemicals or mixtures have sensitizing potential; (2) provides an efficient, uniform approach that promotes consistency when evaluations are done by different reviewers; (3) provides a standard way to convey the rationale and information used to classify chemicals. We applied this approach to more than 50 chemicals distributed among 11 evaluators with varying expertise. Evaluators found the decision trees easy to use and recipients (product stewards) of the analyses found that the resulting documentation was consistent across users and met their regulatory needs. Our approach allows for transparency, process management (e.g., documentation, change management, version control), as well as consistency in chemical hazard assessment for REACH, EC Regulation No. 1272/2008 Classification, Labeling, and Packaging of Substances and the GHS., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The toxicity of tridecafluorohexylethyl methacrylate (6:2 FTMAC), an acrylic monomer used in producing polymeric substances, was evaluated. 6:2 FTMAC has low acute oral and dermal toxicity (LD50>5000 mg/kg), was not a skin or eye irritant, and did not demonstrate skin sensitization potential in a local lymph node assay (LLNA). 6:2 FTMAC was not mutagenic in the bacterial reverse mutation (Ames) test or in the mouse lymphoma assay. 6:2 FTMAC induced structural aberrations in human peripheral blood lymphocytes in vitro in the absence of metabolic activation but not in the presence of S9 metabolic activation. No numerical aberrations were detected under any testing condition. Also, no increase occurred in structural or numerical chromosomal aberrations in an in vivo mouse micronucleus assay in 6:2 FTMAC treated animals compared to controls. 6:2 FTMAC was administered at 0, 100, 500 and 1000 mg/kg/day via gavage to male and female SD rats for 14 days. No test substance-related effects on mortality, clinical signs, body weights, nutritional parameters, or clinical pathology were observed at any dose. Test substance-related increases in liver weights in males and females at all dose levels and thyroid and kidney weights in 500 and 1000 mg/kg/day males were noted. While there was no histopathological correlate for thyroid and kidney weight changes, minimal hypertrophy was noted in liver in males and females at 1000 mg/kg/day group. The changes noted in teeth (altered mineralization; retention of basophilic material) and femur (increased mineralization) in all treated groups were not associated with clinical signs or microscopic changes and were likely related to free fluoride formed from 6:2 FTMAC metabolism. Plasma (3-4-fold) and urine (30-50-fold) fluoride was higher in treated groups versus controls. Therefore, the changes noted in organ weights, teeth, femur, plasma or urine were not considered adverse. In the repeated dose toxicity study, the no-observed-adverse-effect-level (NOAEL) was 1000 mg/kg/day. Based on mean measured concentrations, the 96-h LC50 in fathead minnow was >14.5 mg/L and the 72-h EC50 in Pseudokirchneriella subcapitata was >24.6 mg/L, while the 48-h EC50 in Daphnia magna, based on nominal concentrations, was >120 mg/L. Overall, 6:2 FTMAC is considered to have low toxicity potential based on these studies., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The absorption, tissue distribution, elimination, and metabolism of [1-¹⁴C]-PFHx in rats and mice dosed orally at 2 or 100 mg/kg was evaluated following a single dose or after 14 consecutive doses. Absorption was rapid in rats as evidenced by a short time to maximum concentration (C(max)) of 30 min in male rats and 15 min in female rats at both the 2 and 100mg/kg dose level. The plasma elimination half-life was somewhat longer in males (1.5-1.7 h) than in females (0.5-0.7 h). Absorption in the mouse was also rapid with the maximum plasma concentration occurring between 15 and 30 min after dosing. The maximum concentration was not appreciably different between male and female mice (8 μg equiv./g at 2 mg/kg; ~350 μg equiv./g at 100 mg/kg). The primary route of elimination was via the urine. PFHx was not metabolized in rat or mouse hepatocytes, nor were any metabolites observed after oral dosing in either rodent species. Essentially 100% of the dose was eliminated in urine within 24 h demonstrating that PFHx is readily absorbed and bioavailability approaches 100%, even at a dose as high as 100 mg/kg. The route and extent of elimination was unchanged after 14 days of daily dosing. Tissues were collected at three time points (rat: 0.5, 2, and 24 h; mice: 0.25, 1, and 24 h) after dosing to investigate the tissue clearance kinetics of PFHx following a single dose at 2 or 100 mg/kg. In all tissues except skin, PFHx was not quantifiable 24 h after dosing in both sexes of the two species., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment., (2009 Elsevier Inc. All rights reserved.)

Sodium perfluorohexanoate [NaPFHx, F(CF(2))(5)CO(2)Na, CAS#2923-26-4] was evaluated in acute, 90-day subchronic, one-generation reproduction, developmental and in vitro genetic toxicity studies. In the subchronic/one-generation reproduction study, four groups of young adult male and female Crl:CD(SD) rats were administered NaPFHx daily for approximately 90 days by gavage at dosages of 0, 20, 100, or 500 mg/kg. Selected groups of rats were evaluated after 1- and 3-month recovery periods. Rats selected for reproductive evaluations were dosed for approximately 70 days prior to cohabitation, through gestation and lactation, for a total of about 4 months. The subchronic toxicity no observed adverse effect level (NOAEL) was 20mg/(kg day), based on nasal lesions observed at 100 and 500 mg/(kg day). No effects were observed for neurobehavioral endpoints. NaPFHx was a moderate inducer of hepatic peroxisomal beta-oxidation with a no observed effect level (NOEL) of 20 (male rats) and 100mg/(kg day) (female rats). Elevated hepatic beta-oxidation levels were observed following 1-month recovery in male and female rats at 500 mg/(kg day). No NaPFHx-related effects were observed on any reproductive parameters. The P(1) adult rat NOAEL was 20mg/(kg day), based on reduced body weight parameters, whereas the NOAEL for reproductive toxicity was 100 mg/(kg day), based on effects limited to reduced F(1) pup weights. In the developmental study, female rats were dosed via gavage on gestation day (GD) 6-20 with the same doses of NaPFHx administered in the subchronic study. The maternal and developmental toxicity NOAEL was 100 mg/(kg day), based on maternal and fetal body weight effects at 500 mg/(kg day). NaPFHx is therefore concluded not to present a reproductive or developmental hazard. NaPFHx genotoxicity studies showed no mutations in the bacterial reverse mutation (Ames) assay or chromosome aberrations in human lymphocytes treated with NaPFHx in vitro. The lowest NOAEL from all of the studies was 20mg/(kg day) in the subchronic study based on nasal lesions. Benchmark doses (BMDL10) for nasal lesions were 13 and 21 mg/(kg day) for male and female rats, respectively. The relevance of the nasal lesions to humans is not known., (2009 Elsevier Ireland Ltd.)

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are environmentally widespread and persistent chemicals with multiple toxicities reported in experimental animals and humans. These compounds can trigger biological activity by activating the alpha isotype of peroxisome proliferator-activated receptors (PPARs), ligand-activated transcription factors that regulate gene expression; however, some biological effects may occur independently of the receptor. Activation of the peroxisome proliferator-activated receptor alpha (PPARalpha) modulates lipid and glucose homeostasis, cell proliferation and differentiation, and inflammation. Reported immunomodulation in experimental animals exposed to PFOA and PFOS has included altered inflammatory responses, production of cytokines and other proteins, reduced lymphoid organ weights, and altered antibody synthesis. Mounting experimental animal evidence suggests PPARalpha independence of some immune effects. This evidence originates primarily from studies with PPARalpha knockout models exposed to PFOA that demonstrate hepatic peroxisome proliferation, reduced lymphoid organ weights, and altered antibody synthesis. As human PPARalpha expression is significantly less than that of rodents, potential PPARalpha independence indicates that future research must explore mechanisms of action of these compounds, including PPARalpha-dependent and -independent pathways. This multiauthored review contains brief descriptions of current and recently published work exploring immunomodulation by PFOA and PFOS, as well as a short overview of other PPARalpha ligands of therapeutic and environmental interest.

Repeated high doses of ammonium perfluorooctanoate (APFO) have been reported to affect immune system function in mice. To examine dose-response characteristics in both rats and mice, male CD rats and CD-1 mice were dosed by oral gavage with 0.3-30 mg/kg/day of linear APFO for 29 days. Anti-sheep red blood cell (SRBC) IgM levels, clinical signs, body weights, selected hematology, and lipid parameters, liver weights, spleen, and thymus weights and cell number, selected histopathology, and serum corticosterone concentrations were evaluated. In rats, linear APFO had no effect on production of anti-SRBC antibodies. Ten and 30 mg/kg/day resulted in systemic toxicity as evidenced by decreases in body weight gain to 74 and 37%, and increases in serum corticosterone levels to 135 and 196% of control, respectively. In mice dosed with 10 and 30 mg/kg/day, marked systemic toxicity and stress were observed, as evidenced by a loss in body weight of 3.8 and 6.6 g, respectively (despite a tripling of liver weight), approximately 230% increase in serum corticosterone, and increases in absolute numbers of peripheral blood neutrophils and monocytes with an accompanying decrease in absolute lymphocyte numbers. Immune-related findings at 10 and 30 mg/kg/day that likely represent secondary responses to the systemic toxicity and stress observed at these doses include: decreased IgM antibody production at 10 (20% suppression) and 30 mg/kg/day (28% suppression); decreased spleen and thymus weights and cell numbers; microscopic depletion/atrophy of lymphoid tissue at 10 (thymus) and 30 mg/kg/day (spleen). In summary, no immune-related changes occurred in rats, even at doses causing systemic toxicity. In mice, immune-related changes occurred only at doses causing significant and profound systemic toxicity and stress.

A commercial fluorotelomer-based urethane polymeric dispersion, consisting of polymer, surfactant, and water, was evaluated in subchronic, reproduction, and developmental toxicity studies. The dispersion was administered daily by gavage to rats at dosages of 0, 50, 250, or 1000 mg polymer/kg/day or with 70 mg/kg/day of the sulfonate surfactant. Dose levels of 0, 50, 250, or 1000 mg polymer/kg/day were also used for the reproductive and developmental studies. Nasal olfactory epithelial degeneration and necrosis occurred in all dose groups in the 90-day study. Nasal adhesions were observed only in rats administered surfactant alone. Liver-enzyme alterations at 250 and 1000 mg/kg were considered to be potentially adverse effects. The subchronic no-observed-adverse-effects level (NOAEL) was 50 mg/kg. For the reproduction study, rats were dosed for 10 weeks prior to cohabitation and throughout mating, gestation, and lactation. There were no effects on reproductive function in males or females at any dosage. Thyroid weight was decreased in the 250 and 1000 mg/kg day F(1) groups unaccompanied by microscopic effects. In the developmental toxicity study, female rats were dosed from gestation days 6-20; there was no test-substance-related embryolethality, nor was there any dose-related increase in either fetal malformations. Fetal weight was minimally decreased at 1000 mg/kg/day in the presence of slight maternal toxicity; the NOAEL for developmental parameters was 250 mg/kg/day. The polymeric product was not a specific developmental or reproductive toxin.

EPA guidelines provide a choice in evaluating humoral immune system function in rats and mice immunized with sheep red blood cells (sRBC): an antibody-forming cell (AFC) assay or a sRBC-specific serum IgM enzyme-linked immunosorbent assay (ELISA). Four different laboratories used both methods to detect suppression of the antibody response by cyclophosphamide (CP) or dexamethasone (DEX). Attempts were made to minimize interlaboratory variability through the use of common reagents and vendors; each laboratory used the same source for rodents, immunosuppressive agents, and one sheep for sRBCs, and determined optimal sRBC concentration for immunization and peak day of antibody response in female CD rats and CD1 mice. The CP dose at which statistical significance was first observed in each species was quite similar within each lab using either assay. For DEX, the AFC assay detected significant and greater suppression at lower concentrations compared to the ELISA in both rats and mice. All labs detected DEX suppression using an AFC assay, whereas only one lab detected significant suppression in both species using an ELISA. For both compounds the magnitude of suppression was greater using the AFC assay, and resulted in ID(50) values which were lower in the AFC assay when compared to the ELISA. In addition, cross-species comparisons of ID(50) values suggested rats were more sensitive than mice. These initial experiments with two chemicals indicated that the AFC assay is consistently better at identifying suppression of a T-dependent antibody response across laboratories following xenobiotic exposures in outbred rats and mice. Additional compounds will need to be evaluated before concluding that one method is superior or more sensitive to the other in detecting suppression of the antibody response.

The purpose of this study was to compare the toxicity of linear/branched ammonium perfluorooctanoate (APFO) with that of linear and branched APFO. Linear/branched APFO (approximately 80% linear and 20% branched isomers) was formerly used in the production of commercial products. The extensive toxicologic database for APFO has been developed essentially using this mixture of isomers. The trend now is to use APFO containing only the linear isomer. The current study was performed to determine if the toxicological database developed for the linear/branched isomer is applicable to the linear isomer. To determine the contribution of branched APFO to the toxicity of linear/branched APFO, a form of APFO that was 100% branched was synthesized. Rats and mice were given doses by oral gavage ranging from 0.3 to 30 mg/kg of either the linear/branched, linear, or branched APFO for 14 days. Clinical signs, body weights, food consumption, selected hematology and serum lipid parameters, liver and kidney weights, hepatic peroxisomal beta-oxidation, and serum PFOA concentrations were evaluated. Mean body weights were about 20% lower in rats and mice dosed with 30 mg/kg of linear/branched or linear APFO compared to controls, and 3-5% lower in animals dosed with 30 mg/kg of branched APFO. In rats, all three forms reduced lipids. In mice, all three forms reduced total and HDL cholesterol similarly but triglycerides were increased at lower doses. Increased peroxisomal beta-oxidation activity and serum PFOA concentrations were seen in both species but these effects were least pronounced in rats dosed with the branched material. In rats, serum PFOA levels were 20-51 ppm at Lowest Observed Effect Levels (LOEL) of 0.3-1 mg/kg, based primarily upon lipid parameters. In mice, serum PFOA levels were 10-14 ppm at the LOEL of 0.3 mg/kg, based primarily upon relative liver weight. In both rats and mice, the overall responses to the linear/branched and the linear forms of PFOA were similar, but the branched form appears to be less potent. Based on these results, and for the endpoints evaluated in this study, the toxicological database developed primarily from testing linear/branched APFO is applicable to linear APFO.

1,3-Propanediol (504-63-2) was studied to determine the potential effects following repeated inhalation exposures to rats. Rats were exposed 6 hr/day, 5 days/wk for 2 wk (9 exposures) to vapor or vapor/aerosol mixtures of either 0, 41, 650, or 1800 mg 1,3-propanediol/m(3). In vivo responses were observed or measured daily. Clinical pathology and tissue pathology analyses were conducted after the 9th exposure and on half of each group following an 18-day recovery (nonexposure) period. All rats showed normal body weights. No unusual external signs of response were seen, and no deaths were encountered. Clinical pathology (blood counts, serum chemical parameters) and tissue pathology (gross pathology, organ weights, and histopathology) examinations in the 1,3-propanediol exposed rats were similar to those in the unexposed controls. The highest concentration tested, 1800 mg/m(3), which was the highest concentration that could practically be generated, was the no-observed-effect level (NOEL) for this study. 1,3-Propanediol does not appear to pose a significant hazard via inhalation of either the vapor or a vapor/aerosol mixture.

Isocyanates are low-molecular-weight chemicals implicated in allergic asthmatic-type reactions. Identification of chemicals likely to cause asthma is difficult due to the lack of a validated test method. One hypothesis is that differential cytokine induction (Th1 versus Th2 profiles) in the draining lymph node following dermal application can be used to identify asthmagens and distinguish them from contact allergens. In this study, we compared the cytokine mRNA profiles of six chemicals: toluene diisocyanate (TDI), diphenylmethane-4,4'-diisocyanate (MDI), dicyclohexylmethane-4,4'-diisocyanate (HMDI), isophorone diisocyanate (IPDI), p-tolyl(mono)isocyanate (TMI), and meta-tetramethylene xylene diisocyanate (TMXDI). Whereas TDI and MDI are well-known respiratory sensitizers, documentation for HMDI, IPDI, TMI, and TMXDI is limited, but suggests that HMDI and IPDI may have respiratory sensitization potential in humans and TMI and TMXDI do not. Following dermal exposure of BALB/c mice, all six isocyanates induced cytokines characteristic of a Th2 response. Although LLNAs suggested that the doses chosen for the RPA were immunologically equivalent, the isocyanates tested differentiated into two groups, high responders and low responders. However, two of the low responders (TMI and TMXDI) were further tested and induced higher levels of Th2 cytokine message than dinitrochlorobenzene (not an asthmagen). Further study of these chemicals is needed to determine whether the Th2 cytokine responses observed for these low responders is predictive of asthmagenic potential or represents an insufficient signal.

The object of this study was to evaluate the toxicity of norbornene fluoroalcohol (NBFOH), which is used as an intermediate in the production of fluorinated monomers and polymers. NBFOH was evaluated for acute oral, dermal, and inhalation toxicity, dermal sensitization using the Local Lymph Node Assay (LLNA), mutagenesis by the Ames assay, and subchronic toxicity in a 4-week inhalation rat study. NBFOH demonstrated slight acute toxicity in oral, dermal, and inhalation studies. Approximate lethal doses of 3400 and > 5000 mg/kg for the oral and dermal routes, respectively, and an approximate lethal concentration of 4300 mg/m(3) were determined. NBFOH demonstrated moderate skin irritation, was a severe eye irritant, produced dermal sensitization, but did not cause bacterial mutagenicity either in the presence or absence of S9 activation. Male and female rats were exposed nose only to airborne NBFOH at levels of 0, 410, 1400, and 1500 mg/m(3), 6 h/day, 5 days/week for 4 weeks with clinical and histopathology specimens collected 1 day after the final exposure. Due to the vapor pressure of NBFOH, the 1500 mg/m(3) atmosphere was 27% aerosol and 73% vapor; the 1400 mg/m(3) atmosphere was 5% aerosol and 95% vapor, and the 410 mg/m(3) level was only vapor. No test substance-related mortality or clinical signs of toxicity were observed over the course of the study, and male rats demonstrated significant weight loss and decreased food consumption at 1400 mg/m(3). Male rats from the 1500 mg/m(3) group demonstrated an 11% increase in prothrombin time that was significantly higher than the control value. Examination of fluoride in the urine did not demonstrate a concentration-response relationship, with minimal elevations observed in male rats at all exposure levels and sporadic increases in females. Both male and female rats exposed to 1400 mg/m(3) or greater had squamous metaplasia of the laryngeal mucosa and degeneration of the nasal olfactory and respiratory mucosa. Based on the above findings, NBFOH demonstrates the potential to produce allergic contact dermatitis, and subchronic inhalation studies indicate a no-observed-adverse-effect-level (NOAEL) of 410 mg/m(3).

Exposure to low molecular weight (LMW) chemicals in the workplace has been linked to a variety of respiratory effects. Within the LMW chemicals, one of the major classes involved in these effects are the acid anhydrides. The immunological basis of respiratory hypersensitivity involves CD4+ cells. By virtue of their induction of cytokines typical of CD4+ T-helper type 2 (Th2) cells-interleukin (IL)-4, 10, and 13-respiratory sensitizers may be identified and differentiated from contact sensitizers which induce Th1 cytokines (IL-2 and IFN-gamma). Our previous work suggested that the ribonuclease protection assay (RPA) was useful in identifying the respiratory sensitizer, trimellitic anhydride (TMA), based on quantitative differences in Th2 cytokine mRNA as compared to the contact sensitizer dinitrochlorobenzene (DNCB). Therefore, the purpose of the studies described in this report was to expand the chemicals tested in the RPA. To this end, four acid anhydrides with known respiratory sensitization potential, TMA, maleic anhydride (MA), phthalic anhydride (PA) and hexahydrophthalic anhydride (HHPA), were tested. Although previously determined to induce immunologically equivalent responses in a local lymph node assay (LLNA), the initial dose chosen (2.5%) failed to induce Th2 cytokine mRNA expression. To determine if the lack of cytokine expression was related to dose, LLNAs were conducted at higher doses for each of the anhydrides. The highest doses evaluated (four- to six-fold higher than those used in the initial RPA) gave equivalent proliferative responses for the various anhydrides and were used for subsequent RPA testing. At these higher doses, significant increases in Th2 versus Th1 cytokine mRNA were observed for all anhydrides tested. These results suggest that the RPA has the potential to serve as a screen for the detection of LMW airway sensitizing chemicals. However, the basis for selecting immunologically equivalent doses may require some modification.

Exposure to chemicals in domestic and occupational settings may contribute to increases in asthma and allergy. Airway hypersensitivity (AHS) is T helper-2 (Th2) cell associated, whereas contact hypersensitivity (CHS) is T helper-1 (Th1) cell associated. The distinct cytokine profiles produced by these cells may provide a means of distinguishing respiratory sensitizers from contact sensitizers. In this study, female BALB/c mice were exposed twice on the flanks and three times on the ears using the airway sensitizer trimellitic anhydride (TMA) or the contact sensitizer dinitrochlorobenzene (DNCB). At various times following exposure, total mRNA was extracted from draining lymph node cells and cytokine mRNA profiles analyzed using a multiprobe ribonuclease protection assay (RPA). The Th2 cytokines IL4, IL10, and IL13 were significantly increased in response to TMA compared to DNCB, with optimal detection occurring 14 days following initial exposure. To determine its effect, dose was varied in flank exposures, ear exposures, or both simultaneously. When dose was varied during flank exposures only, TMA induced higher levels of Th2 cytokines than DNCB at all doses tested. DNCB did not induce Th1 cytokines at any dose tested. Variation of TMA dose during both exposures similarly induced Th2 cytokines. Dose only appeared to be a factor when TMA concentration was varied during the ear exposures alone. Thus, these studies suggest that quantitative differences in Th2 responses between TMA and DNCB may be demonstrated over a wide range of doses and these differences may be detected by RPA following dermal exposure to these sensitizers., (Copyright 2002 Elsevier Science (USA).)

There is a concern that certain industrial chemicals found in the environment may mimic or antagonize endogenous hormones and adversely affect the endocrine as well as the immune system. The objective of this study was to determine if exposure of Crl:CD (SD)BR male rats to 17beta-estradiol (17beta-E2), an estrogen receptor agonist, or flutamide (FLUT), an androgen receptor antagonist, would significantly alter the primary IgM humoral immune response to sheep red blood cells (SRBC). This study was conducted in the context of a male in vivo Tier I battery designed to identify endocrine-active compounds (EACs). The Tier I male battery consists of organ weights coupled with a comprehensive hormonal assessment. Rats were dosed by the intraperitoneal route for 15 days with vehicle or 0.001, 0.0025, 0.0075, or 0.050 mg/kg/day 17beta-E2 or 0.25, 1, 5, or 20 mg/kg/day FLUT. Six days prior to termination, selected rats were injected intravenously with SRBC for assessment of humoral immune function. Spleen cell number and spleen and thymus weights were obtained. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. At 0.050 mg/kg/day 17beta-E2, mean final body and absolute thymus weights were significantly decreased to 84 and 65% of control, respectively. 17beta-E2 did not significantly alter spleen weight, spleen cell number, or the primary IgM humoral immune response to SRBC. The no-observed-adverse-effect level (NOAEL) for immune system alteration was 0.050 mg/kg/day 17beta-E2 since the decrease in absolute thymus weight was judged to be secondary to the decrements in body weight. In the Tier I male battery, responses to 17beta-E2 included decreased absolute testis and epididymis weights, decreased relative accessory sex gland unit weights, hormonal alterations (decreased serum testosterone (T), dihydrotestosterone (DHT), and luteinizing hormone (LH), and increased serum prolactin and E2 levels). The lowest-observed-adverse-effect level (LOAEL) for the reproductive indices was 0.001 mg/kg/day 17beta-E2 based on the hormonal alterations seen at this level; no NOAEL was established. Exposure to FLUT did not significantly alter mean final body, spleen, or absolute thymus weights, spleen cell number, or the primary IgM humoral immune response to SRBC. A significant increase (118% of control) in relative thymus weight was observed at 20 mg/kg/day FLUT. The NOAEL for immune system alteration was 5 mg/kg/day FLUT based on the increased relative thymus weights that were judged to be compound-related. In the Tier I male battery, responses to FLUT included decreased absolute epididymis and relative accessory sex gland unit weights and hormonal alterations (increased serum T, DHT, E2, and LH, and decreased follicle stimulating hormone levels). The LOAEL for the reproductive indices was 0.25 mg/kg/day FLUT based on the hormonal alterations seen at this level; no NOAEL was established. Based on these data, the reproductive and not the immune system appears to be the primary target organ of toxicity in young adult male rats treated with either 17beta-E2 or FLUT.

The murine local lymph node assay (LLNA) is a method for the predictive identification of chemicals that have a potential to cause skin sensitization. Activity is measured as a function of lymph node cell (LNC) proliferative responses stimulated by topical application of test chemicals. Those chemicals that induce a threefold or greater increase in LNC proliferation compared with concurrent vehicle controls are classified as skin sensitizers. In the present investigations we have evaluated further the reliability and accuracy of the LLNA. In the context of an international interlaboratory trial the sensitization potentials of six materials with a history of use in topical medicaments have been evaluated: benzoyl peroxide, hydroquinone, penicillin G, streptomycin sulfate, ethylenediamine dihydrochloride, and methyl salicylate. Each chemical was analyzed in the LLNA by all five laboratories. Either the standard LLNA protocol or minor modifications of it were used. Benzoyl peroxide and hydroquinone, both human contact allergens, elicited strong LLNA responses in each laboratory. Penicillin G, another material shown previously to cause allergic contact dermatitis in humans, was also positive in all laboratories. Streptomycin sulfate induced equivocal responses, in that this material provoked a positive LLNA response in only one of the five laboratories, and then only at the highest concentration tested. Ethylenediamine dihydrochloride dissolved in a 3:1 mixture of acetone with water, or in 4:1 acetone:olive oil (one laboratory), was uniformly negative. However, limited further testing with the free base of ethylene diamine yielded a positive LLNA response when applied in acetone:olive oil (AOO). Finally, methyl salicylate, a nonsensitizing skin irritant, was negative at all test concentrations in each laboratory. Collectively these data serve to confirm that the local lymph node assay is sufficiently robust to yield equivalent results when performed independently in separate laboratories and indicate also that the LLNA is of value in assessing the skin sensitization potential of topical medicaments.

A previous study (Ladics et al., 1995) conducted in our laboratory using the known immunosuppressant agent, cyclophosphamide, indicated that a functional assay for assessment of humoral immunity may be conducted in rats in a standard toxicology study. The objective of this study was to further examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats in a standard toxicology study using a chemical, carbon tetrachloride (CCl4), whose principal target organ of toxicity is not the immune system. Specifically, the previous study and this study were done to determine whether the conduct of an assay for assessing humoral immune function would affect standard toxicological endpoints. Male CD rats were untreated or dosed orally for 30 or 90 days, excluding weekends, with vehicle or 12.5 or 25 mg/kg CCl4. Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC) for assessment of humoral immune function. One day prior to necropsy, blood for hematological and clinical chemical measurements was collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and later examined microscopically. One-half of each spleen was used to assess spleen cell numbers and quantitate lymphocyte subsets (Thelper; Tcyt/sup; total T- and B-cells) by flow cytometry. Serum was analyzed for anti-SRBC IgM antibody by using an enzyme-linked immunosorbent assay. Administration of 12.5 and 25 mg/kg CCl4 for 30 days decreased SRBC-specific serum IgM levels 42 and 45%, respectively, while 25 mg/kg CCl4 for 90 days increased SRBC-specific IgM levels by 50%. CCl4 did not alter splenic lymphocyte subset numbers nor the weight nor morphology of lymphoid organs. Exposure to 25 mg/kg CCl4 did increase liver weight and serum sorbitol dehydrogenase levels, as well as produce centrilobular fatty change. SRBC administration did not alter any hematological or clinical chemistry parameters, nor lymphocyte subset numbers. With the expected exception of the spleen (slight increase in number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the rather mild hepatotoxic effects of CCL4 exposure observed in this study. Based on these and previous findings, it appears that a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study without altering standard toxicological endpoints.

The prospective identification of skin sensitizing chemicals is a vital prerequisite for their proper risk management. Traditionally this has been achieved largely by the conduct of guinea pig assays such as the maximization and Buehler tests. These methods are recommended by the Organisation for Economic Cooperation and Development (OECD) and are required by the European Union (EU) for the evaluation of new substances. However, a novel mechanistically based method, the local lymph node assay (LLNA), has been the focus of substantial validation activity in recent years. This material is reviewed in this paper. It is shown that the LLNA has been validated successfully by five interlaboratory assessments as well as by comparisons with guinea pig tests and human data. The method also offers clear advantages to the user in terms of objectivity, time and cost, and delivers important animal welfare benefits. In consequence, it is recommended that the LLNA be formally adopted by the OECD in Guideline 406 and accepted by the EU and US EPA as a method suitable for the classification of the skin sensitizing potential of chemicals.

The local lymph node assay (LLNA) is a method used for the prospective identification in mice of chemicals that have the potential to cause skin sensitization. We report here the results of the second and final phase of an international trial in which the performance of the assay has been evaluated using seven test materials in five independent laboratories. The additional chemicals examined here included compounds which are considered less potent allergens than some of those tested in the first phase of the investigation, and includes hexylcinnamic aldehyde (HCA), a chemical recommended by the Organization for Economic Cooperation and Development (OECD) as a positive control for skin sensitization studies. In each laboratory all skin sensitizing chemicals examined (2,4-dinitrochlorobenzene {DNCB}, HCA, oxazolone, isoeugenal and eugenol) elicited positive responses of comparable magnitude as judged by the derived lowest concentration of test chemical required to elicit a 3-fold or greater increase in the proliferative activity of draining lymph node cells compared with vehicle-treated controls. We observed that sodium lauryl sulphate, considered to be a non-sensitizing skin irritant, also induced a positive response in the assay. Para-aminobenzoic acid (pABA), a nonsensitizing chemical, was negative at all test concentrations in each laboratory. Some laboratories incorporated minor modifications into the standard assay procedure, including the evaluation of lymph nodes pooled from individual mice rather than treatment groups and the use of statistical analyses. The use of statistics did not markedly change the determination of the lowest concentration yielding a positive response. These data confirm that the local lymph node assay is robust and yields equivalent results when performed independently.

The murine local lymph node assay is a predictive test for the identification of skin-sensitizing chemicals. The method has been the subject both of national inter-laboratory studies and of extensive comparisons with guinea pig tests. In the investigations reported here, the local lymph node assay has been evaluated further in the context of an international study comprising five independent laboratories. In addition, the influence of minor modifications to the standard assay procedure on the performance of the test has been examined. The modified procedures investigated were exposure of mice for 4 rather than 3 consecutive days, excision of lymph nodes 4 rather than 5 days after the initiation of exposure and the use of an alternative isotope. All five laboratories, irrespective of whether the standard or a modified protocol was used, were able to identify accurately, and with comparable sensitivity, potassium dichromate and 2,4-dinitrochlorobenzene as skin sensitizers. Using standard criteria, none of the laboratories recorded positive responses with methyl salicylate, a non-sensitizer. In the standard protocol, lymph nodes are pooled for each experimental group and the vigor of responses measured as a stimulation index relative to vehicle controls. A stimulation index of 3 or greater is considered to indicate skin-sensitizing potential. One further modification adopted by three of the laboratories was to analyze nodes from individual animals and, thereby, permit statistical evaluation. This allowed a direct comparison of statistical significance with the conventional stimulation index as criteria for a positive response. The data indicate that, while statistical evaluation may provide, in some instances, for small increases in sensitivity, this may be at the expense of some loss of selectivity. There are, however, insufficient data presently to draw firm conclusions regarding the relative value of statistical analysis. These studies demonstrate that the local lymph node assay is sufficiently robust to accommodate minor procedural and technical modifications without material changes in test performance.

Several proposals have been made with the aim of assisting in the early identification of chemicals with immunotoxic potential. The Organisation for Economic Cooperation and Development is now likely to incorporate enhanced immunopathology into the test guideline for the 28-day rat study, which may be regarded as a Tier I investigation. However, no guidelines have yet been proposed either for how the new data generated will be evaluated, or for how a subsequent risk assessment will be made. In this paper, considerations for the immunopathological assessment of the thymus, spleen, lymph nodes and bone marrow are described, together with comments on haematological and organ weight changes that may be associated with immunotoxicity. Their interpretation will depend on the doses at which changes are manifest, the quantity and quality of the effects observed and the presence and severity of other forms of toxicity. Lastly, risk assessment and the approach to Tier II testing in immunotoxicity is discussed. It is concluded that much of this work must be on a case-by-case basis, but should not in principle differ from the approach adopted for any other type of toxicity identified ina 28-day study.

The objective of this study was to examine the feasibility of conducting an immunotoxicological assay for assessing humoral immunity in rats on standard toxicology study. Male CD rats were untreated or dosed intraperitoneally daily for 30 or 90 days, excluding weekends, with vehicle or 2 mg/kg cyclophosphamide (CY). Six days prior to sacrifice, selected rats were injected intravenously with sheep red blood cells (SRBC). One day prior to necropsy, blood samples for hematological and clinical chemical measurements were collected from each rat. On the day of necropsy standard protocol tissues were collected, weighed, processed to slides, and examined microscopically. One-half of each spleen was used to prepare a single cell suspension in order to assess spleen cell numbers. Serum was analyzed for anti-SRBC IgM antibody using an enzyme-linked immunosorbent assay. A second set of studies was performed to examine further the effect of SRBC administration on lymphoid organ weights using 30- and 90-day study age-equivalent naive male CD rats. Exposure of animals to 2 mg/kg CY for 30 or 90 days resulted in a 28% and 61% decrease, respectively, in SRBC-specific serum IgM levels. CY treatment also caused mild alterations in some leukocytic parameters, with significant decreases of 35% and 33% in white blood cell and lymphocyte counts, respectively, observed in 30-day CY-treated animals receiving SRBC. Injection of SRBC alone did not alter hematological or clinical chemistry parameters. With the expected exception of the spleen (increased number and size of germinal centers), administration of SRBC did not significantly alter the weights or morphology of routine protocol tissues. Furthermore, administration of SRBC did not mask the immunosuppressive effects of CY treatment under the conditions of this study. Based on our preliminary findings, a functional assay for assessing humoral immunity may be conducted in animals on standard toxicology study.

Various chemicals and proteins of industrial importance are known to cause respiratory allergy, with occupational asthma being the most important manifestation of the disease. This paper describes clinical syndromes, mechanisms associated with occupational respiratory hypersensitivity, and methods available currently for the prospective identification of potential respiratory allergens. Certain classes of chemicals are commonly associated with occupational respiratory allergy. There is insufficient information, however, to predict respiratory sensitization potential from analysis of structure alone, although reactivity with proteins is likely to be relevant. As yet there exist no fully validated or widely applied predictive methods or internationally harmonized guidelines. The most promising predictive animal methods are the mouse IgE test and guinea pig models. Work in mice has focused upon events occurring during the induction phase of sensitization following primary encounter with the test chemical. In contrast, guinea pig models have been used primarily to identify respiratory allergens (chemicals or proteins) as a function of elicitation reactions induced in previously sensitized animals. Given the possible serious health manifestations of respiratory allergy, early identification of respiratory sensitizers is urgently required. The two methods should, as a priority, be developed further and the production of a detailed protocol for these methods be undertaken to facilitate further validation. Together, this information will allow for two types of risk assessment associated with respiratory allergy: the risk that exposure to a material will (1) induce sensitization in an individual and (2) elicit allergic reactions in a previously sensitized individual.

The objective of this study was to examine the immunotoxicological effects of the methyl-carbamate pesticide carbaryl via the oral, dermal, or inhalation routes. Male CD rats were exposed to carbaryl 5 d/wk for a 2-wk period. During nose-only inhalation exposures, rats received either 36, 137, or 335 mg/m3 carbaryl in acetone for 6 h. Air only and acetone/air controls were run concurrently. Orally exposed animals received either 1 ml corn oil or 10, 25, or 50 mg/kg carbaryl, while dermally exposed animals received either 2 ml acetone or 100, 500, or 1000 mg/kg carbaryl on their dorsal flank for 6 h. Four days prior to sacrifice, animals from all exposure groups were injected iv with 2 x 10(8) sheep red blood cells (SRBC). The primary immunoglobulin M (IgM) humoral immune response to SRBC was then assessed by measuring SRBC-specific antibody-forming cells (AFC) and levels of serum anti-SRBC IgM antibody, respectively, using the hemolytic plaque assay and an enzyme-linked immunosorbent assay. Individual body weights, spleen, thymus, and liver weights, spleen cell number, and red and white blood cell (RBC, WBC) counts were obtained for each animal. Following nose-only inhalation exposures, dose-dependent decreases in thymus weights, spleen cell number, AFC/spleen, AFC/10(6) splenocytes, and serum levels of SRBC-specific IgM antibody were observed. Significant decreases of 33, 57, and 22% in spleen cell number, AFC/spleen, and thymus weight, respectively, were found at the 335 mg/m3 exposure level. Animals exposed orally to 25 mg/kg carbaryl had a 34% decrease in WBC counts. A 34% decrease in WBC and a 13% increase in RBC counts were observed at the 50 mg/kg oral dose. Significant decreases in liver weights ranging from 11 to 13% were found at all oral exposure levels. Dermal exposure to carbaryl revealed no significant toxicological effects. Results indicate that humoral immune suppression was observed following inhalation, but not following oral or dermal exposures to carbaryl. Immunotoxicological studies evaluating pesticides need to consider relevant exposure routes and dosages for appropriate risk assessment procedures and exposure limits to be established.

In the context of this paper, immunotoxicity is taken to encompass immunosuppression/immunopotentiation and allergy. Over the last 10 to 15 years, well characterized methods for the assessment of altered immune competence have been reported. This has led to proposals for tiered testing schemes. This review examines the suitability of immunotoxicity parameters for inclusion in routine 28-day studies and comments on methods that have been proposed for incorporation within the guidelines issued by the US FDA and US EPA and OECD. It is recommended that the existing OECD Guideline 407 is modified to incorporate total and differential blood cell counts, spleen and thymus weight and histopathology, and draining and distal lymph node histopathology for Tier I level testing. Data so generated will provide a reliable and accurate means of identifying at an early stage potential immunotoxic effects. Tier II testing should be carried out on a case by case basis and only assuming positive results are obtained at Tier I. An increasingly sophisticated understanding of the nature of immune responses to chemical allergens has facilitated the design of novel predictive methods for the identification of sensitizing activity. Opportunities which arise from these new developments in allergy testing such as the local lymph node assay, mouse ear swelling test, and the mouse IgE test should be monitored closely.

Macrophages were isolated from a series of transplanted mouse mammary tumors, originally derived from a single spontaneously arising tumor, to determine whether macrophage content or function correlated with any of a wide range of tumor properties. None of the tumor cell lines differed significantly in susceptibility to killing by MVE-2-activated peritoneal macrophages (cytotoxicity ranged from 40 to 63%). No significant differences were observed in macrophage content among the five tumor lines, nor was there any correlation between macrophage content and tumor weight, time since transplantation, or ability to metastasize. A significant association was observed, however, between a tumor's ability to metastasize to lung spontaneously and the tumoricidal activity of that tumor's infiltrating macrophages. Significant tumoricidal activity was seen when macrophages isolated from every metastatic tumor studied were used, whereas macrophage-mediated tumoricidal activity was observed in only 35% (6 of 17) of nonmetastatic tumors. Macrophage cytotoxicity was associated with spontaneous metastasis and not with lung colony formation per se.

Delta-9-tetrahydrocannabinol (delta 9-THC) and delta 8-THC have previously been shown to suppress humoral immunity in mice. The purpose of these investigations was to determine whether a hyporesponsiveness develops to the immunosuppressive effect of delta 8-THC. BALB/c mice were administered 60 mg/kg delta 8-THC i.p. 2 days after an i.p. immunizing dose of sheep erythrocytes (sRBC). Significant inhibition of direct hemolytic plaque-forming cells (PFC)/spleen was observed on days 3--6, with peak day of inhibition occurring on day 4. The effective inhibiting dose 50 (ED50) measured on peak day of response was 40 mg/kg for PFC/10(6) spleen cells and 38 mg/kg for PFC/spleen. When mice were pretreated daily for 5 days with different doses of delta 8-THC, similar ED50's were detected. Under this pretreatment regimen, 5 or 10 mg/kg produced no immunosuppression. Daily treatment with 5 or 10 mg/kg delta 8-THC for 5 days prior to sRBC and varying doses of delta 8-THC administered 2 days after sRBC resulted in significantly higher ED50's and increased values for the slopes of the dose response curves. These results suggest that a hyporesponsiveness develops to the immunosuppressive activity of delta 8-THC.

This study establishes the responsiveness of mice bearing the Madison 109 lung carcinoma (M109), a tumor relatively resistant to chemotherapy, to the immunomodulator maleic vinyl ether (MVE-2; molecular weight 15,500). BALB/c mice inoculated with 5 X 10(5) M109 cells into the hind footpad developed a primary tumor which metastasized to the lung within 7 days and resulted in death of the host between 36 and 42 days. Early in the disease, weekly intratumor (i.t.) or intravenous administration of MVE-2 (25 mg/kg) inhibited the growth of the primary tumor and significantly prolonged the life span of the host. During the pulmonary metastatic process, systemic administration of MVE-2 alone or MVE-2 coupled with surgical excision or radiotherapy of the primary tumor became decreasingly efficacious. Late in the disease only MVE-2 introduced directly into the metastatic tumor bed by intrapleural injection proved to be effective in prolonging life span. These studies indicate that both the primary and metastatic M109 tumors are sensitive to MVE-2 and suggest that the efficacy of MVE-2 treatment is largely dependent upon its distribution in the tumor-bearing mice.

A 16,500 molecular weight fraction of maleic vinyl ether (MVE-2) induced tumoristatic and tumoricidal activity in peritoneal macrophages of BALB/c and C57BL/6 mice following i.p. administration. Growth of B16 melanoma cells in vitro was inhibited up to 85% by MVE-2-activated, but not resident, peritoneal macrophages. In a tritiated thymidine release assay, B16 melanoma cells, and to a lesser extent Madison 109 lung carcinoma cells, were also sensitive to the cytolytic action of MVE-2-activated peritoneal macrophages. Administration i.v. of MVE-2 resulted in tumoristatic and tumoricidal activity in alveolar macrophages against radiolabeled B16 and Madison 109 lung carcinoma target cells. MVE-2-activated alveolar macrophages significantly inhibited L5178Y lymphoma colony formation following a 48-hr macrophage-tumor cell coincubation. BALB/c mice bearing the lung-metastasizing Madison 109 lung carcinoma footpad tumor were given MVE-2 i.v., using the same dosing regimen that induced alveolar macrophages to be tumoricidal in vitro. Significant increases in life span were observed, suggesting that the antitumor activity of MVE-2 in this tumor system may be mediated by the activation of alveolar macrophages, with a resulting decrease in metastatic growth in the lung.

Intraperitoneal injection of human rIL-1 in a dose of 0.5 microgram daily for 5 d, or 1 microgram daily for 3 d, was capable of causing complete regression of immunogenic SA1 sarcoma growing subcutaneously in syngeneic or semisyngeneic mice. Higher doses of IL-1 were not more therapeutic against the SA1 sarcoma, but needed to be given to cause complete regression of the immunogenic L5178Y lymphoma. On the other hand, the P815 mastocytoma was much less responsive to IL-1 therapy, in that it failed to undergo complete regression in response to doses of IL-1 capable of causing regression of the L5178Y lymphoma. IL-1 caused regression of the SA1 sarcoma when given on days 6-8 of tumor growth, but not when given on days 1-3. This refractoriness of a small tumor to IL-1 therapy suggests that the antitumor action of IL-1 is based on an underlying host-immune response that is not generated until after day 3 of tumor growth. Direct evidence for the participation of host immunity in IL-1-induced tumor regression was supplied by results showing that IL-1 was not therapeutic against the SA1 sarcoma growing in T cell-deficient (TXB) mice, unless these mice were first infused with Ly-2+ and L3T4+ T cells from donor mice bearing an established SA1 sarcoma. In contrast, normal T cells, or T cells from donor mice bearing a YAC-1 lymphoma, failed to provide TXB recipients with the ability to cause regression of their SA-1 sarcoma in response to IL-1 treatment. The results are in keeping with the interpretation that exogenous IL-1, by augmenting the production of tumor-sensitized T cells, converts a subtherapeutic level of host immunity to a therapeutic level. The results suggest, in addition, that IL-1 only stimulates the replication of T cells that are already engaged in the antitumor immune response.

Suspensions of cells from a series of strain BALB/cfC3H mouse mammary tumors, and adherent and nonadherent cells from the tumors, were tested for their ability to increase the mutation rate of Salmonella typhimurium tester strains TA98 and TA100. Significant increases were seen with cells from three of four tumor lines tested on the TA98 strain and with one of four tested on the TA100 strain. The mutagenic activity was due primarily to cells in the adherent fractions which were greatly enriched for macrophages.

The pharmacokinetics and biodistribution of radioiodinated recombinant interleukin-2 (125I-IL-2) was studied after either intravenous (i.v.) or intraperitoneal (i.p.) injection into C57BL/6 mice. Beta-lactoglobulin radiolabeled with 131I served as a control protein. After i.v. injection, 125I-IL-2 preferentially accumulated in the liver and spleen. Liver accumulation was fast, peaking at 5 min, and was followed by rapid clearance. Spleen accumulation was slightly slower, peaking at 15 min. Blood values 1 min after i.v. injection were 22-34% of the injected doses (I.D.)/gram. These values declined quickly over the next hour. In contrast, after i.p. administration no organ showed specific uptake of 125I-IL-2. Blood values after i.p. injection were essentially constant over 3 h and were greater and more sustained than after i.v. administration. Kidney values for both 125I-IL-2 and 131I-beta-lactoglobulin, after either i.v. or i.p. injection, indicated that the major route of clearance for both compounds was rapid loss through the kidneys.

Recent evidence has implicated enkephalins as immunomodulators. Several studies have reported the regulation of tumor growth by methionine enkephalin (ME). However, there has been little effort to relate the immunological significance of enkephalins to the development of anticancer drugs. The present study had three aims: first, to compare the antitumor activity of the synthetic peptide, D-[Ala2]methionine enkephalinamide (MEA), with endogenous enkephalins on PYB6 fibrosarcoma tumor growth; second, to determine whether tumor growth inhibition was mediated by an opiate receptor; and third, to investigate the effects of MEA on selected immune responses. Female B6C3F1 mice were injected i.p. daily for 7 days with 50-4000 micrograms/kg of ME, MEA, leucine enkephalin (LE) or D-[Ala2]leucine enkephalinamide (LEA), beginning 1 day after PYB6 inoculation. ME and MEA, but not LE or LEA, decreased the PYB6 growth rate. The dose of 50 micrograms/kg MEA exerted the maximum inhibition of tumor growth (nearly 72% on day 15 post tumor transplantation). MEA was not directly toxic to PYB6 tumor cells, as evaluated by the measurement of DNA synthesis and cellular ATP levels of PYB6 cells exposed to MEA in vitro. No [3H]-etorphine specific bindings were detected on the cell membrane or sonicates of splenic lymphocytes or PYB6 cells. Therefore, the antitumor activity by MEA is likely mediated by an indirect mechanism. Immunological studies indicated that MEA selectively enhanced the lymphoproliferative response to the T-cell mitogen, concanavalin A, but not to the B-cell mitogen, lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

In murine schistosomiasis, inflammatory macrophages (M phi) are a major cell type of the granulomata that encase eggs of the tropical helminth Schistosoma mansoni as they are deposited in the liver by adult worm pairs. The granulomatous response is vigorous during acute infection (8 wk) and spontaneously undergoes immunomodulation during chronic infection (20 wk). Inflammatory M phi isolated from liver granulomas of 8 and 20-wk schistosome-infected CBA/J mice were assessed for their cytolytic potential. Macrophages from vigorous liver granulomas displayed significant cytolytic activity against 3 mammary tumor lines of strain BALB/cfC3H origin, while leaving untransformed embryo fibroblasts relatively unharmed. The cytolytic activity of inflammatory M phi from immunomodulated lesions was markedly less than that of cells from vigorous lesions at equivalent effector to target (E:T) cell ratios. Granuloma cell preparations depleted of the inflammatory macrophage component were marginally cytolytic at the higher E:T cell ratio; supernatant fluids from cultured inflammatory M phi-enriched cell preparations possessed no tumoricidal activity. The cytolytic potential of the vigorous granuloma M phi was lost subsequent to prolonged in vitro culture. We conclude that inflammatory M phi in S. mansoni egg-elicited granulomas are sufficiently activated to lyse allogeneic tumor cells. The degree of activation appears to correlate directly with the intensity of the granulomatous response.