Scott E. Loveless

Further evaluation of the local lymph node assay in the final phase of an international collaborative trial.

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.

An international evaluation of the murine local lymph node assay and comparison of modified procedures.

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.

Immunotoxicity of perfluorooctanoic acid and perfluorooctane sulfonate and the role of peroxisome proliferator-activated receptor alpha.

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 (PPARα) 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 PPARα independence of some immune effects. This evidence originates primarily from studies with PPARα knockout models exposed to PFOA that demonstrate hepatic peroxisome proliferation, reduced lymphoid organ weights, and altered antibody synthesis. As human PPARα expression is significantly less than that of rodents, potential PPARα independence indicates that future research must explore mechanisms of action of these compounds, including PPARα -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 PPARα ligands of therapeutic and environmental interest.

Respiratory Allergy: Hazard Identification and Risk Assessment

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.

Possible incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats on standard toxicology 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.

Evaluation of the Immune System in Rats and Mice Administered Linear Ammonium Perfluorooctanoate

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.

Absorption, distribution, metabolism, and excretion of [1-14C]-perfluorohexanoate ([14C]-PFHx) in rats and mice

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.

Further evaluation of the incorporation of an immunotoxicological functional assay for assessing humoral immunity for hazard identification purposes in rats in a standard toxicology study.

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 MOUSE IgE TEST: INTERLABORATORY EVALUATION AND COMPARISON OF BALB/c AND C57BL/6 STRAIN MICE

The mouse IgE test is a novel method for the prospective identification of chemicals that have the potential to cause allergic sensitization of the respiratory tract. Activity is measured as a function of increases in the concentration of total serum IgE induced by topical exposure of mice to chemicals; those chemicals that elicit a substantial elevation in IgE are classified as respiratory allergens. The present investigations were designed to evaluate further the utility of the mouse IgE test. For this purpose theassay was conducted in each of fiveindependent laboratories using trimellitic anhydride (TMA), a known cause of respiratory sensitization and occupational asthma, and 2,4-dinitrochlorobenzene (DNCB), a potent contact allergen that is considered not to cause sensitization of the respiratory tract. For these investigations BALB/c mice were used, which are currently the strain of choice for the mouse IgE test. In four of five laboratories, exposure of mice to TMA caused a statistically significant...

Toxicological evaluation of sodium perfluorohexanoate.

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.