Glenn S. Elliott

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.

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.

Subchronic toxicity of cyclohexane in rats and mice by inhalation exposure

Inhalation studies were conducted to determine the potential toxicity and/or potential neurotoxicity of cyclohexane. Groups of rats and mice were exposed to 0, 500, 2000, or 7000 ppm concentrations of cyclohexane vapor 6 hr/day, 5 days/week for 14 weeks. Subgroups of rats and mice were further observed during a 1-month recovery period. Functional observational battery (FOB) and motor activity (MA) behavioral tests were conducted on rats. These tests were conducted prior to the exposure series and during weeks 4, 8, and 13 on non-exposure days. Clinical pathology evaluations were conducted after approximately 7, 13, and 18 weeks. Approximately 14 and 18 weeks after study initiation, tissues from rats and mice were histologically processed and evaluated by light microscopy. During exposure to 2000 or 7000 ppm, rats and mice had a diminished response or an absent response to delivery of a punctate auditory alerting stimulus. Immediately following removal of rats from the inhalation chambers, 7000 ppm males and females and 2000 ppm females displayed a compound-related increase in the incidence of wet and/or stained fur (which occurred in the areas of the mouth, chin, and/or perineum). These signs were transient, were not observed during exposure or prior to exposure the following day, and were not associated with any behavioral or morphological changes. During exposure sessions, mice exposed to 7000 ppm exhibited clinical signs of toxicity which included hyperactivity, circling, jumping/hopping, excessive grooming, kicking of rear legs, standing on front legs, and occasional flipping behavior. Clinical signs of toxicity observed in 7000 ppm mice immediately after exposure included hyperactivity, hyperreactivity, ruffled fur (females only), gait abnormalities, spasms in both rear legs, and excessive grooming (males only). The clinical signs observed in mice during and immediately after exposure were transient, and were not present prior to the subsequent exposure. A few mice exposed to 2000 ppm appeared hyperactive during exposure in the latter portion of the study. There were no compound-related changes in mean body weights, body weight gains, food consumption, food efficiency, or mortality; and there were no ophthalmological abnormalities in rats or mice. In addition, there were no compound-related effects on 37 different behavioral parameters assessed during the FOB or during motor activity tests in rats. Male and female mice exposed to 7000 ppm had slight increases in measures of circulating erythrocyte mass (red blood cells, hemoglobin, hematocrit) and plasma protein concentration (males only). Male rats and male and female mice exposed to 7000 ppm had significantly increased relative liver weights, and 7000 ppm male mice also had significantly increased absolute liver weights at the end of the exposure period. At the end of the 1-month recovery period, absolute and relative liver weights of male and female mice were similar to control. However, relative liver weights of 7000 ppm male rats continued to be significantly higher at the end of the recovery period. Male and female rats exposed to 7000 ppm had a significantly increased incidence of hepatic centrilobular hypertrophy at the end of the exposure period, which was not observed at the conclusion of the 1-month recovery period. No microscopic changes were observed in mice. In rats, the no-observed-effect level (NOEL) for acute, transient effects was 500 ppm based on a diminished/absent response to an auditory alerting stimulus at 2000 ppm and above. The NOEL for subchronic toxicity in rats was 7000 ppm based on the lack of adverse effects on body weight, clinical chemistry, tissue morphology, and neurobehavioral parameters. In mice, the NOEL for acute, transient effects was 500 ppm based on behavioral changes during exposure at 2000 ppm and above. The NOEL for subchronic toxicity in mice is 2000 ppm based on hematological changes at 7000 ppm.

Repeated Dose Toxicity Study (28 Days) in Rats and Mice with N-Methylpyrrolidone (NMP)

Twenty-eight day feeding studies were conducted to evaluate the repeated dose toxicity of NMP, a widely used industrial solvent, in Crl:CD BR rats and B6C3F1 mice. Groups of 5 male and 5 female rats each were fed either 0, 2,000, 6,000, 18,000, or 30,000 ppm NMP; similar groups of mice were fed either 0, 500, 2,500, 7,500, or 10,000 ppm. In vivo parameters, hematology and clinical chemistry parameters, and complete pathology evaluations were conducted after approximately 28 days. Decrements in mean body weight gains, reflecting decreases in food consumption and efficiency, were seen in male rats fed 18,000 ppm and in both sexes fed 30,000 ppm. In rats, clinical chemical changes, indicating possible compound-related alterations in lipid, protein, and carbohydrate metabolism, occurred at 18,000 ppm in males and 30,000 ppm in both sexes. No histopathological changes in rats were judged to be directly related to NMP exposure. Hematological (mild to moderate leukopenia) and histopathological alterations (hypocellular bone marrow, testicular degeneration and atrophy, and thymic atrophy) were judged to be secondary to nutritional and body weight effects in male and/or female rats at 30,000 ppm. In mice, cloudy swelling of the epithelia of the distal parts of the renal tubuli was observed in 4 males and 3 females at 10,000 ppm and in 2 male mice at 7,500 ppm. For both rats and mice, abnormal urine coloration was observed (in mice at 2,500 ppm and above, and in rats at 18,000 ppm and above). The discoloration was interpreted as a sign of systemic availability of the test substance, but not as an adverse effect. The NOAEL was 6,000 ppm for male rats and 18,000 ppm for female rats. In mice, the NOAEL was 2,500 ppm based on the kidney histopathology.

CHRONIC TOXICITY AND ONCOGENICITY OF N-METHYLPYRROLIDONE (NMP) IN RATS AND MICE BY DIETARY ADMINISTRATION

A two-year feeding study in rats and an 18-month feeding study in mice were conducted to evaluate the potential chronic toxicity and oncogenicity of NMP in Crl : CD® (SD)BR rats and B6C3F1/CrlBR mice. Groups of 62 male and female rats were administered diets containing 0, 1600, 5000, or 15 000 ppm of NMP for approximately 2 years. Groups of 50 male and female mice were administered diets containing 0, 600, 1200, or 7200 ppm NMP for approximately 18 months. In vivo parameters were evaluated weekly during the first 3 months of the study, and every other week or monthly during the remainder of the study. For rats, an ophthalmoscopic examination was conducted prior to study start and near the end of the study. Periodically, blood samples were collected from rats and mice for determination of leukocyte differential counts, and from mice for red blood cell morphology. After approximately 2 years of dietary administration in rats and 18 months in mice, all surviving animals were sacrificed. Selected tissues were processed for morphological evaluation. Over the course of the two-year study in rats, test substance-related decrements in body weight and weight gain occurred in 15 000 ppm males and females, which correlated with decreased food consumption and food efficiency. A toxicologically significant, test substance-related increase in the incidence of severe chronic progressive nephropathy occurred in 15 000 ppm males. Several morphological changes noted grossly and/or microscopically were secondary to the increased severity of chronic progressive nephropathy. NMP was not oncogenic in male or female rats at dietary concen trations of 15000 ppm and below. A test substance-related decrease in the percentage of 15 000 ppm males surviving to the end of the two-year study compared to the control group resulted from the higher incidence of severe chronic progressive nephropathy. However, a sufficient population of 15000 ppm rats were at risk for potential oncogenicity, so the lower survival did not impair the ability to detect an oncogenic response in this study. There were no adverse, test substance-related effects on the incidences of clinical observations, ophthalmic observations, or differential leukocyte counts in males or females, or on survival of females at any dietary concentration. Male and female mice administered dietary concentrations of 7200 ppm had significantly increased liver weight, significantly increased incidence of hepatocellular adenoma, and significantly increased foci of cellular alteration in the liver. At 7200 ppm, male mice also had an increased incidence of hepatocellular carcinoma while the increased incidence of hepatocellular carcinoma in female mice fell within the historical control range. In addition, the incidence of hepatocellular hypertrophy was increased in 7200 ppm males. Liver weight and hepatocellular hypertrophy were also increased in 1200 ppm males. There were no adverse, test substance-related effects on the incidences of clinical observations, food consumption, body weight, differential leukocyte counts, red blood cell morphology, or survival in either males or females at any dietary concentration. Under the conditions of the study, the no-observed-effect level for NMP was 5000 ppm for male and female rats, 600 ppm for male mice, and 1200 ppm for female mice.

Induction of coagulation effects by Wyeth-14,643 in CRL:CD®BR rats

A two-year mechanistic bioassay in male Crl:CD BR rats was initiated with 50 ppm Wyeth-14,643 (WY) to investigate the relationship between peroxisome proliferating compounds and Leydig cell adenoma formation. After 154 days, the survival rate in the WY group decreased below control levels. After 300 days, the dose was lowered to 25 ppm for the remainder of the study. Gross examination of WY-treated rats either found dead or euthanized in extremis revealed hemorrhages at several sites. To investigate this observation, blood was then collected on test day 281 from 10 randomly selected control and WY-treated rats and a clinical pathological examination was performed. The WY-treated rats had significantly decreased red blood cell count, hemoglobin, and hematocrit, and elevated platelet counts. In the WY-treated rats, prothrombin times in undiluted plasma were similar to the controls, but were markedly prolonged in 2 of 10 rats when the plasma samples were diluted to 25%. Subsequently, blood was collected prior to sacrificing WY-treated rats which were exhibiting clinical signs of anemia. These rats had prolonged prothrombin times, activated partial thromboplastin time, and thrombin clot time when compared to laboratory historical control data (116.7 vs 13.3, 116.4 vs 13.7, and 42.4 vs 25.7 seconds, respectively). In a subsequent, ongoing study, Vitamin K was added to control and WY-treated diets (100 ppm). No survival differences between control and WY-treated rats occurred through 260 days in this second study. These new data suggest that deaths in the WY-treated group in our initial study were due to a vitamin K deficiency. The role of increased serum estradiol, its effects on blood coagulation, and enhanced hepatic cell proliferation in the vitamin K-dependent coagulation processes warrant further investigation.

90-Day Subchronic Toxicity Study in Rats and Mice Fed N-Methylpyrrolidone (NMP) Including Neurotoxicity Evaluation in Rats

In mice, there were no effects on body weight or food consumption. As observed in rats, mice fed 2,500 or 7,500 ppm exhibited a change in urine coloration which was not associated with morphological changes in cholesterol, triglycerides, calcium, and alkaline phosphatase occurred at 28 days but not 90 days. These changes are thus assessed as being of minor toxicological relevance. Liver weights were elevated in males fed 2,500 or 7,500 ppm and centrilobular hypertrophy was seen in both sexes fed 7,500 ppm. These changes may be regarded as an adaptation process but are clearly related to NMP exposure. Other toxicological endpoints examined were unaffected by NMP. The NOAEL was 3,000 ppm for both sexes of rats based on body weight effects and changes in 3 neurobehavioral parameters (males only) at higher feeding levels. In mice, the NOAEL was 1,000 ppm based on liver responses to higher concentrations.

Repeated exposure inhalation study of pentane in rats

Pentane (CAS No. 109-66-0) is a chemical being used as a co-solvent in a polymer production facility with potential for inhalation exposure in humans. To assess the toxicity of pentane, groups of 10 male rats each were exposed by inhalation, 6 hr/day, 5 days/week for 2 weeks to either 0 (control), 1,000, 3,000 or 10,000 ppm. Five rats per group were killed following the 10th exposure; the remaining 5/group were killed after a 14-day post-exposure recovery period. Parameters investigated were clinical signs of toxicity, functional behavior, body weights, clinical pathology, and gross and microscopic pathology including organ weights. No unusual clinical observations were seen in the pentane-treated rats, and body weights were not altered. Test rats generally exhibited normal behavioral responses in the functional observational battery. Increases in serum calcium and phosphorus concentrations were seen in rats exposed to either 3,000 or 10,000 ppm. These were reversible during the 2-week recovery period. No other clinical pathology changes were observed and no pentane-related tissue pathology was seen in any of the groups. The no-observed-adverse-effect level was 1,000 ppm with reversible clinical pathology changes produced at 3,000 and 10,000 ppm.

Inhalation Toxicity of Cyclododecatriene in Rats

Cyclododecatriene (CDDT, CAS No. 4904-61-4) was tested for its inhalation toxicity in rats following repeated exposures. Male rats were exposed nose-only to CDDT for 6 hr/day, 5 days/wk for a total of 9 exposures over 2 weeks. Particular attention was paid to neurotoxicologic endpoints. Concentrations of 0 (control), 5, 50, and 260 ppm were studied. The 260 ppm chamber contained both vapor and aerosol while the 5 and 50 ppm chambers were vapor only. Four groups of 10 rats each were used to measure standard clinical signs and growth, clinical pathology (including hematology, biochemistries, and urine analysis), and tissue pathology. Another 4 groups of similar size were used for neurotoxicity testing. In the standard toxicity groups, 1/2 of the rats were sacrificed 1 day following the 9th exposure; the other half underwent a 2-week recovery period prior to being sacrificed (recovery group). During the exposures rats inhaling 260 ppm had a diminished or absent response to an alerting stimulus. Irregular respiration and lethargy were observed in these rats immediately following exposure. These signs were rapidly reversible and were not seen prior to the subsequent exposure. Body weights in rats exposed to either 50 or 260 ppm were significantly lower than the corresponding controls. No compound-related clinical pathology changes were seen in any of the test groups and tissue pathology effects only occurred in the nasal tissue. In rats exposed to 260 ppm, minimal degeneration/necrosis of nasal olfactory epithelium was observed in rats examined immediately following the exposure period. This change was not seen in the recovery rats. Functional observational battery (FOB) assessments and motor activity (MA) evaluations conducted after the 4th and 9th exposures on rats from all test groups, and specific neuropathologic evaluation on perfused brain, spinal cord, and skeletal muscle from rats exposed to 260 ppm failed to demonstrate any specific neurotoxicity. Outward signs of sedation were seen at the top level tested. Under the conditions of this test, the no-observed-adverse-effect level (NOAEL) was determined to be 5 ppm based upon a reduced rate of body weight gain in the 50 ppm group. No specific neurotoxicity was detected and the histopathologic response was limited to reversible changes in the nasal epithelia in rats exposed to 260 ppm.

Inhalation toxicity of dimethyl piperidinone

A mixture of 1,3-dimethyl-2-piperidinone and 1,5-dimethyl-2-piperidinone (DMPD) (approximately 63-37 parts by weight) was tested for its inhalation toxicity in rats following 90-day repeated exposures. Male and female rats were exposed whole-body to either 0, 51, 230, or 310 mg/m(3) DMPD for 6 h/day, 5 days/weak for 90 days. Clinical signs, growth, clinical pathology, tissue pathology, neurobehavior, neuropathology, and semen quality were evaluated. No compound-related adverse effects were noted in clinical signs, body weights, food consumption, clinical laboratory evaluations, neurobehavioral evaluations, neuropathology, or sperm counts. Laryngeal changes consisting of minimal squamous epithelial hyperplasia and degeneration/necrosis of the cartilage were present in male and female rats exposed to 310 mg/m(3) both immediately following exposure and after the 1-month recovery period Male rats exposed to DMPD had increased relative kidney weights, increased formation of hyaline droplets and granular casts, and increased incidence of chronic progressive nephropathy. These kidney effects are consistent with increased accumulation of the urinary protein alpha(2 mu)-globulin, which has been well essential for several xenobiotics. The subsequent increased incidence of progressive nephropathy was specific to male rats with the alpha(2 mu) syndrome. Male and female rats exposed to 230 or 310 mg/m(3) had centrilobular hepatocellular hypertrophy, and male rats exposed to 310 mg/m(3) had increased relative liver weights. These liver changes were reversible following the recovery period and were considered not to represent adverse toxicological effects of treatment. Since the male rat-specific renal findings do not connote adversity for man and are net considered relevant to human hazard assessment, the no-observed-effect level in male and female rats was 230 mg/m(3), based on the microscopic changes in the larynx exposed to 310 mg/m(3).