Accounting for Multiple Comparisons in Statistical Analysis of the Extensive Bioassay Data on Glyphosate.

Glyphosate is a widely used herbicide worldwide. In 2015, the International Agency for Research on Cancer (IARC) reviewed glyphosate cancer bioassays and human studies and declared that the evidence for carcinogenicity of glyphosate is sufficient in experimental animals. We analyzed 10 glyphosate rodent bioassays, including those in which IARC found evidence of carcinogenicity, using a multiresponse permutation procedure that adjusts for the large number of tumors eligible for statistical testing and provides valid false-positive probabilities. The test statistics for these permutation tests are functions of p values from a standard test for dose-response trend applied to each specific type of tumor. We evaluated 3 permutation tests, using as test statistics the smallest p value from a standard statistical test for dose-response trend and the number of such tests for which the p value is less than or equal to .05 or .01. The false-positive probabilities obtained from 2 implementations of these 3 permutation tests are: smallest p value: .26, .17; p values ≤ .05: .08, .12; and p values ≤ .01: .06, .08. In addition, we found more evidence for negative dose-response trends than positive. Thus, we found no strong evidence that glyphosate is an animal carcinogen. The main cause for the discrepancy between IARC's finding and ours appears to be that IARC did not account for the large number of tumor responses analyzed and the increased likelihood that several of these would show statistical significance simply by chance. This work provides a more comprehensive analysis of the animal carcinogenicity data for this important herbicide than previously available.

Toxicological evaluation of carcinogenicity of the pyrethroid imiprothrin in rats and mice.

The carcinogenic potential of a non-genotoxic pyrethroid imiprothrin was examined in rats and mice. There was no carcinogenicity in rats up to a maximum dose of 5000 ppm of the diet. There was a higher (p = 0.03) incidence of lung adenocarcinomas at 7000 ppm in males, and females showed an increasing (p < 0.01) trend in the incidence of lung adenomas and combined lung adenoma/adenocarcinomas. Additional step sections of lung demonstrated no significant increases in any tumor at p < 0.05, although an increasing trend with dose was observed among females. We argue that, the 7000 ppm dose exceeded the Maximum Tolerated Dose (MTD) for both sexes, based on systemic toxicity as evidenced by body weight gain reduction (both sexes) and high mortality (females). If the 7000 ppm dose is therefore removed from consideration, there are not significant (p < 0.05) increases in tumor formation. Moreover, a consideration of multiple comparisons reveals that the lung tumor increases observed are totally consistent with what would be expected by chance alone. Based on high susceptibility of this mouse strain for the appearance of lung tumors and the lack of a statistically significant increase in tumors by appropriate analysis, the mouse study does not indicate a carcinogenic effect of imiprothrin, and thus no classification for carcinogenicity is warranted.

A critical examination of the mode of action of quinacrine in the reproductive tract in a 2-year rat cancer bioassay and its implications for human clinical use.

A rat carcinogenicity bioassay (CaBio) of quinacrine was reanalyzed to investigate its mode of tumor induction. Quinacrine's effects in the rat uterus when administered as a slurry in methylcellulose were contrasted with the human clinical experience which uses a solid form of the drug, to determine the relevance of the tumors produced in the rat to safe clinical use of quinacrine for permanent contraception (QS). A review was performed of the study report, dose feasibility studies, and clinical evaluations of women who had undergone the QS procedure. The top three doses of quinacrine in the CaBio exceeded the maximum tolerated dose, and produced chronic damage, including inflammation, resulting in reproductive tract tumors. Chronic inflammation was significantly correlated with the tumors; there was no evidence of treatment-related tumors in animals without chronic inflammation or other reproductive system toxicity. Because such permanent uterine damage and chronic toxicity have not been observed in humans under therapeutic conditions, we conclude that this mode of action for tumor production will not occur at clinically relevant doses in women who choose quinacrine for permanent contraception.

Proper interpretation of chronic toxicity studies and their statistics: A critique of "Which level of evidence does the US National Toxicology Program provide? Statistical considerations using the Technical Report 578 on Ginkgo biloba as an example".

A recent article by Gaus (2014) demonstrates a serious misunderstanding of the NTP's statistical analysis and interpretation of rodent carcinogenicity data as reported in Technical Report 578 (Ginkgo biloba) (NTP, 2013), as well as a failure to acknowledge the abundant literature on false positive rates in rodent carcinogenicity studies. The NTP reported Ginkgo biloba extract to be carcinogenic in mice and rats. Gaus claims that, in this study, 4800 statistical comparisons were possible, and that 209 of them were statistically significant (p<0.05) compared with 240 (4800×0.05) expected by chance alone; thus, the carcinogenicity of Ginkgo biloba extract cannot be definitively established. However, his assumptions and calculations are flawed since he incorrectly assumes that the NTP uses no correction for multiple comparisons, and that significance tests for discrete data operate at exactly the nominal level. He also misrepresents the NTP's decision making process, overstates the number of statistical comparisons made, and ignores the fact that the mouse liver tumor effects were so striking (e.g., p<0.0000000000001) that it is virtually impossible that they could be false positive outcomes. Gaus' conclusion that such obvious responses merely "generate a hypothesis" rather than demonstrate a real carcinogenic effect has no scientific credibility. Moreover, his claims regarding the high frequency of false positive outcomes in carcinogenicity studies are misleading because of his methodological misconceptions and errors.

Using fewer animals to identify chemical eye hazards: revised criteria necessary to maintain equivalent hazard classification.

U.S. Federal Hazardous Substances Act (FHSA) regulations specify eye safety testing procedures and hazard classification criteria for substances regulated by the U.S. Consumer Product Safety Commission (CPSC). Current regulations require up to three sequential 6-animal tests. Testing consistent with the Organisation for Economic Co-operation and Development (OECD) test guideline for eye irritation/corrosion, which specifies 3 animals, can also be submitted to US agencies. However, current FHSA regulations do not provide criteria to classify results from 3-animal tests. An analysis was conducted to determine criteria using results from 3-animal tests that would provide equivalent labeling to FHSA regulations. The frequency that FHSA requirements identify substances as ocular irritants was compared with the frequency that a criterion of either ≥ 1/3 or ≥ 2/3 positive animals would identify these substances. A database of rabbit eye tests was also used to estimate over- and underprediction rates for each criterion. In each instance, a criterion of ≥ 1/3 positive animals more closely matched the expected outcome based on FHSA requirements, while a criterion of ≥ 2/3 positive animals identified far fewer irritants. Using a classification criterion of ≥ 1/3 positive animals provided equivalent or greater eye hazard labeling as current FHSA requirements, while using 50-83% fewer animals.

Safety assessment of allergic contact dermatitis hazards: an analysis supporting reduced animal use for the murine local lymph node assay.

The original Organisation for Economic Co-operation and Development Test Guideline 429 (OECD TG 429) for the murine local lymph node assay (LLNA) required five mice/group if mice were processed individually. We used data from 83 LLNA tests (275 treated groups) to determine the impact on the LLNA outcome of reducing the group size from five to four. From DPM measurements, we formed all possible four- and five-mice combinations for the treated and control groups. Stimulation index (SI) values from each four-mice combination were compared with those from five-mice combinations, and agreement (both SI<3 or both SI ≥ 3) determined. Average agreement between group sizes was 97.5% for the 275 treated groups. Compared test-by-test, 90% (75/83) of the tests had 100% agreement; agreement was 83% for the remaining eight tests. Disagreement was due primarily to variability in animal responses and closeness of the SI to three (positive response threshold) rather than to group size reduction. We conclude that using four rather than five mice per group would reduce animal use by 20% without adversely impacting LLNA performance. This analysis supported the recent update to OECD TG 429 allowing a minimum of four mice/group when each mouse is processed individually.

Investigation of the low-dose response in the in vivo induction of micronuclei and adducts by acrylamide.

Acrylamide is an industrial chemical used in polymer manufacture. It is also formed in foods processed at high temperatures. It induces chromosome aberrations and micronuclei (MN) in somatic cells of mice, but not rats, and mutations in transgenic mice. This study evaluated the low-dose MN response in mouse bone marrow and the shape of the dose-response curve. Mice were treated orally with acrylamide for 28 days using logarithmically spaced doses from 0.125 to 24 mg/kg/day, and MN were assessed in peripheral blood reticulocytes (RETs) and erythrocytes by flow cytometry. Liver glycidamide DNA adducts and acrylamide and glycidamide N-terminal valine hemoglobin adducts were also determined. Acrylamide produced a weak MN response, with statistical significance at 6.0 mg/kg/day, or greater, in MN-RETs and at 4.0 mg/kg/day or greater in MN normochromatic erythrocytes (NCEs). The MN responses at the lower doses were indistinguishable from the concurrent and historical controls. The adducts increased at a much different rate than the MN. When the MN-NCE values were compared to administered dose, the response was consistent with a linear model. However, when hemoglobin or DNA adducts were used as the dose metric, the response was significantly nonlinear, and models that assumed a threshold dose of 1 or 2 mg/kg/day provided a better fit than a linear model. The MN-RET dose-response had greater variability than the MN-NCE response and was consistent with linearity and with a threshold at 1 or 2 mg/kg/day, regardless of the dose metric. These data suggest a threshold for acrylamide in the MN test.

Association of race, age and body mass index with gross pathology of uterine fibroids.

OBJECTIVE: To determine the associations of race, age and body mass index (BMI) with the gross pathology parameters of uterine leiomyomas in premenopausal women undergoing hysterectomy or myomectomy. STUDY DESIGN: Participants (N = 107) were recruited from surgical rosters of the George Washington University (GWU) Medical Center Gynecology Department as part of the National Institute of Environmental Health Sciences Fibroid Study. Tumor data and patient demographics were obtained from clinical reports, pathology forms and interviews. RESULTS: Surgical cases consisted of 78% African Americans, 13% Caucasians and 9% others (non-African American, non-Caucasian or race unknown). This proportion of African Americans was significantly higher than the distribution of GWU health plan participants. Fibroids were localized predominantly within the intramural region. Subserosal tumors were more common in patients with more than 9 tumors. African Americans had the highest mean BMI and mean myomatous uterine weight. CONCLUSION: African Americans were the disproportionate majority coming to surgery for fibroids. The average BMI and uterine weight were greater in African Americans than in Caucasians, although these differences were marginal. Race did not influence the size, location or number of fibroids in these surgical cases. Subserosal tumors were more common in patients with more than 9 tumors.

Variations in phytoestrogen content between different mill dates of the same diet produces significant differences in the time of vaginal opening in CD-1 mice and F344 rats but not in CD Sprague-Dawley rats.

BACKGROUND: The optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical. OBJECTIVES: We conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats. METHODS: Mice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded. RESULTS: The time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets. CONCLUSIONS: The S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogen-sensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays.

A review of large granular lymphocytic leukemia in Fischer 344 rats as an initial step toward evaluating the implication of the endpoint to human cancer risk assessment.

Large granular lymphocyte leukemia (LGLL) is a common fatal disease in aging F344 rats. The current understanding of rat LGLL and a search for mechanistic data/correlations to human leukemia were examined with the goal of improving evaluation of the LGLL endpoint in cancer bioassays as it relates to human cancer risk assessments. The exact cell of origin of the F344 rat LGLL is not fully resolved, although natural killer (NK) cell characteristics were demonstrated in most, if not all cases. Similarities between rat LGLL and a rare human NK-LGLL exist, invalidating claims of no human counterpart, although the underlying etiopathogenesis may be different. There is insufficient data to establish a mode of action of chemical-induced rat LGLL. Evaluation of the National Toxicology Program database revealed only 34 substances (out of over 500 studied) that were possibly associated with increased incidences of LGLL. Of these, only five produced definitive LGLL effects in both sexes; the remaining 29 produced single sex responses and/or only "equivocal" associations with LGLL. Trends of increasing background/variability in LGLL incidence and its modulation by extraneous factors (e.g., corn oil gavage) are key confounders in interpretation. Given that LGLL is a common tumor in control F344 rats, interpretations of bioassays can be improved by increasing the statistical stringency (e.g., p<0.01 over traditional p<0.05), as an indicator of possible carcinogenic effects, but that alone would be insufficient evidence for declaring treatment-related increases. Thus, it was concluded that the evaluation of possible chemically related increases in rat LGLL utilize a "weight-of-evidence" approach.

Analysis of nonlinear regression models: a cautionary note.

Regression models are routinely used in many applied sciences for describing the relationship between a response variable and an independent variable. Statistical inferences on the regression parameters are often performed using the maximum likelihood estimators (MLE). In the case of nonlinear models the standard errors of MLE are often obtained by linearizing the nonlinear function around the true parameter and by appealing to large sample theory. In this article we demonstrate, through computer simulations, that the resulting asymptotic Wald confidence intervals cannot be trusted to achieve the desired confidence levels. Sometimes they could underestimate the true nominal level and are thus liberal. Hence one needs to be cautious in using the usual linearized standard errors of MLE and the associated confidence intervals.

Incidences of selected lesions in control female Harlan Sprague-Dawley rats from two-year studies performed by the National Toxicology Program.

The NTP has a long history of using Fischer rats and has compiled a large database of incidences of lesions seen in control animals. Such a database is lacking for Harlan Sprague-Dawley (SD) rats. The intention of this paper is to report spontaneous lesions observed in female vehicle control Harlan SD rats, and to compare the incidence in 2 strains of rats (Fischer and Harlan SD) used in NTP studies. Female Harlan SD rats served as the test animals for a special series of 2-year studies. Male rats were not used in these studies. Complete histopathology was performed on all animals, and the pathology results underwent comprehensive NTP pathology peer review. The most commonly observed neoplasms in these female control Harlan SD rats were mammary gland fibroadenoma (71%), tumors of the pars distalis of the pituitary (41%) and thyroid gland C-cell tumors (30%). Female Fischer rats had incidences of 44% for mammary gland fibroadenomas, 34% for tumors of the pars distalis, and 16% for thyroid gland C-cell tumors. Fischer rats had a 15% incidence of clitoral gland tumors, while the Harlan SD rats had an incidence of < 1%. In contrast to Fischer F344 rats, the Harlan SD rats had a high incidence of squamous metaplasia of the uterus (44.2%). Squamous metaplasia is not a lesion commonly observed in NTP control Fischer rats. The Harlan SD rats had a very low incidence of mononuclear cell leukemia (0.5%), compared with an incidence of 24% in female Fischer rats.

Upregulation of estrogen receptor expression in the uterus of ovariectomized B6C3F1 mice and Ishikawa cells treated with bromoethane.

In a 2-year NTP bioassay, Bromoethane (BE) was found to induce endometrial neoplasms in the uterus of B6C3F1 mice [; ]. In women, hormonal influences, such as "unopposed" estrogenic stimulus, have been implicated as important etiologic factors in uterine cancer. BE, however, does not affect the serum concentrations of sex hormones in female B6C3F1 mice [] and the mechanism of BE-induced uterine carcinogenesis still remains unclear. In the present study, we examined the estrogenic effects of BE on the uterus of ovariectomized B6C3F1 mice and on Ishikawa cells. Groups of 6 mice were given daily s.c. injections of 0, 100, 500 or 1000 mg BE/kg for 3 consecutive days. Mice treated with 17beta-estradiol served as positive controls. Mice were necropsied 24 h after the final injection, and uteri were weighed and examined histologically and immunohistochemically along with the vagina. Changes observed in the estrogen-treated mice included increased uterine weights, edema and inflammation of the endometrium, increased epithelial layers of the uterine and vaginal lumens and keratinization of the vaginal epithelium. In the BE-treated mice, no such changes occurred; however, immunohistochemical staining of the uterus revealed a significant increase in immunoexpression of the estrogen receptor alpha (ERalpha) in the two higher dose groups. Analysis of mRNA also showed slightly increased uterine ERalpha expression in these groups. Upregulated expression of ERalpha was confirmed in BE-treated Ishikawa cells, in which Western blotting analyses identified an intense signal at approximately 66 kDa, which is consistent with ERalpha. These data suggest that upregulated expression of ERalpha may be important in the induction of endometrial neoplasms in BE-treated mice.

Dose-additive carcinogenicity of a defined mixture of "dioxin-like compounds".

Use of the dioxin toxic equivalency factor (TEF) approach in human risk assessments assumes that the combined effects of dioxin-like compounds in a mixture can be predicted based on a potency-adjusted dose-additive combination of constituents of the mixture. In this study, we evaluated the TEF approach in experimental 2-year rodent cancer bioassays with female Harlan Sprague-Dawley rats receiving 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3 ,4,4 ,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of the three compounds. Statistically based dose-response modeling indicated that the shape of the dose-response curves for hepatic, lung, and oral mucosal neoplasms was the same in studies of the three individual chemicals and the mixture. In addition, the dose response for the mixture could be predicted from a combination of the potency-adjusted doses of the individual compounds. Finally, we showed that use of the current World Health Organization dioxin TEF values adequately predicted the increased incidence of liver tumors (hepatocellular adenoma and cholangiocarcinoma) induced by exposure to the mixture. These data support the use of the TEF approach for dioxin cancer risk assessments.

Gingival carcinogenicity in female Harlan Sprague-Dawley rats following two-year oral treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like compounds.

We evaluated gingival toxicities induced by chronic exposure of female Harlan Sprague-Dawley rats to dioxin and dioxin-like compounds (DLCs) and compared them to similarly induced oral lesions reported in the literature. This investigation represents part of an ongoing initiative of the National Toxicology Program to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. For two years, animals were administered by gavage 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); 3,3',4,4',5-pentachlorobiphenyl (PCB126); 2,3,4,7,8-pentachlorodibenzofuran (PeCDF); 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); a tertiary mixture of TCDD, PCB126, and PeCDF; a binary mixture of PCB126 and 153; or a binary mixture of PCB126 and 2,3',4,4',5-pentachlorobiphenyl (PCB118); control animals received corn oil-acetone vehicle (99:1) alone. A full complement of tissues, including the palate with teeth, was examined microscopically. In the groups treated with TCDD and the mixtures of TCDD, PCB126, and PeCDF; PCB126 and 153; and PCB126 and 118, the incidences of gingival squamous hyperplasia increased significantly. Moreover, in the groups treated with TCDD, PCB126, and the mixture of PCB126 and 153, squamous cell carcinoma (SCC) in the oral cavity increased significantly. This investigation constitutes the first report documenting that chronic administration of dioxin-like PCBs can induce gingival SCC in rats. These results indicate that dioxin and DLCs target the gingiva of the oral cavity, in particular the junctional epithelium of molars.

Toxicology and carcinogenesis studies of dipropylene glycol in rats and mice.

Dipropylene glycol (DPG) is a component of many commercial products such as antifreeze, air fresheners, cosmetic products, solvents, and plastics. Male and female F344/N rats and B6C3F1 mice were exposed to DPG in the drinking water for 2 weeks, 3 months, or 2 years. In the 2-week and 3-month studies, rats and mice were exposed to 0, 5000, 10,000, 20,000, 40,000, or 80,000 ppm DPG. There was no mortality in the 2-week studies. In the 3-month rat study, all animals survived to the end of the study. Liver weights of rats exposed to 10,000 ppm or greater and kidney weights of rats exposed to 40,000 and 80,000 ppm were greater than those of the controls. The incidences of liver and kidney lesions were significantly increased in males exposed to 20,000 ppm or greater and females exposed to 80,000 ppm. Focal olfactory epithelial degeneration was present in all rats exposed to 80,000 ppm. In males, the incidences of testicular atrophy, epididymal hypospermia, and preputial gland atrophy were significantly increased in the 80,000 ppm group. In the 3-month mouse study, three males and one female exposed to 80,000 ppm died. Liver weights were increased, as was the incidence of centrilobular hypertrophy in males exposed to 40,000 ppm and males and females exposed to 80,000 ppm. In the 2-year studies, exposure groups were 0, 2500 (rats only), 10,000, 20,000 (mice only) or 40,000 ppm DPG. Survival of male rats exposed to 40,000 ppm and mean body weights of males and females exposed to 40,000 ppm were significantly less than controls. In male rats, exposure to DPG resulted in increased incidences and severities of nephropathy and secondary lesions in the parathyroid and forestomach. Increased incidences of focal histiocytic and focal granulomatous inflammation of the liver were also observed. In male and female rats, there were increased incidences of bile duct hyperplasia and changes in the olfactory epithelium of the nose. In mice, survival of males and females was similar to controls. Mean body weights and water consumption of males exposed to 40,000 ppm were less than that of the controls. Treatment-related nonneoplastic lesions did not occur in mice. Treatment-related neoplastic lesions did not occur in rats or mice.

Exocrine pancreatic pathology in female Harlan Sprague-Dawley rats after chronic treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin and dioxin-like compounds.

We evaluated the effect of chronic exposure to dioxin and dioxin-like compounds on the pancreas in female Harlan Sprague-Dawley rats. This investigation represents part of an ongoing National Toxicology Program initiative to determine the relative potency of chronic toxicity and carcinogenicity of polychlorinated dioxins, furans, and biphenyls. Animals were treated by gavage for up to 2 years with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3,4,4,5-pentachlorobiphenyl (PCB-126), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a toxic-equivalency-factor (TEF) mixture of these agents; control animals received corn oil-acetone vehicle alone. A complete necropsy was performed on all animals, and a full complement of tissues was collected and examined microscopically. Administration of each of the four compounds was associated with increased incidences of several nonneoplastic changes in the exocrine pancreas, including cytoplasmic vacuolation, chronic active inflammation, atrophy, and arteritis. Low incidences, but higher than those in the historical database, of pancreatic acinar adenoma and carcinoma were seen in the TCDD, PeCDF, and TEF-mixture groups. These results indicate that the pancreatic acini are target tissues for dioxin and certain dioxin-like compounds. Key words: carcinogenesis, dioxin, furans, inflammation, pancreas, polychlorinated biphenyls.

Association of liver hemangiosarcoma and secondary iron overload in B6C3F1 mice--the National Toxicology Program experience.

The literature evidencing the role of iron in promoting a range of neoplasms in humans and animals prompted us to search for a possible association between chemically induced hemosiderosis and hemangiosarcomas in the liver of mice in selected studies conducted by the National Toxicology Program (NTP). Its historical control database was examined for studies in which treatment-related liver hemangiosarcoma was noted; 130 consecutive NTP studies in B6C3F1 mice from Technical Report (TR)-340 to TR-493 were evaluated. Three compounds (2-butoxyethanol, p-nitroaniline, and para-chloroaniline) were associated with a relatively high incidence of Kupffer cell pigmentation consisting of hemosiderin in both sexes; only the male mice developed a relatively low incidence of treatment-related hemangiosarcoma. With a fourth compound (o-nitroanisole), a relatively low incidence (16/50, high-dose males) of chemical-related hemosiderosis was noted, with no associated increase of hemangiosarcoma. Two chemicals (pentachlorophenol and tetrafluoroethylene) increased the incidence of liver hemangiosarcoma in male and female mice, with no increase in Kupffer cell pigmentation. The overall association between liver hemangiosarcoma and Kupffer cell pigmentation was highly significant (p < 0.001). The cause for hemosiderosis in all cases was the erythrocytic hemolytic effect of the compounds. The reason for the sex-increased susceptibility for development of hemangiosarcoma is unknown but may be due to a hormone-related, reduced antioxidative defensive capacity through modulation of the activities of antioxidative enzymes.

Inhalation toxicology and carcinogenesis studies of propylene glycol mono-t-butyl ether in rats and mice.

Propylene glycol mono-t-butyl ether (PGMBE) is used as a solvent in a variety of commercial applications. Male and female F344/N rats and B6C3F(1) mice were exposed to PGMBE by whole-body inhalation for 2 or 14 weeks (0, 75, 150, 300, 600, or 1200 ppm) or 2 years (0, 75, 300, or 1200 ppm); male NBR rats were exposed for 2 weeks. The kidney and the liver were targets of PGMBE toxicity in rats. Renal lesions suggestive of alpha(2u)-globulin nephropathy were observed in male F344/N, in the 2 and 14-week studies, no kidney lesions were seen in NBR rats. In the 2-year study, male rats displayed exposure-related nonneoplastic lesions in the kidney, and may have shown marginal increases in tubular neoplasms. In the liver, the incidences of hepatocellular adenomas occurred with a positive trend in male rats, and may have been related to PGMBE exposure. In mice of both sexes, the major target of PGMBE toxicity was the liver. In the 2-week study, liver weights and in the 14-week study, liver weights and the incidences of centrilobular hypertrophy were increased. In the 2-year study, the incidences of exposure-related hepatocellular adenoma, adenoma or carcinoma combined, and hepatoblastoma occurred with a positive trend, and were significantly increased in 1200 ppm groups. In summary, exposure to PGMBE resulted in nonneoplastic lesions of the kidney characteristic of alpha(2u)-globulin nephropathy, and may have increased renal tubular neoplasms in male rats. Exposure to PGMBE also produced increases in hepatic tumors in male and female mice.