Interim Sacrifices Research Articles

Subchronic and chronic safety studies with genistein in dogs

Genistein is a phytoestrogen that occurs naturally in the diet, especially in soy-based foods. There is widespread interest in phytoestrogens as chemopreventive agents for a variety of diseases and cancers based on epidemiologic evidence. Although soy and its constituents, such as genistein, have been consumed at high levels in several Asian populations without apparent adverse effects, concern has been raised about potential adverse effects due to estrogenic and other activities. The subchronic and chronic safety of genistein were evaluated in the beagle dog including a 4-week study and a 52-week safety study with a 13 week interim sacrifice and a 4 week recovery period. In both studies at doses of 50, 150 and 500 mg/kg/day, genistein was well tolerated. In the 4 week study, except for an increase in uterine weights in female dogs at 500 mg/kg/day, there were no other treatment related findings. In the 52-week study, the primary effects of genistein were observed on the reproductive tract, which included for male dogs: reduced size and/or weight of the testes, epididymus and prostate of 2/2 dogs after 13 weeks of treatment and in 1/4 dogs after 52 weeks of treatment at 500 mg/kg/day. The histological changes observed in the affected dogs at 500 mg/kg/day indicated atrophy of the testes and prostate gland and absent spermatozoa in the epididymus. At the mid-dose of 150 mg/kg/day, although there was a reduction to a lesser extent in testes weight after 13, but not 52 weeks, there were no histopathological changes. In female dogs, the reproductive tract effects included increased uterine weight at 500 mg/kg/day after 13 weeks of treatment, but not after 52 weeks of treatment. There was also a small decrease in ovarian weights at 150 and 500 mg/kg/day after 13 weeks and at 500 mg/kg/day after 52 weeks of treatment. There were no histopathological correlates to the changes in organ weights in female dogs. In the 4-week recovery group dogs, no changes were observed in dogs previously treated for 52 weeks with 500 mg/kg/day of genistein. It is concluded that the administration of genistein to dogs for a period of 4–52 weeks was well tolerated and did not result in systemic toxicity. Effects of genistein on the reproductive tract at very high doses were functional in nature and are of a type that would be expected in view of the relatively weak estrogenic activity of genistein and were considered not adverse effects. In the 4-week study, the no observed adverse effect level (NOAEL) for genistein was considered to be >500 mg/kg/day and the no observed effect level (NOEL) was considered to be 150 mg/kg/day. For the 52-week study, the NOAEL is considered to be >500 mg/kg/day and the NOEL is considered to be 50 mg/kg/day.

ACUTE, SUBCHRONIC AND CHRONIC TOXICITY STUDIES OF A SYNTHETIC ANTIOXIDANT, 2,2'-ISOBUTYLIDENEBIS(4,6-DIMETHYLPHENOL) IN RATS

General toxicity studies on 2,2'-isobutylidenebis(4,6-dimethylphenol)(IBBMP) were conducted using male and female Wistar rats. In the acute test, the oral LD50 values were 119 mg/kg BW in males and 103 mg/kg BW in females. Hypersensitivity, loss of righting reflex and abdominal position were observed. In the subchronic test, rats were fed a diet containing IBBMP at levels of 0, 20, 100 or 500 ppm for 13 weeks with interim sacrifice at 4 weeks (equal to 0, 1.1, 5.5 or 27.9 mg/kg BW/day in males and 0, 1.1, 5.9 or 29.6 mg/kg BW/day in females). In both sexes, there were no changes in general condition, body weight gains and food intakes in all groups. No deaths were observed in all groups. Significant increase in AST was observed in 500 ppm males at Week 4. However, the change was not observed at Week 13. Slight but significant decreases in creatinine were also observed in 100 ppm females at Week 13 and 500 ppm males and females at Weeks 4 and 13. Total cholesterol (T-CHO) was significantly elevated in females of the 500 ppm group at Weeks 4 and 13. Absolute and relative liver weights were increased in 500 ppm of both sexes at Week 4. In females, the increases were also observed at Week 13. However, no remarkable histopathological findings were observed in all treated groups. In the chronic test, rats were fed a diet containing IBBMP at levels of 0, 100, 500 and 1500 ppm for 18 months with interim sacrifices at 6 and 12 months (equal to 0, 3.8, 19.4 or 59.4 mg/kg BW/day in males and 0, 4.3, 20.9 or 67.5 mg/kg BW/day in females). No remarkable changes in general appearance were observed in any rats. Body weight gains, food intakes and survival rates in all treated animals were comparable to those of the control. No remarkable changes in the hematological parameters were observed. T-CHO was significantly elevated in females of the 1500 ppm groups throughout the experiment. Significant increases or tendencies for increase in relative liver weights were observed in the 500 and 1500 ppm animals of both sexes. Increased incidences of swelling in liver cells were observed in 1500 ppm males at 6 months and 1500 ppm females at 12 and 18 months. At 18 months, dose-dependent increases in thickness of basement membrane of renal tubules and Bowman's capsule and cell infiltration to the interstitium of the kidney were observed in males. Significant increases of hyaline cast and basophilic change were also observed in 1500 ppm males. In females, increased incidences of hyaline cast were observed at 500 ppm and higher at 18 months. No other toxicity was apparent. No neoplastic lesions that could be attributed to IBBMP were observed in any organs of either sex. From the result of the chronic toxicity test, the no-observed-adverse-effect level (NOAEL) for IBBMP was concluded to be 100 ppm in the diet (4.26 mg/kg BW/day) in female rats on the basis of induction of hyaline cast in renal tubules at 500 ppm, whereas, in males, only a lowest-observed-adverse-effect level (LOAEL) was given as 100 ppm (3.84 mg/kg BW/day) on the basis of induction of thickening of basement membrane in renal tubules at 100 ppm.

Coalinga Chrysotile: A Short-Fibre, Amphibole-Free, Chrysotile: Part VI - Additional Evidence for Lack of Fibrogenicity

This report describes additional unpublished data that support the notion that Coalinga chrysotile is not fibrogenic. These data are from the same long-term, NTP-NIEHS, chronic chrysotile inhalation study that served as the basis for our earlier publications which concluded that Coalinga chrysotile was not fibrogenic due to its lack of biopersistence. Wagner-scored 12 and 24 month interim sacrifice data from the NTP-NIEHS investigation and an unpublished data set from the comparison MRC study both previously unreported, were recently found in the author’s files. The histology slides and attendant data should have been amongst the materials archived at the NTP but were not present at the time the author reviewed those files in 1995 and 1996. Review of these new materials showed that Coalinga chrysotile did not produce fibrosis either at 12 or 24 months in contrast to the two Canadian preparations which were both fibrogenic even at this early stage of the study. These new data and related testimony greatly strengthen our earlier conclusions and serve to refute earlier apparently contradictory reports. International and Governmental Reports support the conclusion reached in our papers that Coalinga chrysotile is not pathogenic. In fact, the only scientists to criticise our conclusions have been in litigation against Union Carbide.

Bayesian methods for a three-state model for rodent carcinogenicity studies.

The objective of a chronic rodent bioassay is to assess the impact of a chemical compound on the development of tumors. However, most tumor types are not observable prior to necropsy, making direct estimation of the tumor incidence rate problematic. In such cases, estimation can proceed only if the study incorporates multiple interim sacrifices or we make use of simplified parametric or nonparametric models. In addition, it is widely accepted that other factors, such as weight, can be related to both dose level and tumor onset, confounding the association of interest. However, there is not typically enough information in the current study to assess such effects. The addition of historical data can help alleviate this problem. In this article, we propose a novel Bayesian semiparametric model for the analysis of data from rodent carcinogenicity studies. We develop informative prior distributions for covariate effects through the use of historical control data and outline a Gibbs sampling scheme. We implement the model by analyzing data from a National Toxicology Program chronic rodent bioassay.

Extension of Peto's Test by Attribution of Tumor Lethality in the Absence of Cause‐of‐Death Information

AbstractA new statistical testing approach is developed for rodent tumorigenicity assays that have a single terminal sacrifice or occasionally interim sacrifices but not cause‐of‐death data. For experiments that lack cause‐of‐death data, statistically imputed numbers of fatal tumors and incidental tumors are used to modify Peto's cause‐of‐death test which is usually implemented using pathologist‐assigned cause‐of‐death information. The numbers of fatal tumors are estimated using a constrained nonparametric maximum likelihood estimation method. A new Newton‐based approach under inequality constraints is proposed for finding the global maximum likelihood estimates. In this study, the proposed method is concentrated on data with a single sacrifice experiment without implementing further assumptions. The new testing approach may be more reliable than Peto's test because of the potential for a misclassification of cause‐of‐death by pathologists. A Monte Carlo simulation study for the proposed test is conducted to assess size and power of the test. Asymptotic normality for the statistic of the proposed test is also investigated. The proposed testing approach is illustrated using a real data set.

Analysis of DNA adducts in rats exposed to pentachlorophenol.

Pentachlorophenol (PCP) is a widely used biocide that has been reported to be hepatocarcinogenic in mice. Its effects in rats are equivocal, but the liver clearly is not a target organ for carcinogenesis. The carcinogenic effects of PCP in mice may relate to reactive oxygen species generated during metabolism. PCP is known to increase the hydroxyl radical-derived DNA lesion, 8-oxodeoxyguanosine (ohdG), in the liver of exposed mice. To investigate whether the generation of oxidative DNA damage and direct DNA adducts may explain the species difference in carcinogenicity, we have analyzed ohdG in hepatic DNA from PCP-exposed rats. Rats were exposed acutely to PCP for 1 or 5 days. Tissues also were obtained from a 27 week interim sacrifice of the 2 year National Toxicology Program carcinogenesis bioassay. We used HPLC with electrochemical array detection for ohdG analysis. Single or 5 day exposure to PCP (up to 120 or 60 mg/kg/day, respectively) did not increase ohdG. Dietary exposure to 1000 p.p.m. PCP (equivalent to 60 mg/kg/day) for 27 weeks induced a 2-fold increase in ohdG (1.8 versus 0.91x10(-6) in controls). In parallel, formation of direct DNA adducts was analyzed by 32P-post-labeling following nuclease P1 adduct enrichment. We detected two major DNA adducts with relative adduct labeling of 0.78x10(7) adducts per total nucleotides. One of these adducts was found to co-migrate with the adduct induced by the metabolite, tetrachloro-1,4-benzoquinone. We observed differences in DNA adduct formation between acute and chronic studies, with acute studies not inducing any detectable amount of DNA adducts. These results indicated that chronic, but not acute exposure to PCP increased ohdG and direct adducts in hepatic DNA. As the same exposure conditions that enhanced ohdG did not produce liver cancer in rats, the generation of reactive oxygen species, oxidative DNA damage and direct DNA adducts is not sufficient for the induction of hepatocarcinogenesis by PCP in the rat.

CHRONIC INHALATION STUDIES OF TWO TYPES OF STONE WOOL FIBERS IN RATS

A summary is given of the pathology results after long-term inhalation in rats of insulation wool representing the new biosoluble types. The pathology results are compared with previously conducted long-term inhalation study with MMVF21 (traditional stone wool). The biosoluble fiber MMVF34/HT (HT) is characterized by a relatively high content of aluminum and a relatively low content of silica compared to the older MMVF21. HT has a high in vitro dissolution rate at pH 4.5, and a relatively low dissolution rate at pH 7.5. Male Fischer 344 rats were exposed at one exposure level of 30 mg/m3 by nose-only inhalation of a well-characterized fiber test atmosphere. The fibers had been size selected to be largely rat respirable. The negative control group was exposed to filtered air. The exposure duration was 6 h/day, 5 days/wk for 104 wk, with a subsequent nonexposure period lasting until approximately 20% survival in the air control group. Interim sacrifices were performed at wk 13, 26, 52, 78, and 104 to monitor the progression of pulmonary change and fiber numbers. Effectively the main protocol for the previously conducted chronic study with MMVF21 was the same, except that there were three concentration levels (3, 16, and 30 mg/m3). In addition to the endpoints measured in the previous study, slides from both studies were evaluated for collagen deposition using a quantitative morphometric method. The results of the comparative study clearly showed a marked difference in the pulmonary pathogenicity of the MMVF21 and HT in terms of their fibrogenic potential. MMVF21 caused pulmonary fibrosis, but the HT fiber did not. The incidence of tumors for both the HT and the MMVF21 fiber was comparable to the control groups.

Flexible Estimates of Tumour Incidence for Intermediately Lethal Tumours in a Typical Long-Term Animal Bioassay

SUMMARY The estimation of the incidence of tumours in an animal carcinogenicity study is complicated by the occult nature of the tumours involved (i.e. tumours are not observable before an animal’s death). Also, the lethality of tumours is generally unknown, making the tumour incidence function non-identifiable without interim sacrifices, cause-of-death data or modelling assumptions. Although Kaplan–Meier curves for overall survival are typically displayed, obtaining analogous plots for tumour incidence generally requires fairly elaborate model fitting. We present a case-study of tetrafluoroethylene to illustrate a simple method for estimating the incidence of tumours as a function of more easily estimable components. One of the components, tumour prevalence, is modelled by using a generalized additive model, which leads to estimates that are more flexible than those derived under the usual parametric models. A multiplicative assumption for tumour lethality allows for the incorporation of concomitant information, such as the size of tumours. Our approach requires only terminal sacrifice data although additional sacrifice data are easily accommodated. Simulations are used to illustrate the estimator proposed and to evaluate its properties. The method also yields a simple summary measure of tumour lethality, which can be helpful in interpreting the results of a study.

Bayesian Incidence Analysis of Animal Tumorigenicity Data

SUMMARY Statistical inference about tumorigenesis should focus on the tumour incidence rate. Unfortunately, in most animal carcinogenicity experiments, tumours are not observable in live animals and censoring of the tumour onset times is informative. In this paper, we propose a Bayesian method for analysing data from such studies. Our approach focuses on the incidence of tumours and accommodates occult tumours and censored onset times without restricting tumour lethality, relying on cause-of-death data, or requiring interim sacrifices. We represent the underlying state of nature by a multistate stochastic process and assume general probit models for the time-specific transition rates. These models allow the incorporation of covariates, historical control data and subjective prior information. The inherent flexibility of this approach facilitates the interpretation of results, particularly when the sample size is small or the data are sparse. We use a Gibbs sampler to estimate the relevant posterior distributions. The methods proposed are applied to data from a US National Toxicology Program carcinogenicity study.

Chronic study of diacylglycerol oil in rats

The objective of the present study was to evaluate the effects of diacylglycerol oil following long-term administration to rats. Diacylglycerol oil is an edible oil with comparable taste and physicochemical properties of several naturally occurring oils. Diacylglycerol oil can be used as a replacement for any generally used edible oil in the home and has been approved for use in cooking oil in Japan. Male and female Sprague–Dawley rats were divided into four groups and fed low-fat (1.7%) basal diets containing an edible oil composed of rapeseed, corn, high linoleic safflower and high oleic safflower oils at 5.3% (control group 1); an edible oil composed of rapeseed and soybean oils at 5.3% (control group 2); diacylglycerol oil at 2.65% plus edible oil composed of rapeseed, corn, high linoleic safflower and high oleic safflower oils at 2.65% (low-dose group); and diacylglycerol oil at 5.3% (high-dose group) for 2 years. Interim sacrifices were conducted at weeks 30 and 77 and the study was terminated following 105 weeks of feeding. No compound-related effects were noted on clinical signs, body weights, food consumption, cumulative survival rates, hematology, blood chemistry, urinalysis, organ weights or on microscopic non-neoplastic changes. Compared to control group 2, but not control group 1, there was a significant increase in the number of high-dose group females with either benign or malignant epithelial mammary gland neoplasms. These changes were not considered biologically significant, because the tumor incidence was not similar in control group 1 and 2, and the neoplastic findings were not dose related. In summary, the two-year chronic rat study revealed no toxicologically significant or treatment-related effects of diacylglycerol oil consumption at levels of up to 5.3% in the diet.

Mechanisms of extrahepatic tumor induction by peroxisome proliferators in male CD rats.

Wyeth-14,643 (WY) and ammonium perfluorooctanoate (C8) belong to a diverse class of compounds which have been shown to produce hepatic peroxisome proliferation in rodents. From previous work, WY, but not C8, has been shown to produce hepatocellular carcinoma in rats, while C8 has been shown to produce Leydig cell adenomas. In addition, based on a review of bioassay data a relationship appears to exist between peroxisome-proliferating compounds and Leydig cell adenoma and pancreatic acinar cell hyperplasia/adenocarcinoma formation. To further investigate the relationship between peroxisome-proliferating compounds and hepatic, Leydig cell, and pancreatic acinar cell tumorigenesis, a 2-year feeding study in male CD rats was initiated to test the hypothesis that peroxisome proliferating compounds induce a tumor triad (liver, Leydig cell, pancreatic acinar cell), and to examine the potential mechanism for the Leydig cell tumors. The study was conducted using 50 ppm WY and 300 ppm C8. The concentration of WY in the diet was decreased to 25 ppm on test day 301 due to increased mortality. In addition to the ad libitum control, a second control was pair-fed to the C8 group. Interim sacrifices were performed at 1- or 3-month intervals. Peroxisome proliferation measured by beta-oxidation activity and cell proliferation were measured in the liver and testis at all time points and in the pancreas beginning at the 9-month time point (cell proliferation only). Serum hormone concentrations (estradiol, testosterone, LH, FSH, and prolactin) were also measured at each time point. Increased relative liver weights and hepatic beta-oxidation activity were observed in both the WY- and C8-treated rats at all time points. In contrast, hepatic cell proliferation was significantly increased only in the WY-treated group. Neither WY nor C8 significantly altered the rate of Leydig cell beta-oxidation or Leydig cell proliferation when compared to the control groups. Moreover, the basal rate of beta-oxidation in Leydig cells was approximately 20 times less than the rate of hepatic beta-oxidation. There were no biologically meaningful differences in serum testosterone, FSH, prolactin, or LH concentrations in the WY- and C8-treated rats when compared to their respective controls. There were, however, significant increases in serum estradiol concentrations in the WY- and C8-treated rats at 1, 3, 6, 9, 15, 18, and 21 months. At 12 months, only the C8-treated rats had elevated serum estradiol concentrations when compared to the pair-fed control. Histopathological evaluation revealed compound-related increases in liver, Leydig cell, and pancreatic acinar cell tumors in both WY- and C8-treated rats. The data support the hypothesis that the peroxisome-proliferating compounds induce the previously described tumor triad. In addition, both C8 and WY produced a sustained increase in serum estradiol concentrations that correlated with the potency of the 2 compounds to induce Leydig cell tumors (i.e., WY caused a more consistent sustained increase in serum estradiol throughout the entire study, and more specifically at the end of the study, than did C8). This study suggests that estradiol may play a role in enhancement of Leydig cell tumors in the rat, and that peroxisome proliferators may induce tumors via a non-LH type mechanism.

CHRONIC INHALATION STUDIES OF TWO TYPES OF STONE WOOL FIBERS IN RATS

A summary is given of the pathology results after long-term inhalation in rats of insulation wool representing the new biosoluble types. The pathology results are compared with previously conducted long-term inhalation study with MMVF21 (traditional stone wool). The biosoluble fiber MMVF34/HT (HT) is characterized by a relatively high content of aluminum and a relatively low content of silica compared to the older MMVF21. HT has a high in vitro dissolution rate at pH 4.5, and a relatively low dissolution rate at pH 7.5. Male Fischer 344 rats were exposed at one exposure level of 30 mg/m3 by nose-only inhalation of a well-characterized fiber test atmosphere. The fibers had been size selected to be largely rat respirable. The negative control group was exposed to filtered air. The exposure duration was 6 h/day, 5 days/wk for 104 wk, with a subsequent nonexposure period lasting until approximately 20% survival in the air control group. Interim sacrifices were performed at wk 13, 26, 52, 78, and 104 to monitor the progression of pulmonary change and fiber numbers. Effectively the main protocol for the previously conducted chronic study with MMVF21 was the same, except that there were three concentration levels (3, 16, and 30 mg/m3). In addition to the endpoints measured in the previous study, slides from both studies were evaluated for collagen deposition using a quantitative morphometric method. The results of the comparative study clearly showed a marked difference in the pulmonary pathogenicity of the MMVF21 and HT in terms of their fibrogenic potential. MMVF21 caused pulmonary fibrosis, but the HT fiber did not. The incidence of tumors for both the HT and the MMVF21 fiber was comparable to the control groups.

Single-Dose Oral Toxicity Study of a Cross-linked Sodium Polyacrylate/Polyvinyl Alcohol Copolymer in Chickens (Gallus domesticus)

A single-dose oral toxicity study of a grafted copolymer of cross-linked sodium polyacrylate with polyvinyl alcohol was conducted in chickens (Gallus domesticus) to demonstrate this copolymer's safety for use as a hydration medium in recently hatched poultry chicks. Three experimental groups, each composed of 25 male and 25 female 1-day-old chicks, were administered a one-time dose of 0, 3, or 6 g of the hydrated test article by gavage. All chicks were monitored daily for mortality and morbidity during their typical grow-out period of approximately 8 weeks. Interim sacrifices of one animal of each sex from each treatment group were made at 1, 2, 4, and 6 weeks. All surviving animals were necropsied at the end of the study. A single dose of up to 6 g of hydrated copolymer did not adversely affect these animals during their grow-out period. Mortality was comparable across all experimental groups, as no statistically significant survival differences were found. Body weights were also comparable across the three experimental groups at all time points during the study, and no statistically significant differences were detected in mean terminal body weights among the groups. Finally, lesion frequencies were similar across the three experimental groups, with none of the lesions deemed related to administration of the test article. Thus, the safety of this cross-linked sodium polyacrylate/polyvinyl alcohol copolymer has been demonstrated for its intended use.

Chronic Toxicity and Carcinogenic Potential of Tris-(1,3-Dichloro-2-propyl) Phosphate in Sprague-Dawley Rat

The Flammable Fabrics Act of 1953 and its amendments established a need for flame-resistant fabrics. Tris-(1,3-dichloro-2-propyl) phosphate (TDCPP) was briefly used in apparel fabrics to assist in the compliance with federal flammability standards, and continues to be used as a flame retardant in flexible polyurethane foams. A chronic toxicity and carcinogenicity bioassay was conducted in Sprague-Dawley rats to determine the toxicologic and carcinogenic potential of TDCPP after repeated exposure. Four groups of animals, each consisting of 60 male and 60 female rats, received via their diet a daily dose of either 0, 5, 20, or 80 mg TDCPP per kg body weight for up to 24 months. Diets were adjusted after each weekly (first 13 weeks) or biweekly (weeks 14 through 104) body weight and food consumption measurement to achieve and maintain the indicated doses. Ten animals per sex were taken from each group for interim sacrifice at the end of the 12th month. Body weights, food consumption, clinical signs, and hematological and clinical chemistry parameters were measured periodically, and ophthalmoscopic examinations were conducted on all animals. After complete postmortem examination of all animals, microscopic examination of all tissues was conducted for the control and high-dose animals. Liver, kidneys, testes, and adrenal glands were examined from all animals. Mortality was significantly higher and body weights were significantly lower in the high-dose group when compared to control animals. Certain hematology parameters, such as hemoglobin, hematocrit, and total erythrocyte values, were decreased in the high-dose animals. Ophthalmoscope examination revealed no treatment-related changes. Microscopic examination revealed a higher incidence of benign neoplasms and non-neoplastic alterations in several organs of the mid and high dose animals. The no-observed-adverse-effect level (NOAEL) for chronic toxicity and neoplastic activity was the dietary dose of 5 mg/kg/day.

Retrospective evaluation of alpha 2u-globulin accumulation in male rat kidneys following high doses of diisononyl phthalate.

Diisononyl phthalate (DINP), a widely used plasticizer, has been evaluated in two chronic studies in rats and one in mice. In the early 1980s, Exxon found no carcinogenic potential at the estimated maximum tolerated dose (MTD) of 0.6% (307 mg/kg/ day for male rats) administered in the diet of rats for 2 years. A recent study conducted at dietary levels up to 1.2% DINP (733 mg/kg/d for male rats) reported kidney tumors in male rats at the high treatment level, but not in female rats nor mice of either sex. Because these tumors occurred only in male rats, and only at high doses, the male rat-specific alpha 2u-globulin (alpha2UG) mechanism of action was investigated. Technological advances in immunohistochemical staining and computerized image analysis techniques permitted measuring the accumulation of alpha2UG in archived kidneys from the earlier Exxon study. Using archived tissue obtained at the 12-month interim sacrifice, we identified a dose-dependent accumulation of alpha2UG in specific regions of male rat kidneys only. An increase in cell proliferation was confirmed by immunohistochemical detection of proliferating-cell nuclear antigen (PCNA) and was confined to the areas of alpha2UG accumulation. H and E-stained sections revealed tubular epithelial hypertrophy and regeneration, consistent with the immunohistopathology findings. These findings are consistent with the alpha2UG mechanism of tumorigenesis, which is not regarded as relevant for humans. Thus, exposure to DINP produced a dose-dependent alpha2UG accumulation in male rat kidneys, significant at a dietary level of 0.6% and a likely mechanism for the kidney tumors seen only in male rats administered higher dietary levels of DINP.

Chronic Toxicity and Carcinogenicity Studies of Ethylenediamine Dihydrochloride by Dietary Incorporation in Fischer 344 Rats

Ethylenediamine dihydrochloride (EDA·2HCl) was incorporated into the diet and fed to Fischer 344 rats for 2 years at target doses of 0, 0.02, 0.10 or 0.35 g/kg/day (equivalent to 0.009, 0.045 and 0.158 g free EDA/kg/day). Two separate untreated control groups were used. Interim sacrifices were at 6, 12 and 18 months and the terminal sacrifice was at 24 months. Under the conditions of this study, EDA·2HCl was not carcinogenic in the Fischer 344 rat. Most toxic responses were observed at the 12-month sacrifice and thereafter. Reductions in mean body weight gain were observed in high dose group male rats throughout most of the study and in the high dose group of female rats after approximately 18 months. Conversely, there was a slight increase in the mean body weight gain for the medium level female rats from about day 21 until 21 months that was of equivocal biological significance. Increased mortality was observed in the high dose group of both sexes and the mid dose group of female rats. The cause of the decreased survival was unclear, but may have been related to the enhancement of background degenerative lesions such as chronic nephropathy. Throughout the study, male rats from the high dose group had decreased erythrocyte counts, haemoglobin concentration and haematocrit. The cause and biological significance of the haematological changes were unknown. Increased water consumption was observed in the high dose group of both sexes during the latter half of the study. Increased urine volume with concurrent decreased urine specific gravity was generally observed in the high dose group of both sexes in the last half of the study and suggested a possible alteration in kidney function. Altered urine volume and specific gravity persisted to termination in female rats only. Slight increases in absolute and relative kidney weights were also observed in the high dose group of female rats during the latter half of the study. Hepatocellular pleomorphism was observed in the high dose group of both sexes, especially the female rats, and may have contributed to increased mean liver weights observed primarily in female rats from the high dose group. Hepatocellular pleomorphism was first observed in female rats at 12 months but was not observed in male rats until the final sacrifice. Rhinitis and tracheitis were observed with greater frequency in the high dose group of male rats at 12, 18 and 24 months and in high dose group female rats at 18 months. At 24 months, rhinitis, but not tracheitis, persisted at a significantly greater frequency in high dose group female rats. The apparent no-observable-effect level (NOEL) of this study was at the lowest dose level, 0.02 g/kg/day (equivalent to 9 mg EDA/kg/day).

Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile.

Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.

The biopersistence and pathogenicity of man-made vitreous fibres after short- and long-term inhalation.

A summary is given of the biopersistence and pathology after inhalation by rats of two different Man-made Vitreous Fibres, MMVF21 (traditional stone wool) and MMVF34 (HT stone wool), and the results are discussed in relation to biopersistence measured after intra-tracheal instillation. The results are given from a short-term inhalation biopersistence study, a completed chronic inhalation study, and interim results from an on-going chronic inhalation study. In both the short-term and chronic studies, laboratory rats were exposed by nose-only inhalation to well-characterised fibre test atmospheres that had been selected to be largely rat respirable. The short-term inhalation study included groups exposed to aerosols targeted at 150 fibres longer than 20 microns per cm3. The exposure duration was 6 hours/day for 5 days, with subsequent post-exposure periods lasting up to 12 months. For lung burden analyses, interim sacrifices were performed at regular intervals. The ongoing chronic study comprises a group of rats exposed to the MMVF34 fibre at one exposure level of 30 mg/m3. The negative control group is filtered air. The exposure duration is 6 hours/day, 5 days/week for 2 years, with a subsequent post-exposure period lasting until approximately 20% survival in the test fibre group. Interim sacrifices are performed at months 3, 6, 12, 18 and 24 and biopersistence monitored for rats exposed for 3 and 12 months, with subsequent post-exposure periods lasting 6 months. Effectively the main protocol for the previously conducted chronic study was the same, except that there were 3 fibre exposure groups (3, 16 and 30 mg/m3) and no specific biopersistence satellite groups were included. For MMVF34, the inhalation tests of different duration show a similar biopersistence pattern, while the intra-tracheal test gives longer elimination half-times especially for long fibres. The MMVF34 fibre is considerably less biopersistent than the traditional MMVF21 fibre when comparing the calculated elimination half-times after short-term inhalation. When comparing the pathology after 3, 6, 12 and 18 months exposure, MMVF34 showed minor histopathological changes compared to MMVF21. The carcinogenicity and toxicity results of the chronic study with MMVF21 suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently should pose an even smaller risk, if any.

The Biopersistence and Pathogenicity of Man-made Vitreous Fibres after Short- and Long-term Inhaltion

A summary is given of the biopersistence and pathology after inhalation by rats of two different Man-made Vitreous Fibres, MMVF21 (traditional stone wool) and MMVF34 (HT stone wool), and the results are discussed in relation to biopersistence measured after intra-tracheal instillation. The results are given from a short-term inhalation biopersistence study, a completed chronic inhalation study, and interim results from an on-going chronic inhalation stiuly. In both the short-term and chronic studies, laboratory rats were exposed by nose-only inhalation to well-characterised fibre test atmospheres that had been selected to be largely rat respirable. The short-term inhalation study included groups exposed to aerosols targeted at 150 fibres longer than 20 μm per cm3 The exposure duration was 6 hours/day for 5 days, with subsequent post-exposure periods lasting up to 12 months. For lung burden analyses, interim sacrifices were performed at regular intervals. The ongoing chronic study comprises a group of rats exposed to the MMVF34 fibre at one exposure level of 30mg/m3 The negative control group is filtered air. The exposure duration is 6 hours/day, 5 days/week for 2 years, with a subsequent post-exposure period lasting until approximately 20% survival in the test fibre group. Interim sacrifices are performed at months 3, 6, 12, 18 and 24 and biopersistence monitored for rats exposed for 3 and 12 months, with subsequent post-exposure periods lasting 6 months. Effectively the main protocol for the previously conducted chronic study was the same, except that there were 3 fibre exposure groups (3, 16 and 30 mg/m3 and no specific biopersistence satellite groups were included. For MMVF34, the inhalation tests of different duration show a similar biopersistence pattern, while the intra-tracheal test gives longer elimination half-times especially for long fibres. The MMVF34 fibre is considerably less biopersistent than the traditional MMVF2I fibre when comparing the calculated elimination half-times after short-term inhalation. When comparing the pathology after 3, 6, 12 and 18 months exposure, MMVF34 showed minor histopathological changes compared to MMVF2I. The car cinogenicity and toxicity results of the chronic study with MMVF2I suggest that this fibre does not pose a significant health risk to humans and the current results with MMVF34 indicate that this fibre consequently should pose an even smaller risk, if any. © 1998 British Occupational Hygiene Society. Published by Elsevier Science Ltd.

Oral and Dermal Administration of Clarified Slurry Oil to Male C3H Mice

Clarified slurry oil (CSO) is the heavy residual material produced in the fluidized catalytic cracking unit of a petroleum refinery. CSO was adm in istered via gavage or dermal application to male C3H mice (10/ group) at a dose of 1000 mg/ kg/ d, 5 d/ wk, for up to 10 weeks. Interim sacrifices were performed after 2, 4, 8, and 10 weeks of treatment for orally exposed and control mice; dermally exposed mice were sacrificed after 10 weeks. Fifty percent of the “dermal” mice died prior to scheduled sacrifice. Deaths observed in the “oral” group were attributed to apparent misintubations, not to CSO exposure. Mice exposed orally for 2, 4, or 8 weeks (but not 10 weeks) exhibited significantly increased absolute (A) and relative (R) liver weights, while all “oral” mice (including those sacrificed after 10 weeks) had significantly decreased A and R thymus weights when compared to controls. “Dermal” mice had significantly increased R liver weights and significantly decreased A and R thymus weights. After 2 weeks most “oral” mice showed hepatocyte hypertrophy. A few mice showed liver necrosis. Liver findings increased in incidence and severity until 8 weeks of exposure. After 10 weeks, the liver of most “oral” mice showed the same or less discern ible morphologic pathology than was observed after 2 weeks. At 10 weeks, “dermal” mice showed the previously described liver effects, except that liver necrosis was severe, and evidence of fibrosis was observed. In summary, although CSO was hepatotoxic, “oral” mice showed signs of liver recovery after 10 weeks of exposure despite continued treatment. Thus, CSO appears to be more toxic to mice when administered dermally than orally. Humans are more likely to come into contact with these lipids by dermal rather than by oral exposure.