Document n9za7REaKvkL5QojpvnDqN6BR

P"0^JS HATr rt.r!,'U:L Hay * :r f Em'ironmenlal Health Pcrs/n cincs Vol. 53. pp. 27--N. 19S3 Chronic Effects of Dietary Exposure to Amosite Asbestos and Tremolite in F344 Rats by Ernest E. McConnell,* Henry A. Rutter,* Borge M. Ullandf and John A. Moore* Carcinogenesis bioassays of blocky (nonfibrous) tremolite and amosite asbestos alone or in combination with the intestinal carcinogen 1,2-dimethyIhydrazine dihydrochloride (DMH) were conducted with male and female Fischer 344 rats. The minerals were administered at a concentration of 1C1 in pelleted diet for the entire lifetime of the rats starting with the dams of the test animals. One group of amosite rats also received chrysotile asbestos via gavage during lactation. Group sizes varied from 100 to 250 animals. The offspring from mothers exposed to tremolite or amosite asbestos were smaller at weaning than those from untreated mothers and remained smaller throughout their life. The administration of dimethylhydrazine (DMH) did not affect body weight gain, either in amosite-exposed or nonexposed animals. Survival was comparable in the tremolite and control groups. The amosite-exposed rats showed enhanced survival compared to the untreated controls. DMH exposure reduced survival by approximately one year, al though the amosite plus DMH groups survived slightly better than the DMH alone groups. No toxicity or increase in neoplasia was observed in the tremolite-exposed rats com pared to the controls. Significant increases (p < 0.05) in the rates of C-cell carcinomas of the thyroid and monocytic (mononuclear cell) leukemia in male rats were observed in amosite-exposed groups. However, the biological significance of the C-cell carcinomas in relation to amosite asbestos exposure is discounted because of a lack of significance when C-cell adenomas and carcinomas were combined and the positive effect was not observed in the amosite plus preweaning gavage group. The biological significance of an increased incidence of mononuclear cell leukemia is questionable, because of a lack of statistical significance in the amosite group when evaluated using life table analysis, lack of significance when compared to the tremolite control group, and the fact that no toxic or neoplastic lesions were observed in the target organs, i.e., gastrointestinal tract and mesothelium. DMH caused a high rate of (62-74^) of intestinal neoplasia in amosite and nonamositeexposed groups. Neither an enhanced carcinogenic nor protective effect was demon strated by exposure to amosite asbestos. Introduction In November 1973 the National Institute of Environmental Health Sciences and the Environ mental Protection Agency cosponsored a sympo sium on the possible biological effects of ingested asbestos. (7). This conference concluded that a Paucity of definitive data existed concerning the The National Tbxicology Program, National Institute of - "ironmental Health Sciences, P.0 Box 12233, Research VJBle Park, NC 27709. 1 eJton Laboratories America, Inc., 9200 Leesburg Pike, nnh- V . 2218C. effects of ingested asbestos and that specific re search was needed. A subcommittee of the DHEW Committee to Coordinate Toxicology and Related Programs was established to review existing data and to prepare a draft research protocol that would be responsive to the possible public health implication of in gested asbestos. This protocol was widely distrib uted for comment within and outside the govern ment and a public meeting of the subcommittee was held on February 11, 1975. On the basis of the comments received, a revised final protocol was developed which called for the use of long- 10002986 ? * 28 SfcCOXS'ELL ET AL term animal toxicology studies to evaluate the ingestion of several minerals for carcinogenic ef fect. As a result, the National Toxicology Program has investigated the carcinogenic potential of the ingestion of chrysotile asbestos in hamsters and rats, amosite asbestos in hamsters and rats, cro- Table 1. Fiber characteristics of amosite asbestos. Fiber characteristics Surface area, m2(g 4.13 Density, gcm3 3.35 = 0.026 SD Measurements, transmission electron microscopy Fiber count'g 0.3466 x 1010 Median length (pm) 4.37 Range of length, pm 0.85 - 995 Median diameter, pm 0.72 Range of diameter, pm 0.064 - 12.4 Median fiber aspect ratio Kid) 6.4248 Table 2. Chemical-instrumental analysis of amosite asbestos. AI2O3 CaO FeO Fe203 MgO K20 Si02 Na20 MnO Cr203 NiO C02 h2oh2oBenzene extracted organics Content, wt-9 0.42 0.48 34.61 2.24 6.22 0.30 50.36 0.03 2.66 0.03 0.01 0.88 0.15 2.30 0.021 cidolite asbestos in rats and tremolite in rats. All of the studies were to encompass the lifetime of the animal, including exposure of the dams from which the test animals were derived. Crystalline tremolite (not actually in asbestos fiber) was chosen for this study because up to 20 years ago it was a common contaminant of talc which was used in foods and pharmaceuticals. The grinding of tremolite in preparation for its intended use may result in the production of fibers which have the morphology of asbestos minerals. Stanton et al. (2), in reviewing intra pleural mineral deposition studies, speculated that the asbestos mineral hazard question may be directly related to fiber size in contrast to chemi cal composition. Therefore, the study of crystal line tremolite was deemed appropriate because of its past widespread exposure and the fact that it assumes fiber characteristics when ground in the processing of talc. This report represents the results of those stud ies undertaken to determine the effects of tremo lite or amosite asbestos in the diet fed to Fischer 344 rats. In addition, the study was designed to determine ifthe feeding of amosite asbestos modi fied tbe response of a known intestinal carcino gen, 1,2-dimethlhydrazine dihydrochloride (DMH). Reports on chrysotile and crocidolite as bestos will be reported later. Materials and Methods Test Materials Asbestos is a general term applied to certain natural silicates when they appear in a fibrous Table 3. Particle size distribution of amosite asbestos by particle number: SEM. Length interval, pm 0-1.99 2-3.99 4-5.99 6-7.99 8-9.99 10-19.99 20-39,99 Amosite mean width, pm Amosite particles per interval Tbtal amosite particles, Cumulative amosite Amosite, vol-*5* Cumulative volume-'* amosite Number of other particles Amosite particles per length interval by aspect ratio, <* 1:1--2.9:1 3:1-4.9:1 5:1-9.9:1 10:1-19.9:1 20:1-49.9:1 50:1-99.9:1 100:1-199:1 200:1-499:1 >500:1 0.28 57 5.6 5.6 - - 11 12 34 43 11 0 0 0 0 0 0.38 126 12.3 17.9 0.1 0.1 8 0 10 52 34 4 0 0 4 0 0.45 88 8.6 26.5 0.3 0.4 1' 0 6 23 52 18 1 0 0 0 0.45 78 7.6 34.1 0.4 0.8 0 0 5 14 38 41 2 0 0 0 0.48 52 5.1 39.2 0.4 1.2 1 0 2 4 40 54 0 0 0 0 0.52 181 17.7 56.9 2.4 3.6 1 0 0 1 21 64 12 2 0 0 0.51 184 18.0 74.9 5.0 8.6 0 0 0 1 1 30 55 12 1 0 Calculated from particle number data, assuming rectangular cross section with third dimension equal to 1/2 measured width. 0002987 EFFECTS IS RATS OF DIETARY EXPOSURE TO ASBESTOS 29 form. Amosite is a fibrous member of the amphi- bole mineral group, its chemical structure is (Fe2*Mg)7Si8022 (OH)j. Mineral and fiber charac teristics of amosite are shown in Tables 1-3. The amosite sample identified as S-33 was pur chased by the Bureau of Mines from the Atlas Asbestos Company, Montreal, Quebec, Canada. This material is from a mine in the area known as Penge, in the TVansvaal, Republic ofSouth Africa. Not a proper mineral name, amosite is a term used to describe the material from asbestos mines Table 4. Fiber characteristics and chemical-instrumental analysis of tremolite. Fiber characteristics Surface area, m2 g Density, g cm3 Analyses, wt-fr AI2O3 CaO Fe203 MgO K20 Si02 Ka.,0 Ti02 MnO LijO SnO SrO Bi203 co2 h2oh2oBenzene-extracted organics 5.2 = 0.5 2.91 = 0.01 SD 1.57 11.26 0.27 26.71 0.18 54.00 0.80 0.03 0.05 0.02 0.01 0.03 0.01 0.78 0.24 3.73 0.003 in South Africa. To develop homogeneity of the sample the amosite was processed by a single pass through an air jet mill. The temolite sample used in this study was obtained from a single lense from the Governeur Thlc Company, Governeur, NY. This 1200-lb lense was taken from the 500 ft level, American vein, No. 4 footwall stope, lower portion of the footwall bedding. The lense was crushed in a Denver Jaw Crusher and then to minus 14 mesh in a roll crusher. This material was then wheeler-milled at 204C and bagged in 50-lb Kraft bags. The final particle size was nominal minus 325 mesh. Tb develop homogeneity of the sample, approxi mately 960 lb tremolite was blended in a 10 ft3 Vtype blender. Mineral and fiber characteristics of tremolite are shown in Tables 4 and 5. After final blending the samples were weighed to 25 2: 0.5 lb and placed in fiberboard drums. These drums were shipped to a special warehouse at Research Triangle Park, NC. Each drum re ceived a color marking unique to the mineral type. Homogeneity of the samples was verified by fluorescent X-ray spectrography for samples col lected from six randomly selected drums. No sig nificant differences were detected. The homogeneity of the samples and the physi cal and chemical properties of the materials were characterized by the Bureau of Mines, U.S. De partment of the Interior (Supt. of Documents No. I 28.23:8452) and by the Fine Particle Laborato ries, Illinois Institute of Technology Research In- Table 5. Particle size distribution for tremolite by particle number: SEM. Mean width, pm Number of particles per interval of all particles per interval Cumulative H of all particles Tremolite particles per interval 'f of tremolite particles Cumulative Ct tremolite Talc-serpentine particles per inti Other particles per interval Length interval, pm 0-0.99 1-1.99 2-2.99 3-3.99 4-4.99 5-0.99 6-6.99 7-7.99 8-8.99 9-9.99 >10 0.48 59 6.8 6.8 34 5.5 5.5 9 16 0.88 291 33.4 40.2 197 32.0 37.5 72 22 0.97 194 22.3 62.5 128 20.8 58.3 53 10 1.51 106 12.2 74.7 83 13.5 71.8 19 4 2.05 53 6.1 80.8 38 6.2 78.0 11 2 2.19 40 4.6 85.4 27 44 62.4 9 4 2.79 31 3.6 89.0 23 3.7 86.1 8 0 3.29 19 2.2 91.2 15 2.4 88.5 4 0 2.96 9 1.0 92.2 9 1.5 9.0 0 0 3.13 13 1.4 93.6 12 2.0 92.0 1 0 5.22 58 64 100 49 8.0 100 7 2 Tremolite particles Per length interval - aspect ratio, 1 1-29:1 3:1--*9.1 100 92 75 67 76 67 65 66 67 30 35 0 8 22 29 18 30 30 20 22 35 37 o:l-9.9:l 10:1-19.1 20:1--49:1 50 1-99:1 100:1-199 1 0 0 3 4 6 3 5 7 11 35 18 0 0 0 0 0 0 0 7 0 04 0 0 0 0 0 0 0 0 0 04 0 0 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 02 bLp> ala fr aspect ratio obtained from a second set of measurements particles = 871, total tremolite = 61 , total talc-serpentine = \93, and total other = 63. \oo 02?8 i ! \ ; V%". 30 MiCO.XXEIJ. ET At.. stitute, Chicago, IL (Special Report and Adden dum on project L6085, contract N01-ES-5-3157). Copies of these reports are available upon request from the National Toxicology Program. Test Diets The feed used was NIH-31 open formula rodent diet prepared by Zeigler Brothers, Inc., Gardner, PA. TYemolite or amosite asbestos was incorpora ted to a level of 1% by weight into the test diet. All feed was pelleted with a Sprout-Waldron pelleter; the pellets were of oval configuration, 3/e in. by 3/ in. in size. Pelleted feed was packaged in 25lb aliquots in standard paper feedbags which were color coded to minimize the occurrence of feeding errors at the test laboratory. Each lot of blended feed was analyzed for tremolite or amosite asbestos concentrations, pesti cide contamination and nutrient content. 19c Chrysotile (Medium RangeJ Gavage.* The required amount of chrysotile (medium range), a gray powder with lumps, was weighed on a Mettler balance and placed in a beaker. Sterile water (for injection) was added to obtain the desired concentration and the suspension was then mixed in a magnetic stirrer for a short period of time. The suspension was administered by gavage, at a dose level of 0.47 mg/g of body weight, to the amosite and preweaning gavage (PWG) animals from birth to weaning (21 days). Source and Specifications of Test Animals Parental Generation (FJ. Weanling Fischer 344 (caesarean-derived) rats, which were barrier sustained and specific pathogen-free, were pur chased from Charles River Breeding Laborato ries, Inc., Wilmington, MA. These animals consti tuted the F0 generation. On arrival, animals were taken directly to the quarantine area and acclimated to laboratory conditions for approximately 2 weeks. At 24 hr after the animals arrived, eight animals of each sex were selected, sacrificed, and pathogen bur den was determined for each animal. Pathogens examined for included ectoparasites, intestinal parasites, and bacteria. Serological tests were conducted for viruses. After approximately 2 months of quarantine the rats, both males and females, were randomized and divided into test groups by a computerized randomization process and placed on the appropriate designated diets. Animals were to receive 1^5 amosite, but were inadver tently gavaged with l*r chrysotile. After at least 7 days exposure to the appropri ate diets, the rats were placed in breeding cages (one male to two females). During the breeding period, the rats continued to be fed the designated diets; 20 days later (on the average), females were separated and housed individually in polycarbo nate cages (Hazleton Systems, Aberdeen, MD). Males were removed from the breeding cages and re-housed two per cage. Filial Generation (F,). The F0 females were allowed to deliver their Fj litters naturally, and these were reduced to no more than eight pups (four/sex ifpossible) per litter. At birth, the litters from the F0 dams within the control and treated groups were assigned randomly to the corre sponding lifetime feeding phase groups such fhat birth dates were equally distributed. All pups assigned to the amosite and preweaning gavage (PWG) groups were exposed to the PWG phase of the study to assure exposure to asbestos from birth to weaning. At 21 days after birth, the pups were weaned and given a temporary number, then selected, using a random number table, to be placed in their respective groups for the lifetime feeding study. Litters in which only one sex was present were excluded from those animals to be selected. The extra weanlings were discarded. At 8 weeks of age, 1,2-dimethylhydrazine dihy drochloride (DMH) was administered by gavage to a control group and an amosite group every 14 days for a total of five doses. Males received 7.5 mg/kg, and females 15.0 mg/kg, based on a pre vious pilot study (3) which showed that these doses produced an approximate incidence of 15% intestinal neoplasia. Concentrations of DMH in the dosing solutions were determined within one hour prior to dosing and following dosing. The results of these determinations showed that the proper concentration of DMH was present in the dosing solution and had not deteriorated during dosing. Animal Maintenance The control and mineral exposed rats were placed in separate rooms with monitored temper ature and humidity, and a controlled light cycle (12 hr light/12 hr dark). Temperature was main tained at 74 4F and humidity at 50% 10%. The rats were housed three per cage in polycarbo nate cages covered with nonwoven polyester filter sheets and stored on Enviro-racks. Racks and filters were changed approximately once every 2 weeks. Cages and bedding were replaced twice per week. Control and treated diets and tap water EFFECTS IX RATS OF DIETARY EXPOSVRE TO ASBESTOS 31 via automatic waterers were available ad libi tum. Two water samples were collected and sub mitted for asbestos analysis. Stainless steel feed containers were changed once every 2 weeks. The incoming air in the animal rooms was filtered to remove particulate matter. Ten to fif teen changes of room air per hour were provided. Prior to initiation of the study, air samples were collected and analyzed for baseline asbestos de terminations. Additional samplings were col lected approximately every 6 months for analysis to assure personnel safety. Other measures used for personnel protection included the wearing of fully protective disposa ble suits, gloves, boots and bouffant caps and the use of a dust/mist respirator mask. Personnel leaving the animal rooms were required to take showers. In addition, physical examinations, in cluding pulmonary function tests and chest radio graphs, were conducted at the initiation of the study, yearly thereafter, and at the end of the study. Clinical Examinations and Pathology Observations and Records. All animals were observed twice daily for moribund condition and mortality. Recorded weekly were individual body weights; signs of toxicity or pharmacologic ef fects; incidence, size and location of palpable tis sue masses or nodules; and food consumption per cage. Sacrifice and Gross Pathology. Animals were sacrificed when exhibiting any one of these condi tions: palpable masses within the abdominal cav ity (excluding retained testes); masses protruding from the rectum; rectal discharge of bright red fluid (an indication of the presence of a bleeding colonic or rectal neoplasm); large ulcerated masses in the area of the ears or on side of face (Zymbal gland tumors); large subcutaneous masses which have been ulcerated or infected; masses which interfere with breathing and eating or which severely hamper locomotion; huge tissue masses (>10 cm); central nervous system signs accompanied by weight loss (head tilt, circling incoordination, ataxia, paralysis); severe weight oss or emaciation; or comatose or very weak. When the remaining animals of either the con sol and DMH or the corresponding amosite and DMH group of either sex was reduced to 109c of those starting the study, both groups within that sex were killed. When survival or untreated con trol or amosite or amosite and PWG group of Either sex reached 109, all remaining animals of ese groups within that sex were killed. The tremolite-exposed groups were handled similarly. Animals were killed by exsanguination under sodium pentobarbital anesthesia (Nembutal, Ab bott Laboratories, Inc., North Chicago, IL, or Diabutal, Diamond Laboratories Inc., Des Moines, 10). Final body weights were recorded and necropsies performed which included these additional procedures: blood smears taken from animals sacrificed in extremis or terminally sacri ficed, touch preparations made from any enlarged spleen or lymphoid organ. Since the gastrointestinal tract was considered as the target organ prior to the study, it was handled in a manner slightly different from that in standard rodent lifetime bioassays. Prior to placement in fixative, the entire esophagus was opened and examined. The stomach and cecum were opened and pinned with the exterior surface adjacent to paper; 2-cm lengths of duodenum and ileum and two portions of jejunum were placed unopened in fixative. The remaining small intes tine was opened and washed gently with saline and the mucosal surface was then examined care fully using transillumination on a radiograph viewing box. Suspect lesions were processed sepa rately and identified individually as to location. Likewise, the entire colon with anus was opened, examined, and placed on cardboard (serosal sur face down) prior to fixation. The size and location of masses were recorded. Masses greater than 1 mm in diameter were removed as separate speci mens for processing. After fixation and prior to embedding, the colon was "carpet-rolled" starting at the proximal end, with the mucosal surface inward. All tissues were fixed in 109 neutral buffered formalin, sectioned, and stained with hematoxy lin and eosin. Tissues/organs examined micro scopically were: tissue masses, the above-men tioned portions of gastrointestinal tract, mesenteric, celiac, iliocolonic, renal, iliac, mandi bular, cervical, pancreatic and bronchial lymph nodes, mammary gland, salivary gland, thigh muscle, bone marrow (sternum), nasal cavity with turbinates, larynx, trachea, lungs and bron chi, heart, thyroid, parathyroid, liver, pancreas, spleen, kidneys, adrenal glands, urinary bladder, seminal vesicles/prostate, testes/epididymus, ova ries/uterus, brain, pituitary gland, eyes and spi nal cord. Data Recording and Statistical Methods toOOZ"0 The individual animal pathology data on this experiment were recorded in the computerized 32 MKOSXELL ET AL carcinogenesis bioassay data system. The data elements include descriptive information on the chemicals, animals, experimental design, clinical observations, survival, and individual pathologic results. Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (4). Animals were statistically censored as of the time that they died of other than natural causes or were missing; animals dying from natural causes were not statistically censored. Differences in sur vival were evaluated by Cox's (5) life table method. The incidence of neoplastic or nonneoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific ana tomic site (numerator) to the number of animals in which that site was examined (denominator). In most instances, the denominators included only those animals for which that site was exam ined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors) prior to histologic sam pling, or when lesions could have appeared at multiple sites (e.g., leukemia), the denominators consist of the numbers of animals necropsied. For the statistical analyses of tumor incidence data, two methods of adjusting for intercurrent mortality were employed. Each used the classical methods of combining contingency tables devel oped by Mantel and Haenszel (6). The first method of analysis assumed that all tumors of a given type were fatal; i.e., they caused the death of the animal, either directly or indirectly. Ac cording to this approach, the proportions of tu mor-bearing animals in the treated and control groups were compared at each point in time at which an animal died with a particular tumor. The denominators of these proportions were the total number of animals at risk in each group. These results were then combined by the MantelHaenszel methods to obtain an overall probability (p) value. This method of adjusting for intercur rent mortality is Cox's life table method (5). The second method of analysis assumed that all tumors of a give type were "incidental"; i.e., they were merely observed at autopsy in animal dying of an unrelated cause. According to this approach, the proportions of male and female rats found to have tumors in treated and control groups were compared in each of five time intervals: 0-60 weeks, 61-86 weeks, 87-112 weeks, 113-126 weeks and beyond 126 weeks. The denominators of these proportions were the number of animals actually autopsied during the time interval. The individual time interval-comparisons were then combined by the previously described methods to obtain a single overall result (7). For comparisons involving groups receiving DMH (which showed markedly reduced survival), somewhat shorter time intervals were utilized for the incidental tumor test: 0-52 Weeks, 53-78 weeks, 79-92 weeks, 93-116 weeks (males), 93-102 weeks (fe males), beyond 116 weeks (males) and beyond 102 weeks (females). In addition to these tests, one other set ofstatis tical analyses was carried out for each primary tumor: the Fisher exact test based on the overall proportion of tumor-bearing animals 1.8). All re ported p values are one-sided. Except where noted, the three alternative analyses gave simi lar results. Results Establishment of Test Groups The experiment was designed to evaluate the effects of orally ingested tremolite or amosite asbestos during the entire life of the animal, starting from the time the rats were able to eat solid food. For this reason, the mated female rats had been on the test diets for approximately 12 weeks when the first litters were born. To mini mize the chance that the mothers would reject or cannibalize their young, the litters were not han dled during lactation, except for the group receiv ing asbestos via preweaning gavage. Litter size and survivability of offspring were unaffected by the presence of amosite in the diets. The average number of live fetuses born to tremolite-exposed dams was 7.6 versus 7.8 for the con trol groups. The average number of live fetuses born to amosite-exposed dams was 8.5 versus 7.7 for the control groups. Significant mortality was induced in those pups which received the pre weaning asbestos gavage (PWG). The average size of the litters in this group was 3.4 at weaning compared to 7.5 in the non-PWG amosite group. The average weight at birth of the tremoliteexposed pups was 4.7 g versus 4.8 g for the con trols. The average weight at birth of the amositeexposed pups was 4.7 g versus 4.8 g for the controls. The tremolite-exposed offspring were slightly smaller at weaning, 22.8 g versus 26.3 g (control). The amosite-exposed offspring were also slightly smaller at weaning, 23.2 g versus 27.4 g (control). A summary of groups, number of animals and diets for the filial <Ft) animals is presented in Table 6. 10002991 EFFECTS IX EATS OF DIETARY EXPOSCRE TO ASBESTOS 33 Table 6. Summary of distribution and diets: a lifetime feeding study of tremolite or amosite asbestos in rats. Test group Control TVemolite No of animals DMH. mg kg* Male Female 9 of diet Male Female 118 118 250 250 0 1 Control DMH Amosite Amosite * DMH Amosite - PWGb 117 125 250 175 100 117 125 250 175 100 0_ 0 7.5 15.0 1 -1 7.5 15.0 1 -- Gavage with 1,2-dimethylhvdrazine dihydrochloride. bAnimal was inadvertently gavaged during preweaning with intermediate imedium i-range chrysotile instead of amo site. then paralleled the controls (except for DMHexposed rats) for the remainder of the study but the mineral-exposed rats remained smaller throughout the study. Both male and female DMH-exposed groups gained less than their re spective controls. In the tremolite-exposed males and females, the average weekly food consumption was 97*7 that of the untreated controls. In the DMH, amo site, amosite and DMH, and amosite and PWG males, the mean weekly food consumption was 102(7, 102(1, 105*7, and 107*7, respectively, com pared to the untreated control group and 98*7, 101*7, 105*7, and 108*7 that of the untreated control for comparable groups of females. Clinical Signs The incidence of clinical signs occurred at es sentially comparable frequencies throughout the study groups except those that received DMH (see below). No distinct signs of compound effect were noted in any of the tremolite- or amosite-treated animals during the first 52 weeks of study. As the study proceeded, the incidence of clinical signs increased among all the groups. At intervals where there were a large number of moribund sacrificed animals in any one particular group, the clinical signs most frequently observed were supportive of the conditions for moribund sacri fice previously outlined in the Methods section. A comparison of clinical signs observed during the same selected intervals among all the groups revealed a larger number of palpable abdominal masses, tissue masses, and central nervous sys tem signs, a well as red discharge and protruding masses from the rectum in the DMH and amosite and DMH groups. These findings were presum ably due to the administration of DMH since they were not clinically observed with any frequency in any of the tremolite- or amosite-treated groups. Body Weight and Food Consumption Mean body weights were analyzed at selected Intervals: birth, 3, 8, 11, 15, 24, 33, and 60 weeks for the males, and birth, 3, 8,11, 16, 27,48 and 60 weeks for the females by the method of Rao (9). `he data revealed a 13*7 depressed mean body weight gain at weaning in both sexes of the tre'nolite groups and 15*7 in the amosite groups compared to the controls. The depressed weight **ain in the tremolite- and amosite-exposed rats as> more apparent at 8 weeks of age itremo_u.e.33'.f for males and 17*7 for females; amosite: 1 ' for males and 25*7 for females). Weight gain Survival Survival data of intervals prior to the final sacrifice of a group are summarized in Table 7. There were no significant differences in survival between the tremolite-exposed and control groups. Survival of males and females was ap proximately equal until 112 weeks, after which the females tended to live longer. When compared to the survival rates of the untreated control group, the amosite male survival at 118 weeks w'as higher, while amosite and PWG male sur vival was somewhat less. In female rats, the amo site group survival was better than the untreated controls, while the amosite and PWG group was about the same. The survival of both groups of DMH-exposed rats was considerably less than the untreated controls. The amosite plus DMH group was comparable to the DMH alone group. Pathology There were no apparent treatment related neo plasms in the digestive tract of the tremolite, amosite, or amosite PWG groups (Tbbles 8 and 9). Also, no specific type was increased, either at a particular location (e.g., cecum) or in the stom ach, small or large intestine as a whole. In addi tion, the incidences of non-neoplastic diseases of the gastrointestinal tract such as enteritis, diver ticulitis, ulceration or inflammation in general were comparable in the control and tremolite- or amosite-exposed rate (Tables 10 and 11). There were no organs'tissues in the tremoliteexposed rats which showed an increased rate of neoplasia compared to the control groups. Organs which showed an increase in neoplasms in the amosite or amosite PWG groups compared to the control group were the thyroid and hematopoeitic system. The results are as follows. Thyroid. Tbble 12 summarizes the incidence 10002992 i 1 $ i .'j * 1 V; j j 1 \ ;l j ... ^; "s -..i I 34 .\h<V\SELL ETAL Tkble 7. Survival of F344 rats in lifetime oral asbestos study at various time points. Group Control H-emolite Control DMH 1% Amosite 19 Amosite * DMH 19 Amosite + PWG Age, weeks 106 120 146 148 106 120 146 148 106 118 142 146 106 118 142 146 106 118 142 146 106 118 142 146 106 118 142 146 No. alive total no. 98 118 70 118 6 118 " Males Survival, 9 83 59 5 - 206 250 150 250 36 250 - 82 60 14 - 95 117 71 117 7 117 - 81 61 6 - 27 125 16 125 _ - 22 13 _ - 221 250 117 250 35 249 - 88 71 14 - 46 175 26 175 _ - 26 15 _ - 77 100 52 100 6 100 - 77 52 6 - Females No. alive total no. Survival. ^ 97.118 71 118 20 118 14 118 82 60 17 12 207 247 144 247 33 246 22 246 84 58 13 9 92117 62 117 20 116 10.116 79 53 17 9 15125 - _ - 202 246 162246 43 245 28 245 12 - _ - 82 66 18 11 32 174 - - 86 100 56 100 15 100 9100 18 _ - 86 56 15 9 of thyroid C-cell proliferative lesions. A signifi cantly increased incidence of C-cell carcinoma was found in amosite-treated male rats (p < 0.05). This effect was not observed in amosite PWG male rats. Furthermore, the overall incidence of C-cell tumors (adenomas and/or carcinomas) was comparable between control and treated groups. C-cell hyperplasia was equivocally increased in amosite and amosite PWG female groups. Hematopoietic System. A significantly in creased incidence of mononuclear cell leukemia occurred in amosite (p < 0.05) and amosite PWG (p < 0.01) male rats (Ihble 13). However, neither group was significant when compared to the tremolite control group (399fr). This increased inci dence was not observed in amosite-exposed fe males. Miscellaneous Neoplasms Occasionally a somewhat higher or lower rate of commonly occurring neoplasms were observed in amosite treated groups. A statistically signifi cant (p < 0.05) decrease in the rate of neoplasia was observed in the pancreas (Islet cell adenoma), adrenal medulla (pheochromacytoma), thyroid (follicular cell carcinoma) and preputial gland in at least one group of amosite-exposed rats com pared to the controls. Similar observations were not observed in the tremolite-exposed groups. Nonneoplastic Findings A plethora of incidental lesions of aging Fischer 344 rats was found in all groups. Statistical anal yses showed no obvious correlation between the incidence of specific lesion types and the type of treatment. Nonneoplastic lesions that were ob served in more than 591 of the rats in any of the experimental groups are as follows: skin: epider mal inclusion cyst; lung: chronic inflammation (peribronchiolar and perivascular lymphoid cuff ing); spleen: fibrosis, hemosiderosis, extramedul lary hematopoeisis, lymphoid atrophy; lymph 10002993 EFFECTS IX RATS OF DIETARY EXPOSURE TO ASBESTOS 35 Table 8. Number of tremolite-exposed F344 rata with primary epithelial neoplasms of the alimentary tract Control Animals examined Total alimentary Oral pharvnx Papilloma Carcinoma Esophagus Tbtal gastrointestinal Total stomach Nonglandular Papilloma Carcinoma Glandular Polyp Carcinoma Tbtal small intestine Polyp Adca in polyp" Carcinoma Total large intestine Cecum Polyp Adca in polyp" Carcinoma Colon Polyp Adca in polyp" Carcinoma 118 8(71 1(1) 3(3) 4(3) 3(3) 2(2) 1(1) 0(0) 1(1) 1(1) `Values in parentheses are percentages. "Adenocarcinoma arising in adenomatous polyp. Males* TVemolite 250 12(5) 1(0) 1(0) 9(3) 2(11 1(0) 1(0) 3(1) 1(0) 2(1) 4(2' 1(0) 1(0' 2(1) Control 118 3(3) Females* TVemolite 250 7(3) 0(0) HO) 2(2) 5(2) 1(1) K0) 0(0) 0(0) 0(0) 1(1) CKO) 0(0) 0(0) nodes (various): lymphoid or reticulum cell hyper plasia, lymph-angiectasis, hemorrhage, pigmen tation, chronic inflammation; heart: chronic in flammation; liver: degeneration, necrosis, fatty metamorphosis, toxic hepatitis (associated with leukemia), granuloma, angiectasis, pigmenta tion, focal cellular change; bile duct (extrahepatic): chronic inflammation, mucosal hyperpla sia, cysts, fibrosis; pancreas (exocrine): atrophy, hyperplasia, ectopia; pancreas (endocrine): hyper plasia; kidney: chronic progressive nephropathy, cysts, pigmentation; pituitary gland: cysts, an giectasis, hyperplasia; adrenal (cortex): fatty me tamorphosis, hyperplasia; adrenal (medulla): hy perplasia; thyroid: follicular cysts, C-cell hyperplasia; parathyroid: hyperplasia; testes: seminiferous degeneration, interstitial cell hy perplasia; prostate: abscess, chronic inflamma tion, glandular hyperplasia; seminal vesicles: c>sts; ovary: follicular and parovarian cysts; uterus: hydrometra, endometrial cyst; mammary gland: cystic ducts, glandular hyperplasia, galactocele; mesentery: chronic inflammation; eye: cataract, hemorrhage, inflammation, retinal de generation: zymbal gland: cystic ducts; bone: osteopetrosis, exostoses, marrow hyperplasia. Ali mentary tract nonneoplastic lesions are noted in Tables 10 and 11. 1, 2-DimethyIhydrazine Dihydrochloride-Treated Groups Two groups of male and female rats were ex posed to 1, 2-dimethylhydrazine dihydrochloride (DMH) by gavage at levels of 7.5 mg/kg for males and 15.0 mg/kg for females, biweekly for a total of five doses. One group served as a positive carcino gen control and the other received amosite from weaning throughout life. Exposure of rats to DMH or DMH with amosite was associated with a dramatically increased in cidence of neoplasms of the intestinal tract, Zymbal's gland, and liver of male and female rats, and kidney in female rats. It is also noteworthy that survival in the DMH groups was shortened due to the presence of these neoplasms. Tbble 9 summarizes the numbers of rats with primary epithelial neoplasms in the gastrointes tinal tract by specific site and classification. Intes tinal neoplasms, particularly the adenomatous polyps, were often multiple within a given ani mal. 10002994 36 SlcCUSSELL ETAL Table 9. Number of amositc-exposed F344 rats with primary epithelial neoplasms of the gastrointestinal tract. l Untreated control* Amosite* MF MF Animals examined Total gastrointestinal Total stomach Tbtal small intestine `-i Duodenum Carcinoma a Adca in polypb Adenomatous polyp Jejunum i Carcinoma Adca in polypb Adenomatous polyp Ileum Carcinoma Adca in polypb * Adenomatous polyp Total large intestine Cecum j Carcinoma Adca in polypb Adenomatous polyp Total colon Ascending colon Carcinoma Adca in polypb Adenomatous polyp Transverse colon Carcinoma Adca in polypb Adenomatous polyp Descending colon Carcinoma Adca in polypb Adenomatous polyp 117 4<4i l(ll 3(3) 117 2(2) 1(1) 0(0) 2(2) 1(1) 0(0) 1<U 0(0) 1(1) 249 7(3) 2(1) 2(1) 1(0) 250 4(2) KOI 3(D KOI 1(0) 2(1) 3d) 0(0) 3(U 0(0) 1(0) M> 2d i Values in parentheses are percentages. bAdenocarcinoma arising in adenomatous polyp. Amosite P\VG" MF 100 3(3) 0(0) 1(1) 100 3(3i 0(0) nil 1(1) 1(11 2(2) 2' 21 1(1) 111) 2> 21 1(11 1(11 HI) The incidence of gastrointestinal neoplasia was dramatically increased with DMH treatment. However, the incidence appeared to be essentially comparable between groups receiving DMH alone and~those receiving DMH with amosite. Further more, the number of animals with tumors either in the small intestine or in the large intestine was also essentially comparable between DMH alone and DMH with amosite. There was no difference in the time to tumor between the groups. Evaluation of the incidence of the three catego ries of intestinal neoplasia (carcinoma, adenocar cinoma arising in an adenomatous polyp, and adenomatous polyp) by site (Table 14) reveals an increased incidence of duodenal carcinoma (p < 0.05) in the DMH with amosite-treated females, compared to female rats receiving DMH alone. In the jejunum, however, this incidence is reversed, with more carcinomas occurring in the female group receiving DMH alone. In the large intestine the frequency of carci noma arising in an adenomatous polyp and ade nomatous polyps was greatest in the descending colon. In the cecum, the incidence of carcinoma was less in the DMH with amosite-treated group than those treated with DMH alone, in male rats. This effect was not observed in the female group. The appearance of carcinomas in the ascending colon was somewhat greater in DMH with amo site-treated males than in males receiving DMH alone. Adenocarcinoma arising in adenomatous polyp occurred more frequently in the transverse colon of male and female rats receiving DMH with amosite compared to rats receiving DMH alone. Kidney Neoplasms Almost without exception, the renal masses associated with DMH treatment were malignant 10002995 EFFECTS IS RATS OF DIETARY ESPOSl RE TO ASBESTOS 37 Table 10. Incidence of nonneoplastic lesions in the alimentary tract of F344 rats exposed to 1*7 tremolite in the diet.* Males'1 Females'1 Control TVemolite Control TVemolite Animals examined Palate/tongue Inflammation Necrosis Hyperkeratosis Acanthosis Esophagus Inflammation Necrosis Hyperkeratosis Acanthosis Stomach-nonglandular Mineralization Inflammation, chronic Ulceration Necrosis Hyperplasia Hyperkeratosis Acanthosis Stomach-glandular Hyperplasia Small intestine Inflammation Necrosis Ulceration Colon Parasitism Inflammation Necrosis Hyperplasia Cecum Parasitism Inflammation Necrosis Hyperplasia Rectum Necrosis Anus (no lesions) 118 CKO) 0<0) 0(0) 1(U 1(1) 2(2) 9(8) 1(1) 13(1U 19)16) 10(8) 20(17) 3(3) 18(15) 26)22) 7(6) 0(0> 2(2) 0<0) 5)4) 0(0) 0(0) 0(0) 9(8) 1(U 1(1) 0)0) 0(0) 250 0(0) 0(0) KOI 3(1) 0(0) 1(0) 18(7) 0(0) 5)2) 29' 12) 17(7) 46(18) 1(0) 34(14) 54(22' 1)0) 2(1 > 0(0) 1)0) 32(13) 5(2) 3d) 1(0> 2iU 2di 4)2) 0'0) 1(0) 118 0(01 0<0) 0(0) 111' 0(0) 0(0) 3(3 > 0(01 4(3) 25(21) 9(8) 17(4) 0(0) 15(13) 23)19' 3)3' 0(0) 1(1) 0(0) 5(4) 3(3) 1(1) 0i0> 2(2) 4' 4' Kl' 0'0` 0(0) 250 4(2) 1(0! 2(1) 1)0' 1(0) 0(0> 4(2' K0i 2(1) 38(15) 11)4' 31)12) 2(1) 29(12) 45(18) 0(0' 1(0* 3(1) 0(0) 3(U 0(0) K0> K0> K0> 1(0' 3d' 1'0. 0(0' `Incidence of nonneoplastic lesions that occur with a frequency of 1*7 or more in al least or.e group "Values in parentheses are percentages. mesenchymal or mixed malignant tumors. Purely mesenchymal growths were classified according to their morphology (i. e., fibrosarcoma, undiffer entiated sarcoma). Those having epithelial ele ments or epithelial-like elements were classified as mixed malignant tumors. In early stages, these neoplasms appeared as interstitial sclerosing growths near the inner cortex. Collagen forma tion was accompanied by proliferating, baso philic, primitive-appearing cells. Epithelial ele ments consisted of glands, ductlike structures or poorly differentiated solid tubules. The growths Were often massive but rarely metastasized. Table 15 summarizes the incidence of kidney tumors in control and DMH-treated groups. The mgh incidence of renal neoplasms was confined a*most exclusively to treated female rats receiv ing either DMH alone or DMH with amosite (p < 0.01). The incidence rates for the two treated female groups was the same. Renal neoplasms occurred infrequently in male rats. Zymbal Gland Neoplasms Carcinoma was the most commonly observed neoplasm in Zymbal's gland. These neoplasms were composed of proliferating eosinophilic to ba sophilic squamous epithelial cells which formed thick fingers of tissue, masses of keratin and nests of sequestered cells. Some had sebaceous features with formation of sebum. Infiltration of adjacent tissues was not uncommon; however, metastases were rare. Table 16 summarizes the number of control and DMH-treated rats with Zymbal's gland neoplasms. 10002996 "i.uwpimi * - aHw (yaffcaiifVt'mWVii u liMirtirhiir '7 witr.1.1 38 UcCOXXELL ET AL Table 11. Incidence of nonneoplastic lesions in the alimentary tract of F344 rats exposed to amosite asbestos.* Control1* Amosite1* MF MF Ibngue, number examined Esophagus, number examined Hyperkeratosis Stomach, nonglandular, number examined Mineralization Inflammation, chronic Ulceration Necrosis Hyperkeratosis Acanthosis Muscle degeneration Stomach, glandular, number examined Hyperplasia Duodenum, number examined Jejunum, number examined Ileum, number examined Colon, number examined Parasitism Cecum, number examined Rectum, number examined Anus, number examined 117 115 12(10) 117 9(8) 21(18) 13(11) 23(20) 22(19) 31(26) 8(7) 117 615) 117 117 117 117 4(3) 117 117 117 117 117 7(6) 117 3(3) 21(18) 4(3) 13(11) 24(21) 26(221 2(2) 117 2(2) 117 117 117 117 2(2) 117 117 117 249 249 4(2) 249 2UI 56(22 ) 25(101 41(16) 41(16) 62125) 3(1) 249 0(0) 249 249 249 249 17(7) 249 249 249 250 246 7(3) 250 2(1) 60(24) 30(12) 37(15) 56(22) 72(29) 3(1) 250 1(0) 249 249 249 250 6(2) 250 250 250 `Incidence of nonneoplastic lesions that occur with a frequency of 1*1 or more in at least one group. Values in parentheses are percentages. rPWG = preweaning pavage. Amosite PWGb c MF 100 100 12(12) 100 1(11 17(17) 7(7) 15(15) 16(16) 21(21) 0(0) 100 0(0) 100 100 100 100 414) 100 100 100 100 100 6(6) 100 0(0) 18(18) 10(10) 11(11) 17(17) 23(23) 0(0) 100 0(0) 100 100 100 100 8(8) 100 100 100 Table 12. Number of F344 rats with thyroid C-cell proliferative lesions. Control* MF Animals examined Total C-cell tumors C-cell adenoma C-cell carcinoma C-cell hyperplasia 117 27i23l 16(14) 11(9) 21(18) 116 24(21) 14(12) 10(9) 22(19) Values in parentheses are percentages. *p < 0.05 vs. controls (incidental tumor and Fisher exact testsi. Amosite* MF 246 76(31) 26(11) 50*(20> 58(24) 247 65(26) 37(15) 29(12) 71(29) Amosite PWG* MF 100 25(25) 11(11) 14(14) 23(23) 100 29(29) 15(15) 14(141 26(26) f Table 13. Number of amosite-exposed F344 rats with mononuclear leukemia. Untreated control Amosite Amosite PWG MF MF MF Animals examined 117 117 249 250 100 100 Mononuclear cell leukemia* 38(32) 40(34) 106*(42 l 82(33) 49+(49> 34(341 *&1 `Values in parentheses are percentages. *p < 0.05 vs. controls (incidental tumor and Fisher's exact test*. *p < 0.01 vs. controls. Approximately one quarter of all rats receiving Liver Neoplasms DMH alone or DMH with amosite developed Zym- The classification of hepatocellular proliferative bal's gland neoplasms (p < 0.01), while in control lesions was based on the ILAR Monograph (70). animals the occurrence was low (1-3^). The inci Table 17 summarizes the number of control or dence appeared essentially comparable between DMH-treated rats with neoplastic nodules or hep fe- the two DMH-treated groups. atocellular carcinoma. 10002997 Ir'fif', EFFECTS IS FATS OF DIETARY EXPOSl RE TO ASBESTOS 39 Table 14. Number of DMH-exposed F344 rats with primary epithelial neoplasms of the gastrointestinal tract. Untreated control* MF Animals examined Total gastrointestinal Tbtal stomach Ibtal small intestine Duodenum Carcinoma Adca in polypb Adenomatous polyp Jejunum Carcinoma Adca in polypb Adenomatous polyp Ileum Carcinoma Adca in polypb Adenomatous polyp Total large intestine Cecum Carcinoma Adca in po!ypb Adenomatous polyp Total colon Ascending colon Carcinoma Adca in polvpb Adenomatous polyp Transverse colon Carcinoma Adca in polvpb Adenomatous polyp Descending colon Carcinoma Adca in polvpb Adenomatous polyp Colon (other)' Carcinoma Adca in polypb Adenomatous polyp 117 4(4) 1(1) 3(3) 0(0) 117 2(2) 1(1) 0(0) 0(0) 2(2) 0(0) 1(1) 0(0) 1(1) 8l6l 6(5) lili "Values in parentheses are percentages. bAdenocarcinoma arising in adenomatous polvp. 'Colon (otheri = site not identified. DMH positive control* MF 125 92(74) 3(2) 18(14) 124 77(62) 4(3) 14(11) 11(9) 3(2) 2<1) 111) 2ill 81(65) 16(13) ll(9i 70(56) 7(6) 10(8) 2(1) 5(4) 1(1) 8(6) 22(13) 20(16) 34(27) 1(1) 10(8) 4(3) 7(6) 9(7) 22(13) (1) 15(12) 27)22) 4(3) 1(1) DMH with amosite* MF 173 118(68) 2(1) 19(11) 175 114(65) 1(1) 24(14) 13(8) 19(11) 3(2) 2(1) 1(1) 110(64) 6(3) 2(1) 20(12) 3(2) 5(3) 2(1) 21(12) 3(2) 25(14) 40(23) 2(1) 1(1) 101(58) 6(3) 14(8) 7(4) 8(5) 2(1) 20(11) 2(1) 22(13) 31(18) 2(1) 1(1) Table 15. Number of DMH-exposed F344 rats with primary renal neoplasms. Untreated control MF Animals examined Total renal tumors* 117 0(0) 117 1(1) "Values in parentheses are percentages. *p = < 0.01 DMH positive control MF 125 3< 2' 124 49 32 )* DMH with amosite MF 173 4( 21 175 56(32)* A significantly increased incidence of neoplas tic nodules and or hepatocellular carcinomas oc curred in groups receiving DMH alone and in groups receiving DMH plus amosite. Generally, females had a higher incidence ip < 0.01) than males (p < 0.0.')). Miscellaneous Neoplasms In several instances, DMH treatment with or without amosite led to statistically significant decreased incidences of certain spontaneous neo plasms, particularly of the endocrine system 10002998 40 UiCOXXF.LL ET AL Tfeble 16. Number of DMH-cxposcd F344 rats with Zymbal gland neoplasms. Untreated control MF Animals examined Zymbal gland neoplasms* 117 1(1) 117 4(3) Values in parentheses are percentages. *p < 0.01 vs. controls. DMH positive control MF 125 124 33< 26)* 34'27i* DMH with amosite MF 173 175 55(32)* 39(221* Table 17. Number of DMH-exposed F344 rats with hepatocellular neoplasms. Untreated control MF Animals examined Neoplastic nodules* Hepatocellular carcinoma* 117 9(8) 111) 117 4(31 1(11 Values in parentheses are percentages. *p < 0.05 vs. controls. < 0.05 vs. controls (incidental tumor and life table analysis'. ip < 0.05 vs. controls (life table analysis only). DMH positive control MF 125 18( 14'" 9(7)* 124 29(23i* 10(8)* DMH with amosite MF 173 27(15)* 7i4r 175 32(18)* 8(5)+ I These included a reduced number of subcutane or cocarcinogenic effects of DMH and amosite ous fibromas, pituitary adenomas in females, ad asbestos. renal pheochromocytomas, pancreatic acinar cell The clinicopathologic results in this study adenomas and islet cell adenoma in males, mam showed that the ingestion of tremolite or amosite mary tumors, and interstitial cell tumors in male asbestos did not adversely affect the fertility of rats. However, many animals in these two groups the mothers or litter size of the F, bioassay ani died at an early age compared to the untreated mals. The average weight of the offspring at birth controls. from mothers exposed to either mineral was com ( parable to the offspring of nonexposed mothers. Discussion 5 However, the weight of the exposed offspring at weaning was slightly less than the control rats. TVemolite (11) or amosite asbestos (12) was The cause of the decreases in weight during lacta administered at a level of 1% in the diet to male tion is not known. The differences in body weight and female F344 rats for their lifetime, including gain became more apparant between weaning exposure of their dams to the test material. While and 8 weeks of age. While the tremolite- and the tremolite used in this study is considered amosite asbestos-exposed rats paralleled the con crystalline or nonfibrous in its natural form, a trol animals in weight gain, they remained small amount assumes a fibrous character during smaller throughout their life. The mean body the crushing and milling process. However, the weight of the male rats exposed to the chrysotile milling process used in the preparation of the preweaning gavage (PWG) and subsequently to tremolite for this study was identical to what is amosite asbestos was slightly higher than the done in the commercial setting. amosite alone rats. This may be related to the Starting at birth, one ofthree groups of neonate mortality induced in the neonates by the PWG rats from amosite-exposed mothers were given technique which would allow the remaining pups chrysotile asbestos (instead of amosite) by gavage more milk during lactation. Exposure to DMH until weaning at which time they were given the caused a small reduction in body weight gain in 1% amosite diet. For all intents and purposes this female but not in male rats. i.1 group of rats should be regarded as being exposed No clinical signs were observed which could be A to amosite asbestos for their lifetime. Two groups attributed to the ingestion of either mineral. (control and amosite exposed) of weanling rats Starting at 9 months of age, the DMH-exposed 0i were exposed to five biweekly doses of 1,2-di- rats showed signs attributable to DMH-related methylhydrazine dihydrochloride (DMH), a neoplasia, but no difference was noted between knoxvn intestinal carcinogen, to test the promotor the DMH and DMH plus amosite groups. 1000299? I EFFECTS HV JM7S OF DIETARY EXPOSL RE TO ASBESTOS 41 The ingestion of either tremolite or amosite in the diet for the life of the rats did not adversely affect their survival. In fact, survival of female rats exposed to amosite or amosite plus chrysotile PWG was slightly better up to 112 weeks than the controls. Similarly, the survival of male rats ex posed to amosite was slightly better than the untreated controls, although the amosite plus chrysotile PWG group showed slightly less sur vivability. The most plausible explanation for the in creased survival of the amosite exposed rats is their lower weight throughout the study. Yu et al. (13) have shown that rats of lower body weight caused by restricted caloric intake lived longer than rats that were allowed to eat an unlimited amount of food. The survival of the rats (control and amosite) in this study compares favorably with other NTP bioassays (14). At 106 weeks of age (age at end of typical 2-yr bioassay) the percentage of male rats alive in this study was: untreated tremolite con trol, 83%; untreated amosite control, 817*; tremo lite, 82%; amosite, 88%; and amosite plus PWG, 777*. The percentage of female rats alive at this time was: tremolite control, 82%; amosite control, 79%; tremolite, 84%; amosite, 827*; and amosite plus PWG, 86%. Haseman (14) in reviewing the 25 most recent NTP feeding studies found an average of 66% of control males and 737* of con trol females alive at 112 weeks of age. The survival of control groups of males and females was similar at 106 weeks. In most 2-yr studies involving rats, more females generally survive to the end of the study than do males. However, the longer survival of female rats (con trol and tremolite or amosite exposed) was clearly demonstrated after 142 weeks. The ingestion of either tremolite or amosite asbestos over the lifetime of these rats did not cause a biologically significant increase of neo plasms at any anatomic site when compared to the concurrent controls. The gastrointestinal tract was considered a potential target organ based on epidemiological studies in humans (15). The overall incidence of intestinal neoplasms in the control (male 4 and female 2%) and two amo site asbestos groups (male 3 and female 27*; male 4 and female 3%) was low, and there was no significant (p < 0.05) difference between the treated and control groups. Similar observations "ere noted in the tremolite groups and their respective controls. In addition, nonneoplastic le sions of the gastrointestinal tract were not in creased. In summary, amosite asbestos did not C-use any adverse affects in the gastrointestinal tract of either male or female F344 rats. Rais exposed to DMH showed a high incidence (60-70%) of neoplasia of the gastrointestinal tract, primarily in the large intestine. This high rate of intestinal neoplasia was unexpected be cause a pilot study (3) using the same dosing regimen of DMH would have predicted an inci dence of 15 * 5% in this study. In a previous NTP bioassay, hamsters exposed to DMH and chrysotile asbestos also failed to develop the desired rate of intestinal tumors based on a similar pilot study (28). Apparently the neoplastic dose response to DMH is relatively steep and duplication of low rates of intestinal neoplasia are difficult to repro duce. Because of the high background rate of DMHinduced neoplasia, it is not possible to determine with accuracy if amosite had a cocarcinogenic or additive effect in this study. Female rats exposed to DMH and amosite had a higher incidence (11% versus 2%) of neoplasia of the duodenum than the DMH controls. Conversely, they had a lower inci dence (97* versus 1%) of neoplasms of the jeju num; thus the total number of animals with neo plasms of the small intestine was comparable. A similar situation was observed in the large intes tine of male rats. The rats exposed to DMH alone had a higher incidence (13% versus 3%) of carci noma of the cecum but a lower incidence (13% versus 26%) of neoplasms of the transverse colon. The morphologic appearance of the neoplasms induced by DMH were comparable to those de scribed previously in rats exposed to hydrazine compounds <16). In addition, the few intestinal neoplasms which occurred in the control and tre molite- or amosite (no DMH)-exposed rats were of the same morphologic types to those induced by DMH. The neoplasms observed in the kidney, liver and Zymbal's gland of DMH-exposed rats were consistent with those reported for these types of intestinal carcinogens (17). A significantly (p < 0.05) increased incidence of C-cell carcinomas of the thyroid occurred in amosite-treated male rats. This effect was not ob served in the amosite PWG male rats and the overall incidence of total benign and malignant C-cell tumors was comparable between control and treated groups. Therefore, this is not consid ered to be a treatment-related effect. The incidence of mononuclear cell leukemia (synonyms--monocytic leukemia, Fisher rat leu kemia) was elevated in amosite (42%) and amo site PWG (49%) male rats compared to the con current control group (327*). How-ever, the rates were not significant when compared to the tremo lite male control group (397*). This increased inci- 10003000 40, 1 j i - ] ^ -i *-- t ?? X. -t:'.'lI' UrCU.WELI. ET.M. dence was not observed in treated female rats. Coleman et al. (18) reported an incidence of nearly 30# in male F344 rats within the age group of 24--40 months. In 2-yr-old F344 rats, Goodman et al. (19) reported 12# of males and nearly 10# of females had lymphoma/leukemia, a much lower incidence than in these studies. It is apparent from this study and above cited studies that the incidence of leukemia increases rapidly after 2 years of age. In view of considerable varia tion in the incidence of such disorders, the fact that the amosite-exposed male rats survived longer than their concurrent controls and lack of significance when compared to the tremolite con trol group, it is doubtful that the increase in the rate of leukemia is treatment related. More im portantly, an increased incidence of neoplasia was not observed in target organs (GI tract and mesothelium). Even though it is known that cer tain types of asbestos are absorbed through the GI tract (20,22), it is difficult to envision how oral asbestos could cause an increase in leukemia without causing an increase of tumors in the proposed target tissues. In summary, these effects represent only a mod ulation of neoplasms which occur in concurrent control groups and are known to occur in histori cal control rats of this strain. No uncommon or unique neoplasms were observed in any of the tremolite- or amosite-treated groups. In addition, the biological importance of the neoplasms in the absence of target organ neoplasia is questionable. A large variety of nonneoplastic lesions, pri marily lesions of aging, were observed in all groups. There was no obvious correlation between treatment and specific lesions. Therefore, tremo lite or amosite at the level of 1# in the diet did not appear to cause any overt toxicity. Studies on the effects of chronic ingestion of tremolite are not available. However, Stanton et al. (2) showed that the intrapleural inoculation of fibrous tremolite (two types) caused a high inci dence of pleural sarcoma in Osborne-Mendel rats. In contrast, intrapleural studies of tremolite talc failed to show a carcinogenic response in ham sters (22). The tremolite used in the NTP study is a nonfibrous type and more closely resembles that used by Smith (22) than Stanton et al. (2). Other studies involving the long-term inges tion of asbestos are few. Donham et al. (23) re ported equivocal results in F344 rats which were fed a diet containing 10# chrysotile for their lifetime. While they did not observe a statistically significant (p < 0.05) increase in the number of tumors in exposed animals, the authors believed that there was a trend toward increased colon lesions in general, evidence of penetration of as bestos into the colonic mucosa, possible cytotoxic ity to colonic tissues and suggested a possible relationship to peritoneal mesothelioma. Another equivocal study is that reported by Gibel et al. (24), who described an increase in malignant tu mors in the lung, kidney, liver and reticuloen dothelial system but no increase in intestinal neoplasia in Wistar rats fed asbestos filter mate rial (20 mg'day) for a period of 8-14 months. Cunningham et al. (25) reported two studies in Wistar male rats using 1# chrysotile in the diet: one study of 24 months and one of 30 months. These authors concluded that trace amounts of ingested asbestos can penetrate the walls of the gastrointestinal tract, but evidence of carcinoge nicity was inconclusive. Negative results were reported by Gross et al. <26), who fed rats a diet containing 5# chrysotile asbestos for a period of 21 months with no evidence of intestinal neopla sia. Corollary studies to this investigation were conducted in Syrian golden hamsters (27,28). The exposure regimen was similar in that male and female hamsters were exposed to 1# amosite as bestos (same source as the subject study) and short-range or intermediate-range chrysotile as bestos in their diet for their natural life-span. There was no adverse effect on body weight gain or survival, and no asbestos-related neoplasms were observed. Another oral asbestos study in hamsters was reported by Smith et al. (29). They exposed groups of 30 male and female hamsters via drink ing water for lifetime to amosite asbestos, mine tailings, beach rock or Lake Superior drinking water. They did not observe adverse effects on body weight or survival time in any of the groups. A peritoneal mesothelioma, one pulmonary carci noma, and two early squamous cell carcinomas of the nonglandular stomach were found in the hamsters exposed to amosite but the incidence was not statistically significant (p < 0.05). They concluded that the study was essentially nega tive. A subsequent study in rats using similar materials also failed to elicit a carcinogenic re sponse (30). Except for the studies of Donham et al. (23), Smith et al. <2S) and the NTP (11,12,27,28), the other studies were conducted with relatively small numbers of animals. Also some were con ducted for an insufficient period of time to ade quately test the carcinogenic potential of ingested asbestos. A long-term study of amosite asbestos designed to determine the promotor potential of asbestos 10003001 EFFECTS IS RA TS OF DIETAR >' EXPOSl RE TO ASBESTOS 43 was reported by Ward et al. (31). They exposed 6week-old male F344 rats three times per week for 10 weeks to 1 mg amosite asbestos in saline via gavage. Once per week during this same period, half of the rats received subcutaneous injections of 7.4 mg/kg azoxymethane (AOM), a known in testinal carcinogen in animals which produces effects similar to DMH. The rats were allowed to live out their lifespan or untl 94-95 weeks of age at which time they were killed. The authors re ported an intestinal tumor incidence of 66.7# in AOM alone, 77.1# for amosite plus AOM and 32.6# for amosite alone. The authors concluded that while amosite did not significantly add to the incidence of AOM-induced intestinal neoplasia, amosite alone caused a relatively high rate of intestinal neoplasia. However, there was no un treated control group to compare to the treated groups. These results should also be viewed with some suspicion because the authors also reported a 14# incidence of Zymbal gland tumors in the rats exposed to amosite alone. The historical rate of Zymbal gland tumors in the Bioassay Program is 0.34#, indicating that this is a relatively rare tumor (19). However, AOM is known to induce Zymbal gland tumors with a sngle dose of 5.1 mg' kg in male F344 rats producing a 14# incidence of tumors in this organ (17Y, in this study 5.1 mg ' kg AOM also caused a 24# incidence of intestinal neoplasia. A possible explanation for the inci dence of Zymbal gland tumors in the amosite groups would be that they were inadvertently exposed to AOM. If this occurred, these rats would also be expected to show a high incidence of intestinal neoplasms. Conclusions Under the conditions of this lifetime bioassay, tremolite or amosite asbestos was not toxic, did not affect survival, and was not carcinogenic when ingested at a level of 1# in the diet by male and female Fischer 344 rats. While there were significant (p > 0.05) increases in the rate of Ccell carcinomas of the thyroid in male, and mono cytic (mononuclear cell) leukemia in male rats exposed to amosite asbestos compared to un treated controls, their biological significance is questionable because of a lack of response in the concurrent amosite and preweaning gavage group or control group of the corollary study, nonaffect when all neoplasms of that organ are analyzed, lack of significance when examined us ing life table analysis or the absence of neoplasia in target organs. The cocarcinogenic studies us ing 1,2-dimethylhydrazine dihydrochloride (DMH) were considered flawed because of the high rate of intestinal carcinogenesis in both the DMH and amosite asbestos and DMH alone groups. The animal phase of this study was performed at Hazleton Laboratories America, Vienna, VA. This project has been funded with federal funds from the National Institute of Environmental Health Sciences (NIH), Research Triangle Park, NC, under contract #5-2158. Partial funding was pro vided by the Environmental Protection Agency under Intera gency Agreement #D70756. The research described in this paper has been peer and administratively reviewed by the U.S. Environmental Protec tion Agency and approved for presentation and publication Mention of trade names or commercial products does not constitute endorsement or recommendation for use. REFERENCES 1. Proceedings of the Joint NIEHS-EPA Conference on Biological Effects of Ingested Asbestos. Environ. Health Perspect. 9: 113-462 (1974). 2. Stanton. M. F., Layard, M.. Tegeris, A.. Miller. E., May. M.. Morgan, E.. and Smith, A. Relation of particle dimen sion to the carcinogenicity in amphibole asbestoses and other fibrous minerals. J. Natl Cancer Inst. 67: 965-975 (198H. 3. McConnell. E. E,, Wilson, R. E., Moore. J. A., and Haseman. J. K. Dose response of 1.2-dimethylhydrazine and methylazoxvmethanol acetate in the F344 rat. Cancer Letters 8: 271-278 11980). 4. Kaplan. E. L., and Meier, P. Nonparametric estimation from incomplete observations. J. Am. Statist. Assoc. 53: 457-481 (1958). 5. Cox, D. R. Regression models and life tables. J. Roy. Statist. Soc. B34: 187-220 (19721. 6. Mantel. N., and Haenszel, W. Statistical aspects of the analysis of data from retrospective studies of disease. J. Natl. Cancer Inst. 22: 719-748 (1959). 7. Peto, R., Pike, M., Day, N., Gray, R.. Lee, P.. Parish. S., Peto, J.. Richard. S., and Wahrendorf, J. Guidelines for simple, sensitive significant tests for carcinogenic effects in long-term animal experiments. 1ARC. Monographs on the Long-Tbrm and Short-Tbrm Screening Assays for Car cinogens: A Critical Appraisal. World Health Organiza tion, Geneva; Supplement 2.1980, pp. 311--426. 8. Gart, J., Chu, K., and Throne. R. Statistical issues in interpretation of chronic bioassay tests for carcinogenic ity. J. Natl. Cancer Inst. 62: 957-974 (1979). 9. Rao, C. R. Some statistical methods for comparison of growth curves. Biometrics 14: 1-17 (1958i. 10. Stewart, H. L. (Ed ). ILAR monograph: Histologic typing of liver tumors of the rat. J. Natl. Cancer Inst. 64: 179206(1980). 11. NTP. Carcinogenesis bioassav of tremolite in Fischer 344 rats. NTP Technical Report, National Toxicology Pro gram. Research TViangle Park, NC, in press. 12. NTP. Carcinogenesis bioassay of amosite asbestos in Fis cher 344 rats. NTP Technical Report, National Tbxicology Program. Research TViangle Park. NC, in press. 13. Yu, B. P,, Masoro, E. J., Murata, I., Bertrand, H. A., and Lvnd, F. T. Life span study of SPF Fischer 344 rats fed ad libitum or restricted diets: longevity, growth, lean body mass and disease. J. Gerontol. 37: 130-141 (1982) 14 Haseman, J. K Patterns of tumor incidence in two-year cancer bioassav feeding studies in Fischer 344 rats. Fund App). Tbxicol.. ih press. 10003002  Itoli H I m > H lr t `< n i.i..t> ,. A ii> A a llJ U I 44 .\UCUXXEU. AT .4 A 15. Cooper, R. C., Murchio, J. C.t and Paffenbarger. R. S. asbestos filter material following oral intake in experi Asbestos in domestic water supplies for five California mental animals. Arch. Geschwulstforsch. 46: 437-442 countries. Part 11. EHS Publ. No. 79-1, School of Public (1976). Health. Univ. Calif Berkeley, 247 (1979). In: EPA. Am 25. Cunningham, H. M,, Moodie, C. A., Lawrence. G. A., and bient Water Quality Criteria for Asbestos, EPA 4405-80- Pontefract, R. D. Chronic effects of ingested asbestos in 022, U.S. EPA. Washington, DC. 1980. rats. Arch. Environ. Conlam. Tbxicol. 6: 507-513 (1977). 16. Pozharisski, K. M. Morphology and morphogenesis of 26. Gross, P, Harley. R. A., Swinberne, L. M., Davis, J. M. G., experimental epithelial tumors of the intestine. J. Natl. and Green, W. B. Ingested mineral fibres, do they pene Cancer Inst. 54: 1115-1135 (1975). trate tissue or cause cancer? Arch. Environ. Health 29 17. Ward, J. M. Dose response to a single injection of azoxy- 341-347 (1974). methane in rats. Vet. Pathol. 12: 165-177 (1975). 27. NTP Carcinogenesis bioassay of amosite asbestos in Syr 18. Coleman, G. L., Barthold, S. W., Osbaldiston, G. W,, ian golden hamsters. NTP Technical Report No. 249. Foster, S. J., and Jones, A. M. Pathological changes dur National Tbxicology Program. Research TViangle Park, ing aging in barrier-reared Fischer 344 male rats. J. NC. 1982. Gerontol. 32. 258-278 (1977). 28. NTP. Carcinogenesis bioassay of chrysotile asbestos in 19. Goodman, D. G., Ward, J. M., Squire, R. A., Chu, K. C., Syrian golden hamsters. NTP Technical Report No. 246, and Linhart. M. S. Neoplastic and nonneoplastic lesions National Toxicology Program, Research TViangle Park. in aging F344 rats. Toxicol. Appl. Pharmacol. 48: 237-248 NC 11982). (1979/. 29. Smith, W. E., Hubert, D. D.. Sobel. H. J., Peters, E. T., and 20. Cook. P. M.. and Olson, G. F. Ingested mineral fibers: Doerfler, T. E. Health in experimental animals drinking elimination in human urine. Science 204:195-198 (1979). water w-ith and without amosite and other mineral parti 21. Sebastien, P. Masse. R., and Bignon, J. Recovery of in cles. J. Environ. Palhol. Toxicol. 3: 277-300 11980). gested asbestos fibers from the gastrointestinal lymph in 30. Hilding. A. C., Hilding, D. A., Larsen, D. M., and Auf- rats. Environ. Res. 22. 201-216 il980l. derheide, A. C. Biological effects of ingested amosite as 22. Smith, W E. Biological effects of tremolite talc on ham bestos. taconite tailings, diatomaceous earth and Lake sters. U.S Bur. Mines Inf. Circ. IC 8639: 43--48'19741. Superior water in rats. Arch. Environ. Health 36: 298- 23. Donham, K. J.. Berg. J. W., Will, L. A., and Leininger, J. 303 * 1981) R. The effects of long term ingestion of asbestos on the 31. Ward, J. M., Frank, A. L., W'enk, M., Devor, D., and colon of F344 rats. Cancer 45: 1073-1084 (19801. Tarone, R. E. Ingested asbestos and intestinal carcinogen 24. Gibel, W,, Lohs. K. H.. Horn. K. H., Wildner, G. P, and esis in F344 rats. J. Environ. Pathol. Tbxicol. 3: 301-312 Hoffman, F. Investigation into a carcinogenic effect of (I960). I 10003003