Document 6Bxq9qEDe9oKw7D3LE0n4L1K4

Environment E.Ilgren* : B. Chatfield*3.: ; ',/ '. a Consultant Physician, Bryn Mawr, Pa., USA; b Consultant Industrial Hygienist, Toronto, Canada Original Paper Indoor Built Environ 1998;7:18-31 Coalinga Fibre - A Short, Amphibole-Free Chrysotile Part 2: Evidence for Lack of Tumourigenic Activity PLAINTIFF'S EXHIBIT Keywords Short fibre chrysotile Coalinga Jeffrey UICC/B Tumours Abstract Controversy continues to surround the biological activity ofshort fibre chryso tile largely due to a lack of`pure exposure' situations available for study: most human exposures are confounded by concomitant long fibre and/or amphibole exposure. This report presents the morphological and morphometric findings of a lifetime inhalation study of F344 rats exposed to three types of chrysotile. Fibres from the first sample, from Coalinga, Calif., are almost all less than 5 pm in length and do not contain amphibole types of asbestos. The other two, from Quebec, Canada, are a sample from the Jeffrey mine and the UICC/B standard. These are both long fibre preparations with a minor degree of amphibole contamination. Animals exposed to these fibres displayed no tumours above control levels following exposure to Coalinga chrysotile but gave significant tumourigenic responses with both types of Canadian fibres. Introduction The two major theories often cited to explain asbestosrelated disease, the `Stanton' [1,2] and the `amphibole' [3, 4] hypotheses, would lead one to assume that short, amphibole-free chrysotile is the least tumourigenic form of asbestos. This proposal, previously put forth by Davis [5], is supported by earlier studies especially those using injection techniques [5,6], Early inhalation studies [7-11] demonstrated that short fibre chrysotile was less biologi cally potent than long. However, the inability of re searchers to obtain sufficient quantities of `pure' short fibre chrysotile, devoid of significant numbers of long fibres, confounded interpretation ofthe experiments. One of the first long-term inhalation studies to use a highly purified, short fibre chrysotile sample was conducted by Platek et al. [12], who exposed rodents and primates to a preparation produced by intense milling. Although no tumours were obtained, other workers [6] felt that the study could have been confounded by a loss ofcrystallini ty induced by the milling with a consequent loss ofbiolog ical activity [13], Davis and Jones [6] attempted to cir cumvent this problem by using a short chrysotile fibre sample prepared without milling but were unable to remove many of the long fibres, and a significant tumour response was still obtained. Although Davis and Jones [6] concluded that the shorter chrysotile fibres might have had some carcinogenic activity, they cast doubt on this interpretation since the highly purified, short fibre amosite preparation, used in one of their other inhalation studies [ 14], failed to produce any tumours. This was fur ther confirmed by a reassessment based on transmission electron microscopy of all of Davis' inhalation studies [15] which led these authors to conclude that fibres less than 5 pm in length were not tumourigenic. The present study is based upon a lifetime investiga tion of F344 rats exposed by inhalation to Coalinga chry sotile and two Canadian long fibre preparations, which KARCFR IV/*\I\VJEIV 1993 S. KaiserAG, Basel L420-326X/98/0071-00 IBS 15.00/0 Fax+4161 306 12 34 E-Mail karger@karger.eh Accessible online at: www.karger.comhttp://BioMedNet.com/karger Dr. E.B. llgren, MD, DPhil Consultant Physician Suite No. 503,830 MontgomeryAvenue Bryn Mawr, PA 19010 (USA) Tel.+1 (610) 525 5960, Fax+1 (610) 5201156 was performed at the National Institute of Environmental Health Sciences (NIEHS) and the National Toxicology Program (NTP) between 1978 and 1980. Coalinga chryso tile, a naturally occurring, amphibole-ffee, short fibre chrysotile, is probably the mineralogicallypurest sample ever to be tested experimentally and contains virtually no long fibres [Chatfield and Ilgren, in preparation]. The other two chrysotiles are from Quebec, Canada, and are samples from the Jeffrey mine and the UICC/B standard [16]. In the first part of this investigation we demonstrated that Coalinga chrysotile was not fibrogenic [16]. Here, we are able to show clearly that it also lacks tumourigenic poten tial. Taken together, these findings further support our ear lier proposal [16] that short fibre chrysotile is innocuous and should be regarded and regulated as a nuisance dust. Materials and Methods Information pertaining to the source of materials including data and animals, the chrysotile preparations, exposure conditions, ani mal sacrifice, tissue distribution and sample preparation, as well as morphometric and statistical analyses are as described by Ilgren and Chatfield [16], Histologically, the tumours reported here were classi fied after McConnell et al. [17] as adenomas, adenocarcinomas, squamous cell carcinomas or adenosquamous carcinomas. For brevity, from hereon the different chrysotile samples are referred to by their descriptors. Table 1. Incidence ofpulmonary neoplasia in F344 rats on lifetime test exposed to Coalinga, UICC/B and Jeffrey chrysotile fibres : Treatment :. v ' ./V: ; ~ Control [1998] ,/' Control [1984, MRC] Control [1984, NIEHS] Control [1984, NIEHS] Control [1984, NIEHS] Coalinga [1998] U1CC [1998] UICC [1984, MRq UICC [1984, NIEHS] Jeffrey [1998] Sex Total arumals with tumours ; v ;,.j ; , ; ; ; . f- ; ;7ai M 30 F 30 / nZ 'BAHfX'Ad :;:.;Ca;. .iJ-Meso". .total' hi: ' 27 0 0 0 0 2(7.4) 26 0 0 0 0 0(0) M 28 24 1 0 0 0 0(0) F 28 24 0 0 0 0 0(0) M 30 27 0 1 2 0 3(10) F 30 26 0 0 0 0 0(0) M 2,320 F 2,320 _ - nd 35 nd 18 25 10 0 60(2.7) 0 28 (1.3) M 529 F 529 - - nd 3 nd 1 12 7 1 16(2.7) 0 8 (1.5) M 30 27 3 0 2 0 2(7.4) F 30 24 3 0 0 0 0(0) M 30 29 2 1 8 0 10(35) F 30 25 1 1 3 0 4(12) M 28 24 3 1 6 0 7(29) F 28 24 2 0 5 0 5(21) M 30 29 2 3 6 0 9(31) F 30 27 3 1 l 0 2(7.4) M 30 25 1 1 9 0 10(40) F 30 24 2 0 1 0 1 (4.2) Pulmonary neoplasia = most severe (unquestionable) lesion; nl = number of animals at risk at the start ofthe study; n2 = number ofanimals examined histopathologically at the end of the study; BAH = bronchiolar adenomatous hyperplasia (see McConnell et al. [17] for discussion); Ad = adenoma; Ca = carcinoma; Meso = mesothelioma. Figures in parentheses indicate percentages. MRC=Medical Research Council Pneumoconiosis Unit, Penarth, UK; NIEHS National Institute ofEnvironmental Health Sciences, Research Triangle Park, N.C., USA; 1998 = this study (Ilgren and Chatfield, 1998) the data for which have been put in bold typescript; 1984 study of the control and UICC/B-treated animals from this investigation previously assessed by McConnell et al. [17] and Wagner et al. [18]; the results from Coalingaand Jeffrey-treated animals in these studies have not previously been presented. Short Fibre Coalinga Chrysotile - Lack of Tumourigenicity Indoor Built Environ 1998;7:18-31 19 Table 2a. UICC/B-treated males and females - lifetime test: non-pulmonaiy neoplasia Case Animal Fibrosis Primary ,, Secondly . Eztrapulmonary tumours ' No. No.' . score ' tumour " tumour ' . ` . : Males(.16.6%, 5/30, probably lethal non-pulmonary tumours; 33%, 10/30, lung tumours) 3 130 7 AdCa(BAH) none testicular, ?mesothelioma 4 113 5 Ad none pituitary, testicular S 177 5 AdCa none none 6 203 5 none none adrenal, malignant ganglioneuroma (2 cm) 7 207 6 +d none none none 8 152 5 none none none 10 204 5 (BAH) none testicular 11 210 5 none none testicular 12 241 6 Ad none none 13 168 5 none none testicular 14 105 4 none adrenal? metastases to liver, pancreas, lung 15 36 6+d none none thymic lymphoma with metastases to liver, kidney, lung 16 211 6+d none none SqCa mouth; fibroma neck 17 219 5+d none none testicular 18 28 5 none none testicular 19 40 5 AdCa none thymic adenoma; probably metastases from lung to cervical spine (metastasis) 20 82 6+d (BAH) none none 22 133 6 none none testicular 23 99 6 none none testicular 24 72 6 none none none 25 56 5+ d AdCa none pancreatic islet cell (small); pituitary 26 212 6 +d AdCa none pituitary 28 79 5+d none none synovial sarcoma, arm; leukaemic infiltration liver 29 234 6 +d AdCa none none 30 32 6 none none none l(L)1 176 - AdCa none subcutaneous fibroma; leukaemia spleen, lungs 2 (MS) 236 - AdCa n.d. 9(A) 117 - none none n.d. 21(A)2 224 - n.a. n.a. n.d. 27 (L) 225 - none none leukaemia lungs, liver ` Females (%3.3%, 20/24, probably lethal non-pulmonary tumours; 12.5%, 3/24, lung tumours) 1 304 ** 6 (BAH) mammary glands splenic leukaemia; mammary AdCa, metastases 2 459 6 none none n.a. - largely cannibalized 3 325 6 none none liver leukaemic infiltration; splenic lymphoma, pituitary Ad 5 323 6 + dust none none pituitary 6 364 4 AdCa none uterine/ovarian tumour (gross; no micro) 7 390 6+d none none cut keratoma; sarcoma,? site; pituitary 8 411 6 none none sebaceous gland; Ad; pituitary; ovarian cyst 9 548 6+d none uterus uterine AdCa; pituitary 10 321 5+d none none adrenal Ad; leukaemia; uterine (gross) 11 361 6 (?)!! none pituitary Ad; probably uterine and fallopian tube AdCa 13 496 4 none uterus uterine AdCa; pituitary M 371 5 +d none n 478 5 none uterus** pancreas vaginal Ca; leukaemia; uterine (gross) pituitary, pancreatic Ca widely metastatic 16 398 6 +d none neuro- abdominal sarcoma, widely metastatic sarcoma 17 313 5 +d none none none 18 387 5+d AdCa uterus uterine AdCa, widely metastatic 19 439 5 +d none uterus uterine AdCa, widely metastatic 20 381 5 none none none 21 435 5 +d none none mammary tumour (gross); uterine AdCa, massive 23 318 5+d none none fallopian tube Ca 24 410 5+d adenoma none pituitary, uterine and fallopian tube Ca 26 363 5+d none none liver leukaemia; caecal nodule 22 (L) 345 - none none lymphosarcoma, widely metastic 25(A) 329 - none none uterine AdCa, massive 20 IndoorBuilt Environ 1998;7:18-31 Ilgren/Chatfield Table 2b. Jeffrey-fibre-treated males and females - lifetime test: non-pulmonary neoplasia Case Animal' Fibrosis ' Primary . Secondary Extrapulmonary tumours No. . No. score:-; tumour , ; tumour . . F Males (8.3%, 2/24, probably lethal non-pulmonary tumours; 36%, 9/25, lung tumours) 1 178 4+d AdCa none testicular; jejunal nodule 2 84 6 +d (BAH) none pituitary Ad 3 153 5 +d AdSqCa none pituitary Ad 4 141 6 +d none none splenic lymphoma 5 208 7+d none none colonic nodule 6 173 6 +d none none none 7 44 6+d AdCa none testicular (mesothelioma) 8 114 4 none none tumour in inguinal area (no histopathology) 9 166 5 none none splenic lymphoma 10 209 6+d none lymphoma pituitary Ad; thymic lymphoma in liver 11 27 4 +d none none none 12 185 5+d none none splenic lymphoma 13 53 5 AdCa none splenic lymphoma; testicular 14 137 6 +d none none probable adrenal pheochromocytoma 15 214 5 none none thymic lymphoma; jejunal cyst; testicular 16 198 6 none none splenic lymphoma; testicular 17 144 7 AdCa none testicular 18 159 6 +d none none none 19 52 6+d AdCa none lymphoma; testicular 20 142 7+d SqcellCa none testicular 21 184 6 +d none none pituitary 22 216 6 AdCa none splenic lymphoma 24 141 4 none none none 25 192 6 +d none none splenic lymphomain liver, testicular 26 237 6+d AdSqCa none pituitary adenoma Females (50%, 13/ 26, probably lethal non-pulmonary tumours; 3.9%, 1/26, lung tumours) 1 479 5 AdCa none none 2 388 6 none ovarian thymiclymphoma; pheochromocytoma; ovarian AdCa and ++ hepatic metastasis 3 504 6 +d none none none 4 358 6 none none splenic lymphoma (6 cm) in liver 5 352 6 +d none skin cutaneous tumour, 1 cm, nearjaw 6 489 5 none none uterine AdCa (6 cm) 7- 462 4 none none splenic lymphoma (probable) 8 412 6 none none none 9 502 7 none fallopian tube none 11 480 6 none none ovarianAdCa (8 cm) 12 433 5 none none none 13 374 7 none none splenic lymphoma (massive) and uterine AdCa (massive) 14 468 4 none uterus uterine AdCa invading spleen, liver, ovaries 16 493 6 none none none 17 389 6+d none none none 18 344 6 +d none none splenic lymphoma (massive) 19 485 6+d none none none 20 515 7+d none none ovarian AdCa (4 cm, probable) 24 399 6+d none uterus uterine AdCa (large); splenic lymphoma (probable) 22 326 6+d none none splenic lymphoma (probable) 23 408 7 +d none none none 24 512 6+d none pancreas pancreatic AdCa with invasion ofliver, stomach, uterus 25 317 6+d none uterus splenic lymphoma and uterine AdCa (massive) with metastasis to adrenal gland 15 487 MS (none) (none) uterine and preputial AdCa (probable) 26 341 MS (none) (none) no information - necropsy report missing Ad = Adenoma; AdCa = adenocarcinoma; BAH = bronchiolaradenomatous hyperplasia (see McConnell et al. [17] for discussion); d = dust; Sq = squamous; Ca = carcinoma; L = leukemia; n.d. = not detected; n.s. = not stated; A = autolysis; n.a. = not available;!! = lung tumour reported in necropsy report but not found in slides; MS = missing slides. 1 Cases on lifetime test that could not be read due to autolysis. 2 Cases on lifetime test that could not be read due to leukaemia. Short Fibre Coalinga Chrysotile - Lack of Tumourigenicity Indoor Built Environ 1998;7:18-31 21 Table 3. Alveolar epithelial type I cell volume (mm3), number (X 106) and surface area (pm2) in F344 rats . Sex Control . .. . . Coalinga - ' - UICC/B Jeffrey ' ; Cell volume 3 months 12 months 24 months Cell number 3 months 12 months 24 months Cell surface area 3 months 12 months 24 months M 81 6 U23a 93 8 952 0.080.01cm3[a] 0.110.001cm3[a] n.d. 0.090.00acm3[a] 1,469 116 pm3 [a] 2,523 156a pm3 [a] n.d. l,877109apm3[a] F 787 806 635 795 M 833 1132a 11312a 1557a*b>c F 57 7 648 67 8 1339a,b,c M 848 U415 1167 121 22 F 666 668 795 87 8C M 53 1 [a, b, c] 43 4 [a] F 395[b, c] 363 M 626 [b, c] 57 2 F 474 [b,c] 383 M 597 [b, c] 463 563 43 3 575 44 1 675a>c 48 3 [a, c, d] 32 2 [c, d] 1024a>b*c[c, d] 604c[c, d] 749a'c[c] M +F M+F M+F 8,919+941 [e] 8,013562 [e] 9,189538 [e] 9,898 581 [e] 8,832 276 [e] 8,480 777 [e] 8,348 544 [e] 9,259 418 [e] 7,412 605 [e] 6,200 660a[e] 6,090 569a[e] 6,608 604a[e] All data are means SEM; n = 4 for each group and time period; ap < 0.05 when comparing to the age-matched control values using Duncan's multiple-comparison test after first testing for significance using a one-way ANOVA; b p < 0.05 when comparing the group exposed to Jeffrey chrysotile with all other treatment groups;c p < 0.05 when comparing one treatment group to its corresponding treatment group ofthe preceding time point; n.d. = not done. [ ] = References in which these data have previously been presented in part or wole are replicated here for the sake of comparison: a = these data were in the quarterly report `Effects of Chronic Exposure to Airborne Environmental Agents', August 1 to October 31, 1978, Becton Dickinson Research Centre, CS Stone; there are small discrepancies between these and those presentedby Pinkerton [ 19]; b = Pinkerton et al. [21], table 7; c=Pinkerton et al. [22], table 3; d = Pinkerton et al. [23], figure 1; e = NTP archives data obtainedbythe author (E.B.I.) in December 1995. Results The incidence of primary pulmonary tumours in Coalinga-treated animals on a lifetime test (3.9%, 2/51) was the same as that of untreated controls (3.8%, 2/53; table 1). In contrast,^2% of the animals exposed to Jef frey and 26% of those exposed to UICC/B had primary pulmonary tumours: 11/49 (10 carcinomas, 1 adenoma) and 14/54 (12 carcinomas, 2 adenomas), respectively (ta ble 1). There were no mesotheliomas and only one in stance of distant metastasis from a pulmonary primary tumour. The lungs of all tumour-bearing, UICC/B- and Jeffreytreated animals demonstrated evidence of fibrosis with scores between 5.0 and 6.0. The 2 Coalinga-treated, tumour-bearing animals had fibrosis scores of 3.0 which were probably overestimates due to concomitant uraemic pneumonitis and leukaemic infiltration. The incidence of primary pulmonary tumours was greater in males than females in both the Jeffrey and the UICC/B treatment groups (table 1). By contrast, the inci dence of non-pulmonary tumours was higher in females than males (tables 2a, b). The incidence of non-pulmo nary neoplasia was not treatment related. The incidence of tumours in the interim sacrifice ani mals could not be determined precisely since some of the records were missing. However, for the 24-month interim sacrifice animals, the available data [19,20] strongly sug gest an absence of tumours in the untreated controls, probably no more than 1 tumour-bearing Coalingatreated animal and 4 Jeffrey-treated tumour bearing ani mals in addition to the 2 UICC/B-treated rats with tumours already known to exist in that group [17]. Examination of the lungs of the Coalinga-treated rats sacrificed at 3-, 12- and 24-month intervals by electron microscopy did not reveal ultrastructural epithelial ab- 22 Indoor Built Environ 1998;7:18-31 Ilgren/Chatfield Table 4. Alveolar epithelial type II cell volume (mm3) and number (x 106) ofF344 rats ' .Sex ' Control . - Coalinga'. . . UICC/B Jeffrey Cell volume 3 months 12 months 24 months Cell number 3 months 12 months 24 months M 369 13214a 7912 0.04 0.01 cm3 [a] 0.130.01acm3[a] n.d. 436104|im3[a] 974133anm3[a] n.d. F 25 1 686a 66 7a M 30 4 824 10014a F 20 3 56llb 709a M 289 62 15 962ia F 18 5 299C 41U M 797 [b, c] F 497[b, c] M 587[b,o] F 56 5 [b, c] M 578 [b,c] F 504[b, c] 131I0a 80lla 1153a 927a 118 50 619C 121 10a 949 1149a 774 1107a 96lla 123427* 0.1240.03* cm3 [a] 9374176* pm3 [a] 6745* 156437* 9947*.c 73426* 70416*-c 127416* [c,d] 7545* [c#d] 220429*-h-qc, d] 13847*-h-qc, d] I24413*-C[c, d] 116412*-c[c, d] All data are means SEM; n = 4 for each group and time period;a p < 0.05 when comparing to the age-matched control values using Duncan's multiple-comparison test after first testing for significance using a one-way ANOVA; b p < 0.05 when comparing the group exposed to Jeffrey chrysotile with all other treatment groups;c p < 0,05 when comparing one treatment group to its corresponding treatment group ofthe preceding time point; n.d. = not done. [] = References in which these data have previously been presented in part or whole are replicated here for the sake ofcomparison: a = these data were in the quarterly report `Effects of Chronic Exposure to Airborne Environmental Agents', August 1 toOctober31,1978, Becton Dickinson Research Centre, CS Stone-there are small discrepancies between these and those presented by Pinkerton [19]; b = Pinkerton et al. [21], table 7; c = Pinkerton et al. [22], table 3; d = Pinkerton et al. [23], figure 1. normalities. In contrast, prolonged exposure to Jeffrey and UICC/B resulted in dramatic ultrastructural changes in type II epithelial cells at 12 and 24 months which included megalamellar bodies and markedly dilated rough endoplasmic reticulum cistemae (also, for further description of changes noted on untreated and Jeffreytreated animals see Pinkerton [19], NTP [20] and Pinker ton et al. [21-23] respectively). These qualitative electronmicroscopic findings-clearly discriminate Coalinga from the Canadian fibres and were confirmed morphometrical ly using data parameters related to changes in the epithe lium as a whole and its type I and type II cellular constitu ents. Changes in the thickness of the whole epithelium, referenced here as alterations in `total epithelial volume', are not shown for the sake of brevity but are available upon request from the authors. Data referable to changes in the number, surface area and volume of type I and type II cells are shown in tables 3 and 4. The time course of events within the epithelium and its cellular components during and following exposure to each ofthe three chryso- tiles is classified here as `acute', `subchronic' and `chronic' events for alterations taking place after 3, 12 and 24 months of exposure, respectively. Over the first 3 months of exposure, acute changes (see above) were generally noted within the total epithelial vol ume in response to all three types of chrysotile largely due to type II epithelial responses. After 12 months of expo sure, all three chrysotile types significantly increased the total epithelial volume though this was far greater for Jef frey than for UICCYB and Coalinga. Again, this was large ly due to type II cell responses. Although the degrees of cellular changes observed with Coalinga and UICC/B were generally similar, UICC/B increased type II cell vol ume significantly over controls whilst Coalinga did not. By contrast, at 12 months, Jeffrey dramatically increased both type I and type II cell responses, not only above con trol values, but also above the preceding 3-month time point and the other two treatment groups as well. The Jef frey-induced type II cell responses were greater than those noted in type I cells. By 24 months, Coalinga was the only Short Fibre Coalinga Chrysotile - Lack of Tumourigenicity Indoor Built Environ 1998;7:18-31 23 fibre that failed to produce a persistent, significant in crease in any epithelial cellparameter. In contrast to Coal inga, the majority of epithelial changes noted following exposure to Jeffrey remained significantly above con trols by 24 months, indicative of persistent injury. Some Jeffrey-related, epithelial parameters significant at 12 months fell to non-significant levels by the end of the study suggestive of regression or resolution. By 24 months, the majority of UICC/B-related epithelial tissue measurements were also significantly greater than con trols, but the pattern of epithelial changes noted following cessation of exposure was clearly different to those seen with the other two fibre types. Thus, the pattern of changes in the type I and II epithelium in the UICC/B treatment groups displayed, for the most part, either no change or progression. The latter was evident in the signif icant increase in type I cell numbers over the preceding 12-month values (table 3). In fact, there was only one measured parameter, type I cell volume, that was not sig nificantly greater than in controls by the end of the study period in the UICC/B treatment group. Throughout the 24-month time course, the severity ofthe observed epithe lial changes following UICC/B was greater than that noted with Coalinga but less than with Jeffrey. Irrespective of fibre type, changes which occurred in the type II epithe lium were more pronounced than those seen in type I cells. This is reflected in percent changes in the number of alveolar type I and type II cells for the three different fibres. Although the type I cell responses appear to be gen erally attenuated, the differential responses in type I cell surface area (table 3) still serve to distinguish Coalinga from the two Canadian fibres. Finally, the type II cell responses in females appeared to be less than those noted in males irrespective offibre type. Survival and Tumour Formation Statistical analysis (F test for two-sample variance) did not demonstrate significant differences in length of sur vival between control and/or Coalinga treated animals with or without primary and/or secondary tumours. How ever, when the UICC/B and Jeffrey male and female ani mals without primary tumours were compared with those with primary neoplasms, the observed reduction in sur vival duration just missed attaining statistical signifi cance (p = 0.08). Moreover, when UICC/B- and Jeffreyexposed females were examined separately with regard to secondary neoplasms, there was a suggestion that the sec ondary tumours also had reduced survival though again this failed to attain significance [p=0.14 (UICC) and 0.23 (Jeffrey)]. Discussion Our findings demonstrate that long-term, high-dose exposure to Coalinga fibre (a short, amphibole-free chrysotile) does not induce either tumour formation or persis tent epithelial responses. In contrast, the two long fibre Canadian samples tested are both tumourigenic and capa ble of inducing severe attendant epithelial abnormalities. These results were based upon our own analysis of ani mals on lifetime test, plus careful review of interim sacri fice data, analysed both morphologically using light mi croscopy and morphometrically with electron microscop ic methods, provided to us by Drs. Kent Pinkerton and Gene McConnell. Other Studies with Coalinga Chrysotile The investigation by Muhle et al. [24] is the only other inhalation study of Coalinga chrysotile, and they too failed to find tumours. These workers chose Coalinga as their `positive' control since they were unable to obtain sufficient amounts of Canadian chrysotile for an inhala tion bioassay and they stated that `when we started our inhalation experiment we expected a carcinogenic poten cy comparable to that of the UICC chrysotile samples'. Muhle [pers. commun., 1988] was indeed very surprised that `significant tumour rates were found neither in the inhalation experiment nor after intraperitoneal injection of 0.5 mg'. Although Muhle et al. [24] did not use long fibre chrysotile in these experiments, they did employ a concurrent crocidolite positive control. Unfortunately this also failed to induce tumours, a finding that led some [25] to argue against the claim of Muhle et al. [24] that Coalinga lacked carcinogenic potential. However, Muhle et al. [24] have countered this by stating that `the high rate of bronchiolar alveolar hyperplasia of 74%, one case of squamous metaplasia, and one adenocarcinoma may in dicate a tendency of the crocidolite fibres used to induce neoplasms'. We would support this interpretation and suggest that the high percentage of short fibres in the UICC crocidolite preparation [26] was the most likely explanation for the very low tumour rates noted by the German group and by others [27, 28]. Oberdorster [29] has argued that Coalinga's lack ofcarcinogenicity is due to its low biopersistence. The evidence to date suggests that Coalinga's failure to persist is due more to its short length and increased mechanical clearance than to intrinsic hy persolubility [30] though further research in this area is clearly warranted [see Chatfield and Ilgren, in prep., for detailed discussion]. 24 IndoorBuilt Environ 1998;7:18-31 Ilgren/Chatfield Table 5. Intraperitoneal injection studies with Coalinga, UICC/A (Rhodesian) and UICC/B (Canadian) chrysotile Source : :n ..Sek; Dose rag Study Tumor yield . : duration : / weeks Authors) : - ' . Year; Coalinga Coalinga Rhodesian Canadian Rhodesian Coalinga Canadian Canadian Canadian Rhodesian Coalinga Rhodesian 50 F 0.5 50 F 0.5 50 F 0.5 24 M 0.5 33 M 0.5 50 F 1.0 50 F 1.0 36 F 1.0 32 F 1.0 32 F 1.0 50 F 3.0 24 M 2.5 116 142 142 99 99 131 112 103 51 51 131 144 6(2/32) 6.3(2/32) 56.3(18/32) 91.7 (22/24) 81(26/33) 2(1/50) 62 83.35(27/32) 84(27/32) 84.4(27/32) 10(5/50) 97.1(22/24) Muhle et al. [24] Pottetal. [31] Pott et al. [311 Davis [33] Bolton et al. [34] Rittinghausen et al. [32] Rittinghausen et al. [32] Pott et al. [31] Muhle et aL [24] Pottetal. [31] Rittinghausen et al. [32] Davis [35] 1987 1987 1987 1984 1983 1991 1991 1987 1987 1987 1991 1988 Coalinga = Fibre from the Coalinga deposit mined by Union Carbide Corp. (Calidria Tl4). Muhle et al. [24], and other German workers [31, 32], conducted three separate, well-controlled injection stud ies with Coalinga and Canadian chrysotiles all of which have convincingly demonstrated Coalinga's lack of carci nogenicity (table 5). All of these studies utilized large numbers of animals (n = 50) and dose levels low enough to avoid non-specific, high-dose, mass effects [29, 36-38]. An Italian group, represented by Maltoni and his col leagues [39, 40], found results opposite to those of Muhle and his colleagues. However, their chrysotile sample was labelled `California', not Coalinga, and could thus have been from one of the many serpentine ore bodies in that State. Even assuming that the sample was Coalinga, the data of Maltoni et al. [39] are severely undermined by a total lack of `internal' and `external' consistency. Thus, in 1982, Maltoni et al. [39] obtained tumour yields of 10 25% with their `California' sample whilst in 1988, Mal toni and Minardi [40} induced a 72.5 % incidence presum ably with the same sample. The major difference was that the earlier study ran for 67 weeks whilst the later one lasted 104. However, study duration is probably not suffi cient to account for the dramatic difference in tumour yields since Davis et al. [41 ] and Davis and Jones [6] pro duced tumours well in excess of 50% by 67 weeks with six different types of chrysotile using the same dose (25 mg) and the same mode of administration. Large inconsisten cies have also been noted in other chrysotile injection studies conducted by Maltoni et al. Thus, in their 1982 study, untreated Canadian and Rhodesian preparations induced 0-5% tumours whilst, in their 1988 investiga tion, tumour yields of 80% were obtained. Some of this could be attributable to non-specific mass dose effects from the enormous 25-mg inoculum. Oberdorster [29] notes that `large injected doses, even of short fibres and lower doses of large, non-fibrous particles can readily overwhelm and block' clearance mechanisms so that the dosage must be `appropriately considered' if this mode of administration is to be used in an attempt `to identify a potential hazard ofa fibre for inducing mesothelioma'. As Oberdorster [29] also points out, `the dose defines the mechanism', `the challenge (being) to reach an agreement as to what is unacceptably high'. As the maximum total fibre dose (250 mg) recommended by the International Co-Operative Research Program for injection, normal ized over the lung's surface area, would weigh more than an entire rat's lung ifgivenby inhalation [29], it is obvious that such doses are grossly excessive. We would argue that the same is true for the maximum recommended amount of 50 mg per single injection and that this should not exceed 10 mg (see Ilgren [36] for comparative tumour yields at this.dose level). If this is done, the `possibility of the tumours being due to secondary events and mecha nisms associated with high-dose toxicity' [29] will be greatly lessened. Still, the discrepancies in the data of Maltoni et al. are so great that factors other than dose must also be operative though at the present time these are not apparent. For example, the low (0-5%) tumour yields obtained by Maltoni et al. are totally inconsistent with every other intraperitoneal injection chrysotile study ever performed in the rat at the 25-mg dose level (68- Short Fibre Coalinga Chrysotile - Lack of Tumourigenicity Indoor Built Environ 1998;7:18-31 25 100%, [36]). Finally, a New York/Japanese collaborative group, represented by Suzuki and Kohyama [42, 43], assessed the carcinogenicity ofCoalinga using the injection method. Although these workers were able to induce tumours with Coalinga, their studies are subject to serious criticism. Firstly, the use of the mouse is problematical since mineral fibre injection studies employing this species give far less reliable results than those which use the rat [Davis, pers. commun., 1994]. Secondly, the studies of Suzuki [42] and Suzuki and Kohyama [43] are confounded by supra-maximum-tolerated-dose effects [29] indicated by the high number of very sick or dead mice (192 of 586) foundbefore 7 months. Suzuki and Kohyama [43] also rec ognized that they should have used much lower doses to avoid these untoward effects stating that `animals receiv ing large doses ofasbestos... died ofperitoneal fibrosis fol lowed by intestinal obstruction before the tumour was induced. To perform long-term observations of the devel opment and course ofexperimental peritoneal mesothelio ma, doses of the carcinogenic mineral fibres must be care fully chosen'. The high percentage of tumours with sarco matous histology, a variety more apt to be associated with damage-related, reparative effects and scarring than the epithelial form, is also probably another manifestation of the very high doses employed by these workers. Again, Suzuki and Kohyama [43] would appear to concur with this stating: `It is not known why, unlike human mesothe lioma, the epithelial form is so rare and the fibrous form so common in the induced mouse mesothelioma, although the severe focal fibrosis'tvhich constantly occurred around the mesothelium might be related to the high incidence of fibrous malignant mesothelioma.' By contrast, Rittinghausen et al. [32, 44] found that 80% of their Coalingainduced tumours were partially or totally epithelial, a find ing consistent with the lack ofobserved, untoward mortali ty (median life-spans for Coalinga identical to controls [24, 32]) and the lower doses employed (0.5-3.0 mg). The stud ies of Suzuki and Kohyama were also confounded by tech nical problems such as the accidental administration of fibre into the abdominal wall, diagnostic problems, as in the inability `to differentiate plasmacytomas from meso thelioma', and viral infection. maintained in a manner identical to those of McConnell et al. [17] and observed tumour yields similar to ours (ta ble 1, see 1984 MRC). Small discrepancies in tumour yields between our own analysis and those of McConnell et al. [17] and Wagner et al. [18] are potentially attribut able to interobserver variation. This is well supported by the findings of 13 independent pathologists from 9 insti tutions1 who reviewed 20 tumour cases from the compar ative study [17]. Diagnostic variation was particularly notable (Appendix). Wagner [45] and Wagner et al. [46] appear to be the only other group to have administered UICC/B to rats via inhalation using approximately the same duration and intensity as was used in the present study. The incidences of primary pulmonary tumours noted by Wagner [45] (30%, 8/23) and Wagner et al. [46] (22%, 5/23) were con sistent with those noted by us. However, 3 mesotheliomas were reported by Wagner [45] whilst none were found either by us in this study, by McConnell et al. [17] or by Wagner et al. in other studies [18, 46]. One of our cases displayed marked pleural invasion from an underlying pulmonary neoplasm thus resembling the type of periph eral tumour described by McConnell et al. [ 17] `that could have been confused with mesothelioma if the primary tumour had not been observed' (also note case 18 and the footnote in the Appendix). McConnell and Adkins [47] were the only other group to study Jeffrey fibres via inha lation and used the same exposure duration and intensity as the present investigation. They found a far higher inci dence of bronchiolar adenomatous hyperplasia and lung tumours (30 and 20%, adenomas and carcinomas com bined, no mesotheliomas, respectively) than we did, and it is presently not possible to reconcile the observed differ ences with our own findings. Sex-Related Differences The incidence of primary pulmonary tumours was greater in males than females (table 1). This finding is not only consistent with that of McConnell et al. [17] but also with the sex-related differences noted in many of the mor phometric epithelial responses seen in this study and in Other Comparable UICC/B and Jeffrey Inhalation Studies McConnell et al. [17] examined the same untreated controls and UICC/B treated groups as we did for this report and found nearly identical tumour incidences in both cases (table 1, see 1984 NIEHS). Wagner et al. [18] also examined untreated and UICC/B-treated animals 1 Drs. Wagner (MRC), Cobb and Hulse (AEA, Oxford), Smith (Los Alamos), McConnell and Boorman (NIEHS), Hollander (the Netherlands), Lamb (Scot land), Kevsser and Smith (Rutgers), Kotin (JM). Kuschner (SUNY) and Mohr (Hannover) were brought to the MRC Pneumoconiosis Unit (Penarth, UK) on December 2,1981, to review 20 cases from rats exposed to UICC/B, rockwool and glasswool with and without resin. Their diagnoses were subsequently tabu lated and a final classification given after the meeting. The appendix gives the findings of pathologists from three ofthe institutions, but the full data sets are available upon request from the authors. 26 Indoor Built Environ 1998;7:18-31 Ugren/Chatfield various interstitial changes previously observed by Ilgren and Chatfield [ 16]. Neither Wagner et al. [ 18] nor Wagner [45] noted sex-related differences in the incidence of lung tumours, although the former failed to stratify their data by fibre type. Nonetheless, Wagner [45] did find that the distribution of adenocarcinomas, squamous cell carcino mas and the number of mesotheliomas differed between the sexes. Wagner et al. [46] did not state the sex of the animals used whilst McConnell and Adkins [47], Davis et al. [14,27, 48] and Davis [49] used only males. The sexrelated differences in tumour incidence noted by us and McConnell et al. [17] are difficult to explain. The compar atively high incidence of malignant, non-pulmonary tu mours in females might `competitively' inhibit the growth of a primary lung tumour [50] or simply kill the animal before one has time to grow. Also, the high incidence of tumours arising in endocrine productive and/or respon sive tissues, e.g. ovarian, uterine, pituitary adenomas and carcinomas, noted at autopsy might reflect important conditioning, age-related hormonal changes. These might also account for sex-related differences in lung tumour incidence since hormones can directly and indirectly affect the growth of normal [51] and neoplastic pulmo nary tissue [52]. Survival: Tumour Lethality, Metastasis and Competing Causes ofDeath McConnell et al. [17], in their analysis of the same untreated and UICC/B-treated animals, reported that `ap proximately half of the fats with pulmonary tumours died from mononuclear cell leukaemia (monocytic leukaemia, Fischer rat leukaemia)' and thus claimed that the `pulmo nary tumours grow relatively slowly and may not always be the cause of death'. In this study, there was a strong suggestion that the primary pulmonary tumours reduced survival. Fibrosis was also previously shown to reduce survival independently of tumour formation, and indeed the two may thus be. working in concert. Since definite metastasis from a pulmonary primary was only noted in one instance in our study, and not observed at all by McConnell et al. [17] in their review of the same group of animals, it is unlikely that distant spread of tumour was an important cause of death. McConnell and Adkins [47], in their study of Jeffrey-treated animals, also failed to find metastasis from the primary pulmonary tumours al though metastasis was noted, almost in a dose related fashion, by Wagner [45] and Wagner et al. [46] following exposure to UICC/B (carcinomas: 1/4 at 3 months; 2/5 at 6 months; 5/12 at 12 months; 3/5 at 24 months). Morphological and Morphometric Correlation of Tumour Formation with Epithelial Changes The failure to observe tumours after exposure to Coa linga clearly correlates with the lack of persistent, mor phometrically detectable, epithelial changes. Conversely, the significant tumour formation noted with the two Canadian fibres was associated with severe, persistent epithelial changes. This was particularly so for type II cells attempting to repair injured type I epithelium. The acute epithelial changes noted with Coalinga were not manifes tations of injury but reversible, non-specific, high-dose effects [29]. Thus, exposure to exceedingly high levels of Coalinga chrysotile from 3 to 12 months, even though it was continuously applied, was stillnot able to prevent epi thelial resolution and healing from taking place during this period of time. In contrast, continuous exposure to Jeffrey over the same 3- to 12-month time period pro duced dramatic epithelial abnormalities that frequently progressed to eventual irreversible, neoplastic changes. In contrast to either Coalinga or Jeffrey, exposure to UICC/ B produced clear progression of type I epithelial changes over the final 12 months of the study. This temporal pat tern ofepithelial changes resembled the interstitial abnor malities previously shown to be unique to this fibre type [16] which again emphasizes the interdependence of epi thelial and interstitial responses noted by others [53]. Possible Potentiation ofLong Fibre Related Effects by Short Fibre Davis [54] has stated that `human fibre exposures asso ciated with workplace dust will always be to a mixture of fibres and particulate materials of several kinds'. Since short fibre chrysotile willfrequently make up a substantial proportion of many such occupational exposures, it is important to ask if it could contribute to fibre carcinogen esis bypotentiating the effects oflong fibre. Davis [54] has indicated that particle-mediated potentiation could occur either by producing a modified tissue reaction and/or by modifying fibre clearance rates through macrophage over load and the alteration of fibre transport. Although the long-term, high-dose administration of insoluble, nonfibrous, nuisance dusts (reviewed by Oberdorster [29] and Ilgren [37]) is able to induce lung tumours, the findings of this report and others [12] indicate that this would not be the case for short fibre chrysotile either for primary pul monary neoplasms or mesothelioma. Indeed, Oberdorster [29] has argued that `in the rat, any persistent particle at sufficiently high lung burdens appears to induce lung tumours' though only `in association with lung particle overload' of the sort that occurs `at extremely high partic- Short Fibre Coalinga Chrysotile - Lack of Tumourigenicity IndoorBuilt Environ 1998;7:18-31 27 ulate lung burdens' (also see Lehnert et al. [55]). However, as short fibre chrysotile does not persist in the lung [6,24, 56] but is largely, if not totally, cleared, this would not apply to this fibre type. Oberdorster [29] has also stressed that solubility is also probably more important for hu mans than rodents in this regard stating that: `because mechanical clearance rates for highly insoluble particles are slower in humans by a factor of 8-10 compared to rats, it can be predicted that high solubility rates of fibres have a much greater impact on lowering the overall bio persistence of fibres in human lungs than in rodent lungs'. As for the potentiation of fibre carcinogenesis and specifi cally mesothelioma induction [48, 54], the evidence as sembled to date would indicate that this can only be brought about by insoluble particles able to persist within macrophages (e.g. TiC>2) and/or by those which directly affect the pleural lymphatic endothelium (e.g. quartz) so as to enhance pleural fibre transport. As it is `highly unlikely* that short fibre chrysotile can cause overload [38], it is equally unlikely that short fibre chrysotile will potentiate long-fibre-mediated mesothelioma induction. Its inability to cause overload is largely related to rapid mechanical clearance and short length, possible hyperso lubility (see above) and a lack of pleural lymphatic endo thelial cell toxicity. Short fibre chrysotile should therefore be regulated as a `nuisance dust' but must be distinguished from those non soluble `nuisance dusts' able to induce pathogenic effects either when administered alone under long-term, highdose conditions or in association with long fibres. Relevance ofthese Findings to the `Amphibole Hypothesis' and Mesothelioma Induction The failure to observe mesotheliomas in this study was due, in no small part, to the absence of amphibole in the Coalinga fibre [Chatfield and Ilgren, in prep.] and the presence of only very low amounts in the two Canadian samples. Although Wagner et al. [46] failed to find tremolite in their UICC/B" sample (only some talc), `traces of tremolite' were found in their sample from the Bells mine (Quebec), one of the eight `contributors' to the UICC/B standard (also see Addison and Davies [57]). Wagner [45] did not mention the amphibole content of their UICC/B sample, but Wagner et al. [58] indicated that tremolite had been found in a crude ore grade 7 sample from the Bells mine and also in the lungs of rats dusted with the `super-fine asbestos' sample from the Canadian Norman die mine that had been used by these same workers in their earlier study of this sample [46]. As both Bells and Normandie were used to produce the UICC/B standard sample, further analysis of UICC/B-exposed rat lungs would probably have revealed significant tremolite con centrations though many of these lung tissues no longer appear to be available for study [58], The level of tremol ite in the first 1974 UICC/B sample may have been higher than that of the later ones, an idea consistent with the batch variation described by Elmes [26]. The general fail ure to observe mesotheliomas in rats exposed to UICC/A (Rhodesian chrysotile), as opposed to UICC/B [59], is probably related to the lower amounts of tremolite in the former (tremolite probably <0.05% [60,61]) than the lat ter (probably <1.0% [60, 61]; though see Wagner et al. [58]). Nonetheless, UICC/A, despite its reduced tremolite content, may still be able to induce mesotheliomas in cir cumstances that greatly facilitate fibre release and local deposition (e.g. with unusual chrysotile preparations like exp. WDC, [41 ]) or that markedly enhance fibre transport to the pleura (e.g, when co-administered with quartz [48]). The relative thinness of the rat's visceral pleura [48] and biological consequences of the comparatively short life-span of the rodent [62] are also probably important contributory factors to the `anomalous' induction of me sothelioma with chrysotile in rodents exposed via inhala tion. Nonetheless, long-term exposure to short fibre chry sotile contaminated withvery low levels oftremolite simi lar to the material composition of many end products (also see Sebastien et al. [63] for evidence that tremolite levels are reduced during the processing ofchrysotile ore) is not able to induce mesotheliomas in humans. Good support for this comes from other investigations such as the NIOSH rat and primate inhalation study where long term, high-dose exposures to short fibre chrysotile failed to produce tumours even though the animal lungs con tained significant amounts of tremolite [Salomon and Stettler, pers. commun.]. Sluis-Cremer [59] also stated that `unless there is evidence to the contrary, no one can be certain that pure chrysotile fibres can cause mesothe lioma when inhaled at all. Final proofthat chrysotile fibre can cause mesothelioma can only come by the experimen tal inhalation of absolutely pure chrysotile'. The present study, in failing to find mesothelioma with such a pure form of chrysotile, thus goes a very long way to support Sluis-Cremer's proposal. The evidence in support of the `amphibole hypothesis' is far clearer in humans than in animals though the con straints of space preclude detailed discussion of this sub ject here. Although several recent review articles claim a clear, causal link between chrysotile and mesothelioma [64-66], their claims are based upon numerous false assumptions (see rebuttal in Mossman and Gee [67]), 28 JndoorBuilt Environ 1998;7:18-31 Dgren/Chatfield which include the contention that asbestos usage and con sumption patterns are reliable indicators of exposure, that lung tumours are appropriate `surrogate measures' ofme sothelioma incidence and that pleural fibre burdens and `early events' can predict mesothelioma risk. There has probably never been an attributable, clinically and patho logicallyproven case ofmesothelioma in any manufactur ing industry, e.g. cement, friction products, or textiles, amongst the many tens of thousands of workers where chrysotile alone has been used. The McDonalds [68-71] have clearly shown, in critical, comprehensive analyses of the available data over the last 20 years, that small amounts of amphibole are sufficient to account for the appearance of mesotheliomas in such settings. Those less inclined to believe this should be reminded of the follow ing parable [Browne, personal commun., 1997]: `A man drank whisky and water the first night and awoke with a hangover; the next night he drank gin and water, and again awoke with a hangover; the third night vodka and water with the same result. The fourth evening he decided he must avoid water since this was clearly the cause ofhis symptoms.' Appendix Independent pathologists' review (the full data set is available from the authors) . Wagner :; McConnell, . Kotin/ Diagnoses .. (MRCPn) :: Boorman .: \ Kuschrier 1: ; "reclassified . (NIEHS):! ' V; . (JM/SUNY) : after meeting 1 UICC/B 2 UICC/B 3 UICC/B 4 UICC/B S UICC/B 6 UICC/B 7 UICC/B 8 UICC/B 9 UICC/B 10 Rockwool 11 Glasswool/sR 12 Rockwool 13 Glasswool/sR 14 Glasswool/sR 15 Glasswool/sR 16 Glasswool/sR 17 Glasswool/sR 18 Glasswool/sR 19 Rockwool 20 UICC/B AdCa Ad AdCa+Ad AdCa - SqCa Ad(?) - SqCa Ad Ad(dys) Ad Ad Ad Ad AdCa AdCa + SqCa AdCa Ad Ad AdCa not sent AdCa AdCa AdCa + Sq diff - - AdCa + Sq diff AdCa or Ad hyper AdCa AdCa - not sent - AdCa AdCa + Sq diff AdCa or mesothelioma2 AdCa or Ad hyper AdCa or Ad hyper AdSqCa - AdCa metaplasia metaplasia AdSqCa metaplasia metaplasia AdSqCa metaplasia metaplasia metaplasia metaplasia - metaplasia AdCa (prob) AdSqCa AdCa metaplasia metaplasia AdCa BAH AdCa+BAH AdSqCa AdSqCa BAH AdSqCa Ad Ad + BAH Ad BAH BAH BAH AdCa AdSqCa Aden2 BAH AdCa+BAH MRC Pn. = MRC Pneumoconiosis Unit, Penarth, UK; NIEHS = National Institute of Environmental Health Sciences, Research Triangle Park, N.C., USA; JM = Johns Manville Company; SUNY = State University ofNew York, N.Y., USA. Ad = Adenoma; Ca = carcinoma; Sq = squamous; BAH = bronchiolar alveolar hyperpla sia; diff = differentiation; hyper = hyperplasia; dys = dysplasia; - = no lesion found; prob = probably; / = or; sR = without resin. 1 Selected 4 out ofthe 9 groups (see text). 2 Smith (Los Alamos), Hollander (TVO) and Smith (Fairleigh) diagnosed this case as mesothelioma. Short Fibre Coalinga Chrysotile - Lack of Tumourigenicity Indoor Built Environ 1998;7:18-31 29 References 1 Stanton M, Wrench F: Mechanisms of meso thelioma induction with asbestos and fibrous glass. J Natl Cancer Inst 1972;48:797-821. 2 Stanton M, Layard M, Tegeris A, Miller E, May M, Morgan E, Smith A: Relation of particle dimension to carcinogenicity in amphibole as bestos and other fibrous minerals. J Natl Can cer Inst 1981;67:965-975. 3 Pott F: Asbestos use and carcinogeneity in Ger many and a comparison with animal studies. 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