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Asbestos Contamination of Parenteral Drags is not now known whether it occurs as the result of other routes of administra Abstract. Chrysotile asbestos has been found in approximately one third of tion, although ingestion may be suspect the samples from two sets of 17 widely used parenteral drugs. in view of some evidence of increased Appropriate filtration procedures for sterilization and removal of foreign mat Another reason for chrysotile's effec tiveness as a filter is the possibility of its incidence of gastrointestinal cancer among asbestos workers (9). In the ex perimental animal, it has been demon ter from parenteral solutions became fragmentation in solution. The fibers are strated that parenteral administration necessary several decades ago (/). The not unit structures, but rather bundles (typically, of 10 to 30 mg) of all vari pharmaceutical industry found asbestos of ultramicroscopic fibrils, each 200 to eties of asbestos can produce neoplasms filters useful and effective for this pur 400 A in diameter and 1000 A to (10). Such neoplasms have appeared at pose, and they have become widely used. several micrometers in length (3). Un the site of injection of fibers. Other Unfortunately, we have found that treated, the fibers in a filter can fragment studies have also shown that adminis such filters add a contamination of their into a much larger number of fibrils, tered fibers are readily disseminated own to the filtrate and that, at least in the number of effective filtering elements from the injection site, both hcmatog- the past 3 years, parenteral solutions being vastly increased and their surface enously and along draining lymphatic used for intravenous, intramuscular, and area, concomitantly extended. channels (11). intraperitoneal therapy in the United Serious health hazards have been An investigation of a number of par States have often contained measurable identified with asbestos exposure (4); enteral drugs has been undertaken. Sin amounts of asbestos. these include pulmonary fibrosis and a gle-dose vials of widely used drugs for The physicochemical properties of variety of neoplasms (such as broncho parenteral injection, taken from the asbestos make it highly suitable for fil genic carcinoma, pleural and peritoneal pharmacy stock of Mount Sinai Hospi tration, and it is therefore widely used mesothelioma, and perhaps other neo tal during 1969 and again about 1 year in chemical, food, drug, and other in plasms). Once thought to be limited to later, were examined for their asbestos dustries. The individual fibers and fibrils heavily exposed asbestos workers, these content. Analysis for chrysotile as are both fine and strong, ranging in neoplasms are now known to occur bestos was made by using optical and diameter from 200 A to tenths of with much less exposure, such as that electron microscopy. micrometers and in tensile strength experienced by those living in the house Samples for analysis were obtained from 20,000 to 60,000 kg/cm2. hold of an asbestos worker (5), working by filtering the contents of each vial Hydrated magnesium silicate miner in shipyards in which asbestos is used through Millipore AA membrane filters als, they tend to be chemically inert in some areas (6), or merely living (diameter 13 mm, pore size 0.8 /xm). and resist attack by most chemicals (2). within 800 m of an asbestos plant (3, 7) Drugs received from manufacturers as There are several varieties of asbestos or in the vicinity of asbestos operations powders were prepared with sterile (chrysoule, amosite, crocidolite, antho- (*) water, which in turn had been filtered phyllite, tremolite), and special proper Such human disease has been the through membrane filters (pore size ties of each can be utilized for specific result of inhalation of asbestos fibers. It 0.8 pm); drugs received as solutions filtration purposes. Thus, crocidolite has been used for gas mask and cigarette filters. Chrysotile is widely used in food processing industries (sugar, lard, beer, and so forth). For pharmaceutical use, chrysotile again has been the asbestos variety of choice. These filters are used at many points in the manufacturing process, from the preparation of raw materials, through the intermediate steps, to the final filtra tion of the product. The latter is im portant: in order to minimize <n.prevent bacterial growth (and the presence of bacterial toxins), it is often desirable to complete production procedures within 8 hours or less. Speed of final filtration may be critical in such production schedules. Chrysotile allows rapid filtra tion without reduction of efficacy. Fig. 1 (a) Chrysotile asbestos observed by polarized light microscopy from a singlevial sample of tetracycline. This particular fiber was 1 mm in total length (magnifica tion x 1300). (b and c) Chrysotile ob served by electron microscopy in ampicillin and vancomycin, respectively (magnifi cation x 25,000). 14 1ULY 1972 171 *cre filtered with no additional diluUob. While the affective pore sue jf the membrane flJten used is larger ihno the diameter of many ol the asMato* fibers of interest, it was found that the surfacecharge properties of the fillers and the asbestos fibers, as well is the circuitous path through a filler.* Silo* virtually complete coUecuoo of til isbestot ma terial At tbe ume of preparation of the drug samples, cootrol simples were prepared with filtered sterile water. One tuefl control was processed with each of four drug samples All were prepared in a laminar-flow, filtered air hood, and strict dean-room procedures were fol lowed it normal room iir may provide a ready source of contamination. Samples were prepared for optical microscopic examination by mourning one quarter nf the membrane filter with collected material on a microscope slide. The filter waa cleared for ie*mg with a 10 percent solution of membrane fil ter material in a I 1 mixture of di ethyl oxa.ate aod dimethyl phthaJate The cleared 61 ter was wanned by meaoa of polarized light cmcro^rtpy *t a nmg. nification of * 400 All birefnogent fibers longer than 5 i*m and with a length-to-width ratio greater than 5 were counted Whenever possible, identification of chrysotiie asbestos was made on the basis of ex tinction angJe and morphology. This identification could be made with rea sonable auurioce only for those fiber* longer than about 100 For identification of asbestos by elec tron rmcrovnpy, one half of the filter and collected maieniJ u uhed in a low-temperature ac.ivated oxygen asher t*2). The residue was dispersed by grinding for 2 to 5 minutes in a 1 per cent solution of nitrocellulose dissolved iq amyl acetate, and allowed to dry A, known fraction of the dried film was irajis/ened to a 200-meih. Forrovar- coaied eleettoo microscope gnd. The prepared grids were then scanned at a magnification of x 42 000, and the number of ashetos fibrtls and their size were determined id two to four gnd square* f 100 by 100 In some selected cases, the residue from a par tially ashed membraoe filter along with collected residue was mounted directly on the electron rrucroacope gnd. In aJJ cases* tbe identification of chrysoide asbestos was made oo the basis of tti unique morphology (J). Other tabula* minerals, such as hailoysKe, exist in nature, but ibeir pre^a ui these samples can be ruled out oo tbe basis of size, geologic rarity, and abeence from manufacturing processes for par enteral drop. Dunog 1969, 16 sample* were (canned optically, and all fibers longer than 5 -ra were recorded. Fibers longer than lOO uun that could be identified with reasonable certainry as asbestos were ooted. The results of thi> snalysia are given in Table 1 {13) Many fibers and panicles, in addition to asbestos, were present in the filtered samples. They included cellulose fibers, starch granules, and loorgnmc and organic fibers of unidentified composition Moreover, in optical scanning, numer ous fibers were seen that were consistent with asbestos, but idenhficatioa could not be made reliably and they were 126090015 TaBfe l Fibrous parucwlaus in umpla o( pununi flrvai obtained durine two eeriodj of tunc. Obeer-rtuoa rre ntde by optical nu- cmwope <0) or tiectroo menmopc iE> Tbe opecal driemuaaikM were side oo oae qinner of each sample. tbe ekcuoomKiosccpe deui- mma'ion* on 104 of etch temple AJl dnjp e-cre labeled for tntraeenouj use ecep in^te "lenufied by IM for icitrarmitcolsr or IP fo/ jurxperitnoeU Amount; of >mplea are ui gram* unieu specified otbermae "Yea" ifter optical dua indicates Uiei tome fibers oi U>e iid- pie were denufed i cHnwmle by optical micmacopr. "Ink o*n? Qben seen io all sample* could tne been asbeetce. the majority m ui XU -xf--- of 27 x IO*' i or low for ihe eelmiaied DIMS at -- cooJd have resulted from Uie analysis background; A. Dumber fibers by O, Dm* <) Quysotik fibnfc by I97MI IV) Aabevtoa, tftjfnttr) aSuT (1a^p4ie3 1970-7! Sodtua ampkdUfl Ccphaferidine Wwiimn crpbiloiho ChlortmptmKari CoriBMft <IM> lioceaycie HQ ^diun fifihit;JUn Nocoyein sulfate* (lM> Mcotayaa julfau* (IP) Sodium oxacillin Suvptgoycn wttate* (IM) Srapunyoa wlfalc* (JM) $o]fiB#iins3k diolamina TeenerdiDc RQt Tetneydice HOt VaacomyOn HC1 Potauium peruoIM G Sodium peQit iltia 0 Wales control 202S : i 2 49 91 51 IQ 24 < I 101 57 SI 2* 35 11 15 9 6 24 7 14 It * 7 16 7 1 14 4 1 >100 >100 10 15 35 U 10 12 27 lOt * f^iKate aalrw e< mW Ckv amulaemRis of iba mb* dm* rPupbcw maiyM* oc tampiM as uas lo< W uaH oaaufvnurti { Acr*# at * taspfn ranmi Iroo t 2 x ift- io 21 x lb-* * scopes. ' - *** ST0060925 not recorded as asbestos. Figure la shows a portion of a chrysotile asbestos fiber found by optic*! microscopy. To confirm the presence of asbestos, 6 of the 16 samples taken in 1969 and a duplicate of one of the positive samples were scanned by electron mi* croscopy. In five of the seven cases, in cluding ail those in which asbestos fibers were identified optically, concen trations of asbestos several times those in background controls were found. Examples of asbestos found in two samples by means of transmission elec 1. See, (or example, L. C. Milkr, in Setter at Lmtt Vobamt Farenmrtt Sohaiom, Smtauat Symposium Froettdin$r (Government Printm* Ottce, Wnhmiton. D.C, 1967), p. 6. X S. Spiel end J. P. Lctneweber, Environ. Rat. X 166 (1969). ). A. M. Lenntr. I. /. SeUkofl, A. Suuc, Arch. Environ. Httitk n. Ml (1971). 4. I. J. Sellkofl. (, CRsa, E. C. Hammond. /. Amur. tied. Am. IN, 22 (1964). 3. M. L Newhouee end H. Thompson, BrU. J. tnd. tied. n. 261 (1963). 6. P. O. Harriet, Ann. Oeeup. Brt- 1L 133 (1961); 1. ScumpUun, BrU. J. tnd. tied. 2B, 39 (1971). 7. I. Lieben and H. Piauwtu, Arc*. Environ. Health 14. 339 (1967). I. t. C. Waancr, C. A. SIsox P Marcfcand. Bril. J. tnd. tded. 17, 260 (1960). * ,9. L 1. SeUkoC, E. C. T1m........ I J. rw. j Amor. tded. Am. 64 106 (1961). 10. W. E. Smith, L. Millar. R. B. Ehoaar. B. D. Hobeit, Ann. N.T. Acad. Set UB, 454 (1963); S. C. Weiner, Nature 196, 110 (196Z). 11. F. I. C. Roc, R. L Carter, M. A. WaMn, J. S. Harriniton, Int. I. Cmetr X 621 (1967); X- Keneiawn, XLC. RMeck, R. L. Carter. F. 1. C. Ron, BrU. 1. Cmetr M, 96 (1970); A. Holmes and A. Mortal. HmvreU Report AEREJ3B9 (Atomic Enertr leeeenh Eatab- Uafcmmt, Harwell. Bertaidre, 1967). IX C. Berkley, X Chora, I- X SeUkoft W. E. Smith. Ann. N.T. Aend. Set 11X 4S (1965). 13. Them date wen prreenred at t aamtaar of the Food and Dru Adminietradoo on 7 October 1969 end at dm annual meedni at the Parenteral Oral Ataodadon in New York City on 30 October 1969. 10 March 1972; revired 13 May 1972 m tron microscopy are shown in Fig. 1, b and c. During fall 1970 and spring 1971, a second set of drug samples was inves Crustacean Color-Change Hormone: tigated to ascertain whether asbestos might still be found in typical par Ammo Add Sequence and Chemical Synthesis enteral drug preparations. In this sec ond study, only ultramicroscopic anal ysis was undertaken of the residue from filtered drugs. The results shown in Table I indicate that contamination of parenteral drugs by asbestos was still a Abstract. The blanching hormone of the prawn, Pandaius borealis, is pGlu-LeuAsn-Phe-Ser-Pro-Gly-Trp-NHv Its structure was settled by a combination of mass spectrometry and Edman-dansyl analysis of a thermolysin fragment. Confirmation of the structure was obtained by chemical synthesis from amino acids. This neurosecreted hormone is active in picogram amounts when tested in shrimps. common occurrence. Here, the quality of asbestos in 6 of 17 samples signifi cantly exceeded background levels. The amounts of asbestos estimated in some of these samples exceeded a mi crogram and were much higher than those reported for other environmental circumstances. Asbestos concentrations measured in ambient air, for example, are typically in the range of nanograms Many crustaceans have the ability to ebange their body color in order to match their background. These color changes are brought about by hypodermai chromatophores, that is, specialized cells containing movable pigment granules and having richly ramified cell processes. Crustacean chromatophores are controlled via neurosecreted hor G^.mGIYo Leuj itPheo.ggPiOi oiSezi.isTrpioo (J), which accounts for 89 percent of the weight of the hormone. The hormone is electrophoreticaily immobile at acid, neutral, and alkaline pH (<5), in accordance with its blocked NHo-terminus, and proving that none of its carboxyl groups is free. Digestion of the hormone (23 pg) per cubic meter. Contamination with nanograms or micrograms of asbestos may be evaluated with the knowledge that 10- g of asbestos might represent mones, which are released from nerve endings in the sinus gland, a neurohemai organ located in the eyestalks of most decapod crustaceans (1). with thermolysin and fractionation of the digest on a column of Sephadex G-25 gave two major peptide fragments. Upon acid hydrolysis, one of these 10s fibrils of a size typically seen in One color-change hormone, the gave aspartic acid, glutamic acid, and drugs (400 A in diameter by 1000 A blanching (red-pigment-concentrating) leucine in about equimolar proportions. in length). It should be noted that nega hormone, has been isolated from eye- Since no free NHj-terminal group was tive or indefinite results for a particular stalks of the prawn, Pandaius borealis obtained by the DNS-C1 method (7), sample do not guarantee the absence (2). This hormone, which is active in this peptide was an NH2-terminal frag of asbestos in the drug lot from which shrimps in picogram amounts (J), is ment of the hormone. Acid hydrolysis the sample was taken. A single vial is present in very minute amounts- in the of the other peptide yielded about equi an inadequate sample of a large produc- crustacean eyestalks (J) and, therefore, molar amounts of glycine, phenylala tion run. Moreover, the drugs" sampled only about 90 pg of the pure blanching nine, proline, and serine. In addition, represent only a small fraction of those hormone has been available for our it contained tryptophan, which was de on the market. On the other hand, the studies of its structure. The hormone termined by ultraviolet spectroscopy. It finding of asbestos in one third of was found to be a small peptide with a had a free NH2-terminus, and analysis the single vials that were examined in blocked NH2-terminus (2), and we by the Edman-dansyl method, essenti this investigation over a 1-year period now report the deduction of its com ally as described by Gray and Smith indicates significant asbestos contami plete structure, which we have con (8), established its sequence as Phe- nation of some parenteral drugs at this firmed by chemical synthesis. Ser-Pro-Gly-TrjvNHj. The COOH- time. Quantitative amino acid analysis (4) terminus was identified by omitting the W. J. Nicholson on 8 pg of the isolated hormone (after hydrolysis after the DNS-CI treatment C. J. Maggiore hydrolysis in 6M HQ at 110C for 24 which followed a four-cycle Edman I. J. Seldcofp hours at reduced pressure) and de degradation. The DNS product so ob Mount Sinai School of Medicine of the termination of its tryptophan con tained was identical to a reference DNS- City University of New York, tent by ultraviolet spectroscopy (2) tryptophan amide when compared in New York 10029 gave the following composition: Asp121- polyamide thin-layer chromatography 14 JULY 1972 173