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Int J Anat Res 2020, 8(2.3):7557-63. ISSN 2321-4287 7557

Original Research Article

Neeta Chhabra.

ABSTRACT

Corresponding Author: Dr Neeta Chhabra, Associate Professor, Department of Anatomy, GSMedical College and Hospital, Nh-24, Near Railway Station, Pilkhuwa, Hapur -245304, India.Mobile: 9811113296E-Mail: [email protected]

Introduction: Formaldehyde is not just a human carcinogen but the cause of many other unwanted hazards;respiratory distress, red eyes, etc. Occupational health authorities throughout the world are therefore likely toincrease the strictness of regulations for the use of formaldehyde within anatomical disciplines.

Aim: This study evaluates the effectiveness of Phenoxetol (Phenoxyethanol) as suitable alternative to formaldehydefor long term preservation of cadavers.

Results: The soft consistency, colour and flexibility, of cadavers preserved in Phenoxetol, was found to be suitablefor dissection & demonstration purposes.

Conclusion:Phenoxetol proves to be a viable replacement of formaldehyde.Its efficacy as a preservative andfixative is supposed to be better than formalin. In addition to avoiding the well-known hazardous effects offormaldehyde, the use of Phenoxetol also prevents the pungent and irritating smell associated with formalin andother local and systemic manifestations of its toxicity.

KEY WORDS: Formaldehyde, Phenoxetol,Preservation, Cadavers.

INTRODUCTION

International Journal of Anatomy and Research,Int J Anat Res 2020, Vol 8(2.3):7557-63. ISSN 2321-4287

DOI: https://dx.doi.org/10.16965/ijar.2020.158

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Quick Response code International Journal of Anatomy and ResearchISSN (E) 2321-4287 | ISSN (P) 2321-8967

https://www.ijmhr.org/ijar.htmDOI-Prefix: https://dx.doi.org/10.16965/ijar

DOI: 10.16965/ijar.2020.158

Associate professor, Department of Anatomy, GS Medical C0llege and Hospital, NH-24, NearRailway Station, Pilkhuwa, Hapur, India.

Received: 27 Apr 2020Peer Review: 27 Apr 2020Revised: None

Accepted: 15 May 2020Published (O): 05 Jun 2020Published (P): 05 Jun 2020

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RG JournalImpact: 0.21*

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LONG TERM PRESERVATION OF CADAVERS: SUBSTITUTION OFFORMALIN WITH PHENOXYETHANOL: NEED OF THE HOUR

Cadaveric dissection has been the paradigm ofanatomy teaching since the Renaissance, anddefining experience of medical teaching sincethe 16th and 17th centuries [1,2]. The practiceof cadaveric dissection helps students to graspthe three dimensional anatomy and concept ofinnumerable variations. Dissection has also beencalled “sharp end” of medical education asthrough dissection, students are able to get thefeel of the human tissues and structures of thehuman body [3,4]. Cadaver and museumspecimens help the medical students to learnregional and topographic anatomy structure

including vessels, nerves and muscles of humanbody[5]. A comparison of educational toolsrevealed that human bodies have distinctproperties and that there are no viable alterna-tives[6].For the use of human bodies in dissection rooms,the appropriate preservation of the cadaver isthe most important prerequisite i.e. the cadavershould be safe from harm, destruction ordecomposition. This is achieved by treating thecadaver with special chemicals, and the proce-dure is called Embalming. Embalming leads toa long-term preservation of structure withminimal shrinkage and distortion of tissue and


also prevents over-hardening. The most commonchemical used for this purpose is Formaldehydewhich has been used as a fixative in anatomy,pathology, and in embalming since the 20thcentury [7]. But Formaldehyde has been shownto cause mutation in various primitive organisms[8,9]and in cultured mammalian cells [10,11].Furthermore, inhaled formaldehyde causednasal carcinoma in rats and mice and subcuta-neously injected formaldehyde caused sarcomain rats [12,13]. Recent epidemiologic surveysamong embalmers and industrial workersexposed to formaldehyde may indicate anincreased cancer risk even to humans [14].Formalin has an odour that many students andfaculty members find unpleasant. Chemicalhypersensitivity has been attributed to exposureto formaldehyde or other volatile compounds.Students develop many troublesome symptomslike nausea, headache, and gastrointestinaldisturbance during cadaver dissection orcomplain of disorders due to irritation of themucous membrane of the eye and nose [15].The anatomists, technicians, and students in thelaboratories are continually exposed to Formal-dehyde during their dissection course. The levelof exposure depends on the duration of timespent in the dissection hall and the workingenvironment [16].In developed countries, The Occupational SafetyHealth Administration (OSHA), formaldehydestandard and equivalent regulations in statesprotect workers exposed to FA and apply to alloccupational exposures to FA. The permissibleexposure limit (PEL) for FA in the workplace is0.75 ppm of air measured as an 8-hourtime-weighted average. The standard includesa second PEL in the form of a short-termexposure limit of 2 ppm which is the maximumexposure allowed during a 15-minute period [17].At concentration above these permissiblelimits, protective measures should be employedlike giving respirators, putting administrativecontrols, exposure monitoring and other requiredprocedures. Very few literature has beenpublished on indoor FA concentrations inMedical Colleges of India. Reports from othercountries have reported FA levels aboveprescribed standards that could be harmful tothose exposed.Several western countries have

restricted the use of formalin. But in India thereare no standards for air monitoring of formalde-hyde levels in dissection hall and also there areno rules governing the use of formalin and of itsinclusion in occupational health hazards. More-over FA level monitoring, cadaver positioningand ventilation strategies are also not rigorouslyinspected by any medical regulatory bodiesacross the world including India.Some laboratories have already found ways ofabandoning the use of formaldehyde andphenol for long-term preservation of specimensfor dissection and museum purposes. Mostwidely used among such methods is theimmersion of previously formaldehyde andphenol-fixed specimens in alcohol solutionsranging in concentration up to 75% [18]. Thisprinciple was reinvestigated by Bjorkman andChristensen in 1982 who found the atmosphericformaldehyde concentration was lowered tobelow 0.5 ppm when the specimens had beenextracted for about 3 months in 20-50 % etha-nol. However, ethanol in high concentrations isexpensive, flammable, and evaporates rapidly.Inaddition, most tissues were subjected to exces-sive hardening and loss of natural colours inthese concentrations. In lower concentrationsthe antimicrobial action of ethanol may beunsatisfactory [19].Another method described by Owen andSteedman (1956, 1958) was use of phenoxetol[20]. They found various zoological specimens,previously fixed by any desirable method, to bewell preserved for years in a solution of 1%propylene phenoxetol (1-phenoxy-propan-2-01) alone or in combination with 0.2%”Nipa”esters (“Nipastat” or mixture of alkyl esters of4-hydroxybenzoic acid). This technique wasrecommended for general use by Spence 1967,and Steinmann et al in 1975reported goodresults on veterinary medical and zoologicalspecimens. But was not reported to be used onhuman material [18,21]. Frolich et al carried outa study in 1984 with phenoxetol as long termpreservative where they found that thematerial had remained soft and flexible with aconsistency and colour retention suitable fordissection and demonstration purposes for upto10 years. Fungal attacks were rare and nobacteria could be grown from such specimens.

Neeta Chhabra. LONG TERM PRESERVATION OF CADAVERS: SUBSTITUTION OF FORMALIN WITH PHENOXYETHANOL: NEED OF THE HOUR.


Microscopical structure of most tissues alsoremained satisfactory after 5years in 1%phenoxyethanol [22].Tandon et al in a study carried out in 2014 foundphenoxetol to be a viable replacement ofFormaldehyde for long term preservation ofcadavers. Moreover it was observed thatstudents found experience with cadaverspreserved in phenoxetol very pleasant as it hasa fruity smell as compared to the offensive odourof formaldehyde [23,24].In view of above an attempt was made toimprove the environment of our dissection hallby substituting formaldehyde with phenoxy-ethanol (ethylene glycol mono phenyl ether) orphenoxetol, a non-toxic chemical for long termpreservation of embalmed cadavers.

washed thoroughly with tap water and kept ondissection hall tables for 24 hrs to remove thefixative by evaporation. They were then trans-ferred to a tank filled with 1% Phenoxetol inwater. The capacity of our tank is 1400 litres andtaking water displacement by cadavers into con-sideration, the tank was filled only with 900 litresof tap water and nine litres of phenoxyethanolwas poured into it thereby making 1% solution.The cadavers were left in this solution for aperiod of at least 3-4 months before they wereused for the purpose of dissection. The solutionwas prepared in the month of May and cadav-ers were taken out for dissection in September.During this period the tanks were regularlyobserved for any discoloration of fluid and signsof fungal infection. During the dissection sched-ule the bodies remained on stainless steel tablesfor the week, covered with sheets soaked in 1%Phenoxetol. During weekends the bodies werere-immersed in the tanks having 1% Phenoxetol.Phenoxetol in the tanks was renewed once inthe month of December, when the solution wasdiscoloured by exudate from the cadavers andagain after 3 months in the month of March. Thebodies were thus treated by a minimum of twosuccessive baths of 1% Phenoxetol to removemost of the remaining free aldehydes left by thefixative solutions. Dissected specimens werealso stored in 1% Phenoxetol and were used fordemonstration purposes.

MATERIALS AND METHODS

The present study was conducted at GSMedical College & Hospital Pilkhuwa, Hapur. 10cadavers (7 males & 3 females) between 56 to87 years of age were taken for the study. All thecadavers were those who had opted tovoluntarily donate their bodies before death formedical research purpose. These cadavers hadbeen embalmed 4-6 months earlier by single-point embalming. The embalming fluid as perbelow composition was injected in the commoncarotid or femoral artery using embalmingperfusion pump.Composition of Embalming fluid:Formalin: 2000 mlMethanol: 1000 mlGlycerine: 500 mlPhenol: 500 mlSodium citrate: 15 gmSodium borate: 15 gmThymol: Few crystalsWater: To make it to 8000-1000 mlAfter the initial vascular injection the cadaverswere observed for 24-48 hrs and supplementaryinjections of the same fluid was given locallywherever the initial injection proved unsatisfac-tory. The cadavers were then transferred to tanksfilled with 4% formaldehyde and stored therefor a period ranging between 4 to 6 months. Afterthis period the cadavers were taken out and

RESULTS

In the present study we found that the cadaversand dissected specimens preserved in 1%phenoxetol were good for dissection purposes.The unpleasant and intrusive smell of formal-dehyde and phenol traditionally found in dissec-tion rooms was markedly reduced. Much of therigidity of the tissues caused by the initialaldehyde fixation disappeared after some weeksin Phenoxetol and all tissues gain a flexible,partly elastic, consistency not unlike theconsistency of fresh unfixed tissue, and wellsuited for dissection and demonstrationpurposes, especially for demonstration ofmovements across joints. The colour retentionof tissues was good and the tissue remainsaesthetic even after longer use as prosections.There was no sign of microbial or fungal infec-tion in cadavers. We are of the opinion that 1%



Phenoxetol enhances life like tissue coloursrather than disturbing them. As far as cost ofthe solution is considered only one can of 25litres of Phenoxetol with an approximate costof Rs 13,000 was sufficient for whole yearwhereas the expenditure on formaldehyde isaround Rs 5,000. Thus cost factor of phenoxetolis not a big issue taking into consideration itslow toxicity compared to formaldehyde.

DISCUSSION

Formalin has been the most widely acceptedchemical for preservation of cadavers fordissection purposes, as it is economically anexcellent preservative and fixative solution. Thebenefits of using formalin, its cost effectiveness,and its proper fixation and efficient preserva-tion of tissue, have to outweigh its adverseeffects on the health of those working withit.Moreover formalin has been recently classi-fied as carcinogen which is even suspected ofcausing adverse reproductive and developmen-tal effects[25]. To limit the exposure of forma-lin various methods are being followed like ven-tilated dissection halls, use of exhaust fans anduse of gloves during dissections, still theobserved FA levels in dissection halls are foundbeyond the prescribed guidelines laid down byregulatory bodies. This is considered to beharmful for students and teachers health[26].Since formalin is toxic, an alternative and a saferfixative detection is important. Phenoxyethanol,discovered by Frolic et al in 1984 has been foundto be an excellent alternative to formalin. It isalready being used for long term preservationof cadavers by few medical colleges with goodresults [22].Phenoxyethanol is a glycol ether with knownbactericidal and antifungal properties with anoily colourless appearance and a characteristicpleasant odour [27]. It is a widely used preser-vative to prevent bacterial and fungal contami-nation, especially in cosmetic industry and forpharmaceutical products. It was tried early inlocal treatment of burns and wounds, especiallyagainst Pseudomonas aeruginosa or Pyocyaneainfections, because it proved to show little skinor tissue irritation on local application as a so-lution or ointment. It has shown low toxicitywhen injected subcutaneously, intra-peritoneal

or intravenously in various laboratoryanimals [28]. Phenoxetol is chemically relatedto propylene phenoxyethanol, which was used,by Owen and Steedman in 1958 as a preserva-tive for zoological specimens. Both substanceshave much the same antibacterial activity.Propylene phenoxyethanol though more effec-tive against moulds and yeast, should ideallybe preferred for preservation purposes. Sincephenoxetol gives fully satisfactory results as apreservative and is cheaper than propylenephenoxyethanol, phenoxetol is commonly used[20]. In a study carried out by Burke and Sheffnerin 1976 it was proved that phenoxetol as pre-servative gives satisfactory results from a mi-crobiological point of view and also for long-termstorage of human bodies for dissection purposeswhen compared with more conventional preser-vative techniques [29].Though Phenoxetol is combustible, it must bepreheated before ignition can occur (NPFA FireRating 1) and a harmful contamination of theair will not or only very slowly be reached onevaporation of this substance at room tempera-ture. Concerning potential health risks, its usehas been attributed to irritation, sensitization,and allergic contact dermatitis at low incidencerates in large cohort studies [30,31].The most important advantage of Phenoxetolpreservation still seems to be its low toxicity,as mentioned above. Skin contact which shouldbe avoided using the phenyl-mercury compoundseems to present no problem with phenoxetol[32]. On the contrary, Phenoxetol is recom-mended as an antiseptic ingredient in medicalcreams and ointments [24].Despite its widespread use for many years,contact allergy to Phenoxetol has been veryrarely described [7]. Erythema and scaling thatoccurred on intradermal injection of phenoxetolin rabbits were not associated with gross orhistopathologic changes and were not consid-ered toxicologically significant [32]. Few casereports exist on PE causing pain, headache,tremor, and central nervous system depressionwhen swallowed intentionally or inhaled in largequantities. Consequently, standard safety pre-cautions must be followed including room ven-tilation, protective gloves, clothing and safetygoggles when handling Phenoxetol [33].



cannot be used for initial fixation purposes as ithas no ability to arrest autolysis, and unlessother proper fixatives are used, the tissues willdecompose rapidly [22]. But recently publishedPhenoxyethanol-based embalming (“Crosado”technique) has shown to give reliable andreproducible results for the anatomical fixationover the period of nearly two decades of use atthe University of Otago. They have proved thatphenoxyethanol based technique is suitable forembalming human tissues for a variety ofdissection-based courses and prosections,providing a suitable basis for plastination andhistology. A major advantage of the techniqueis its potential to reduce the amount of formal-dehyde to a minimal content, while at the sametime being robust enough to be performed out-side of the anatomy setting. The specimensresulting from PE-based fixation were durablewhen used in the dissection course and provideda level of pliability to position the cadaver fordissection. There was no unpleasant or intru-sive smell as formaldehyde based or Thielembalming may cause, and the tissues remainaesthetic even after longer use as prosections.In fact the PE-based fixation complementsexisting embalming techniques and provides analternative to the use of hazardous chemicalsor minimizes its application (33). Brenner in2014 documented that with phenoxyethanol themuscles remain pliable and joints freely mobilecompared to formalin which causes extreme ri-gidity [7].Our objective to replace formaldehyde as longterm preservative with a less toxic alternativewas thus achieved. It must be stressed howeverthat any of such alternatives will have itsadverse effects too. Some animal experimentshave shown that oral or parenteral administra-tion of 0.5 to 2% phenoxetol can causehaemolysis in rabbits and liver and reproduc-tive toxicity in Swiss CD-1 mice [32,39].However, phenoxetol being a high boiling liquidwith a low vapor pressure, exposure to vapoursto a concentration sufficient to cause sucheffects is unlikely at room temperature [40]. Acase of an immediate hypersensitivity reactionhas been reported by Bohn and Bircher in2001[41].Limitations of the use of phenoxetol seem to be

The results of this study are in agreement withthose reported by Owen and Steedman in 1958on zoological samples and by Steinmann et al in1975 on veterinary and zoological samples[20,21]. Three other studies on human cadav-ers by Frølich et al, Tandon et al and Wineskiand English highlight the potential of PE inlowering the exposure to formaldehyde andphenol. They described that soft tissue pliabil-ity can be recovered by partly removing theformaldehyde and phenol from their fixatives[22,23,34]. Their findings are in large agreementwith the results presented here, where increas-ing pliability is found when tissues are exposedto the PE-based conservation fluid.From an anatomical point of view, Phenoxetolgives superior results to those of most otherpreservatives. Sehdev et al in 1963 proved thatthe colour retention of tissues preserved inphenoxyethanol was good and the tissueconsistency softer and more flexible than thatachieved by ethanol preservation [35]. Thiel in1992 and Eisma et al in 2013 observed that sinceno mineral salts are used in the preservativefluid, other embalming methods using such saltswill definitely be superior in terms of colour re-tention [36,37]. Smell and mucosal irritation maybe suspected at formaldehyde concentrationsas low as 0.15 ppm (38). In a study carried outby Tandon et al on medical studentsphenoxyethanol caused little skin irritation ascompared to formaldehyde and phenol.They reported few cases of mucosal irritationor smell with phenoxyethanol which may be dueto the formalin used during embalming ofcadavers [23].A second aspect of PE preservation is the onsetof bacterial or fungal growth. Though its onsetonly occurs under extreme storage conditions,for example, direct sun exposure of storagecabinets with condensing water washing outchemicals or contaminations introduced bynormal skin commensal micro-organisms of theusers or air-borne fungi. These can be success-fully taken care of by improvements in storagetemperature, thorough cleaning with Trigene andPE/Arquad-75, to treat and prevent contamina-tion [33].Earlier studies by Frolich et al had stressed thatPhenoxetol or propylene phenoxyethanol



requirement of large amount of chemical andits prohibitory pricing. In a very recent reviewarticle by Brenner et al, Phenoxetol has beendescribed as relatively inexpensive, non-flam-mable, slow to evaporate, effectively antimicro-bial and an excellent tissue preservative andsoftener [7]. Our studies are in agreement withBrenner et al as use of phenoxetol only slightlyincreased the overall expenditure on preserva-tive. Thus by replacing Formalin with phenoxetol,our efforts to improve the working conditions inthe dissection hall will benefit the students,teachers and the staff in charge of the embalm-ing procedure itself. The prolonged exposure ofthe latter group to formaldehyde fumes in theembalming room seems equally important toconsider. We believe that a reduced exposureof this group to formaldehyde vapours must besought via the reduction or replacement offormaldehyde as primary fixative and improvedventilation at critical places within the embalm-ing room. Towards addressing this issue the Thielmethod of embalming or its modification (Eismaet al, 2013) or Crosado technique can be triedand evaluated subsequently.CONCLUSION

The acute toxic effects of formalin are wellknown in medical fraternity. But the occupa-tional hazard due to FA is not given utmostconsideration despite of necessity due to lackof regulations either from governing authoritiesor regulatory bodies. An excellent alternative toformalin is phenoxyethanol. In our experiencePheno-xetol proves to be a viable replacementof formaldehyde. Its efficacy as a preservativeand fixative is supposed to be better thanformalin. In addition to avoiding the well-knownhazardous effects of formaldehyde, the use ofPhenoxetol also prevents the pungent andirritating smell associated with formalin andother local and systemic manifestations of itstoxicity. It is simple to use and adds only a smallcontribution to the overall cost of preservative.At present initial fixation of cadavers is beingdone with Formalin. It is recommended thatinitial fixation may also be slowly replaced anddone with phenoxetol. Thus with proper guid-ance and effort we may be able to give studentsan odour free and harmless environment in thedissection hall.

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How to cite this article:Neeta Chhabra. LONG TERM PRESERVATION OF CADAVERS: SUBSTITUTIONOF FORMALIN WITH PHENOXYETHANOL: NEED OF THE HOUR. Int J AnatRes 2020;8(2.3):7557-7563. DOI: 10.16965/ijar.2020.158


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