The Potential Cancer Risk Associated with the Use of Ethylene Oxide

as a Sterilant for Cord Blood Processing Systems


Byron E. Butterworth, Ph.D., Fellow ATS

Consultant for ThermoGenesis Corp.


John R. Chapman, Ph.D.

Vice President, Scientific Affairs

ThermoGenesis Corp.


March 26, 2007


Executive Summary

We want to make the agency aware that the current FDA guidance entitled “Class II Special Controls Guidance Document: Cord Blood Processing System and Storage Container, Toxicity of Chemical Sterilants,” as written may present a serious potential cancer risk to cord blood transplant recipients.  In the guidance document, the FDA allows a maximum residual level of ethylene oxide of 5 mg/device.  We strongly urge the FDA to reconsider this position due to the following considerations.

·         Stem cells are long lived, continuously dividing cells that will repopulate the bone marrow and give rise to all hematopoietic cells for the rest of the transplant recipient’s life.  As such, no procedure is acceptable that might introduce mutations that could lead to cancer in these cells.

·         Ethylene oxide is a potent, direct acting mutagen and clastogen that has been demonstrated to induce hematopoietic cancer in mice, rats and human beings.

·         The direct exposure of stem and progenitor cells to ethylene oxide greatly increases the induction of mutations resulting in a higher risk of cancer for the eventual transplant recipient.

·         For direct acting mutagens, there is no recognized “no effect” dose in any FDA accepted cancer risk assessment model.

·         This safety issue results from the use of ethylene oxide as a sterilant and can be simply eliminated by using either gamma irradiation or steam

·         Prudence dictates that any units that have already been archived in cord blood banks that were exposed to residual ethylene oxide should best not be used without full notification of the transplant physician and patient.    


Nature and use of Stem Cells in Cord Blood

Cancer chemotherapy causes sufficient cytotoxicity to the bone marrow to eliminate malignant bone marrow cells, but leaves the patient with no functioning normal hematopoietic cell population.  Bone marrow transplantation has been used historically to reconstitute the hematopoietic cells for such patients.  The use of the stem cells in umbilical cord blood for hematopoietic reconstitution in patients with hematological malignancies has emerged as a viable alternative to bone marrow transplantation.  After intravenous injection of cord blood to the patient, hematopoietic stem cells migrate to and repopulate the bone marrow.  These stem cells will be long lived, continuously dividing cells that will create all hematopoietic progenitor cells for the rest of the patient’s life.  Cell proliferation is initially involved in repopulating the bone marrow and subsequently in producing new cells over a lifetime.  Therefore, it is particularly critical for these cells that no promutagenic DNA adducts are formed (such as those produced by covalent binding of ethylene oxide) that would yield mutations upon cell division.  Further, exposure to chemical or physical agents that cause mutagenic changes should be avoided to the greatest degree possible to eliminate the potential creation of precancerous or cancerous mutations that may be expressed in these progenitor cells.


Genotoxic Properties of Ethylene Oxide

The direct-acting alkylating properties of ethylene oxide that make it a good sterilant also make it a potent mutagen.  The compound is inherently DNA reactive without the need for metabolic activation.  A National Library of Medicine search yields 107 articles for “ethylene oxide and mutagenicity” and 47 articles for “ethylene oxide and leukemia”.  Ethylene oxide is mutagenic in the Ames bacterial mutagenicity assay (Victorin and Stahlberg, 1998) and in human diploid fibroblasts (Kolman et al., 1992).  Ethylene oxide induced DNA damage in a variety of human cells in culture including lymphoblasts (Adam et al., 2005).  Ethylene oxide yielded DNA adducts and mutations in various cells including bone marrow in exposed rats and mice (Recio et al, 2004; Rusyn et al, 2005; Sisk et al., 1997; Walker et al., 1997).  Cytogenetic damage was observed in bone marrow cells of rats exposed to ethylene oxide (Lorenti Garcia et al.,  2001).  Workplace exposure to ethylene oxide resulted in increases in the frequency of sister chromatid exchanges in exposed individuals (Laurent et al., 1983).


Mutagenic and Carcinogenic Susceptibility of Stem Cells to Ethylene Oxide

Current FDA guidance proposes the allowance of 5 mg of residual ethylene oxide per cord blood processing device (FDA, 2007).  Of particular concern is the fact that this residual ethylene oxide will come into direct contact with the stem cells.  Exposure of human beings to ethylene oxide increases the risk of mortality from lymphatic and hematopoietic neoplasms (Hogstedt et al., 1979a, 1979b; Stayner et al., 1993).  Ethylene oxide has been shown to produce various cancers in rats, including leukemia (Snellings et al., 1984; Lynch et al, 1984).  A different spectrum of ethylene oxide induced tumors was seen in exposed mice, including lymphomas (NTP, 1987).  In summary, the fact that neoplasms of the hematopoietic system are commonly produced by this agent support the need prevent its use in the manufacture of disposables used in the processing of cord blood units for transplantation.


 In vivo, hematopoietic stem and progenitor cells in the bone marrow are partially protected by  competing  ethylene oxide binding sites in proteins and other molecules of tissues and blood followed by their, detoxification, metabolism, and/or excretion.  In the case of cord blood stem cells being prepared and stored for transplantation, the naked cells are exposed directly to the reactive ethylene oxide.  The potency of this mutagenic agent will be maximized when present in the environment of the cells as a residue of the sterilization process.  Presumably, the genotoxic risks will be far greater than estimated in epidemiology studies and whole animal risk assessment models.  The following observation emphasizes the seriousness of this issue.  Mouse lymphoma cells grown on glass were compared to cells grown in polycarbonate culture flasks sterilized by either autoclaving or exposure to ethylene oxide.  The mutation frequency increased 6- to 14-fold in the ethylene oxide treated flasks while no increase was observed in the autoclaved flasks (Krell et al., 1979).


There is No “Safe” Dose of Ethylene Oxide

The current FDA guidelines specify a maximum residue limit of ethylene oxide of 5 mg per cord blood processing device (FDA, 2007).  Approximately 50 to 150 ml of cord blood would dilute the ethylene oxide and then reside in a concentration of about 0.1 to 0.03 mg/ml (100 to 30 mg/ml) of ethylene oxide.  We are unaware of any data demonstrating the safety of ethylene oxide at this or any other residual level for the processing and storage of human stem and progenitor cells.


Some relevant quantitative comparisons are instructive.  Kolman et al., 1992 measured the induction of mutations in cultured human diploid fibroblasts at 2.5 mM (110 mg/ml) ethylene oxide, which is about equivalent to the potential concentration of 100 mg/ml that might be found in a cord blood preparation.


Adam et al., 2005 measured ethylene oxide induced DNA damage in cultures of human lymphocytes and breast cells at a concentration of 20 mM (0.88 mg/ml).  The potential concentration of 100 mg/ml that might be found in a cord blood preparation is over 100 fold higher than that DNA damaging concentration.


Various risk assessment evaluations conclude that exposure levels of about 1 ppm or below of airborne ethylene oxide would not be expected to produce a significant increased risk of leukemia in an exposed population (Austin and Sielken, 1988; Kirman et al., 2004; van Sittert et al., 2000).  A continuous 1 ppm exposure to airborne ethylene oxide for an individual would result in a steady state blood level of 0.009 mg/ml (Fennell and Brown, 2001).  Thus, the potential concentration in the cord blood under the current guidelines of 100 mg/ml would be some 11,000 fold above the estimated safe blood level.


The FDA maximum allowable residual level of ethylene oxide of 5 mg/device is 100 to 1,000 times higher than the alkylating agent residuals introduced by pathogen reduction technologies for cellular blood products, which are yet to be approved for human use in the United States.


For direct acting mutagens, there is no recognized “no effect” dose in any FDA or EPA accepted carcinogen risk assessment model (EPA, 2005).  FDA guidance is extremely restrictive in allowing clinical trials with drug candidates that exhibit even minimal genotoxic activity (FDA, 2006).  Without question, the direct exposure of cord blood hematopoietic stem and progenitor cells to any concentration of the DNA-reactive mutagen ethylene oxide would impose an avoidable risk to transplant patients.


Use of Gamma Irradiation or Steam is Strongly Recommended

The safety issue of ethylene oxide can be eliminated simply by the use of gamma irradiation or steam for the sterilization of all products used for cord blood processing and storage.  It is our belief that manufacturers of adult peripheral blood collection sets have already made this transition to gamma irradiation or steam for citrate filled collection bags, and have discontinued the use of ethylene oxide.  We strongly encourage the FDA to adopt the position that all stem cell processing devices and storage containers be sterilized by gamma irradiation or steam.


In addition, we question whether any units that have already been archived in cord blood banks that were exposed to residual ethylene oxide should be used in patients without extensive testing to assure their safety.  At the very least, the transplant physician should be made aware of whether the stem cell product that they are selecting has been exposed to ethylene oxide.  This transparency in information will allow the physician to make an informed choice regarding product safety.





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