Antimicrobial Preservatives Part Three: Challenges Facing Preservative Systems

Sunday, January 1, 2012

Introduction

The third article in this series deals with the many external challenges that face the pharmaceutical scientist tasked with developing preservation systems for multi-use oral, topical and parenteral medicinal products. This article provides views on safety, regulatory, public relations issues and utilizes two case studies to highlight the very real issues encountered whilst trying to preserve multi-use pharmaceutical products.

Safety and Side Effects of Preservatives

An ideal preservative should be active only against microbial organisms, with little or no adverse effects on mammalian cells. This is a challenging remit and, in reality most preservatives have some effect against both microbial and mammalian cells.

Alcohols are generally considered to be safe. However, benzyl alcohol is not recommended for use in parenteral products due to fatal toxic syndrome in low weight neonates [1, 2]. Sensitization to benzyl alcohol is generally low in topical products [3]. Similarly, the long chain alkyl alcohols, cetyl and stearyl alcohol, are infrequent sensitizers [3]. 2-Phenylethanol can be mildly irritant to skin, eye and mucous membranes [4-5].

Carboxylic acids, e.g. benzoic acid can be gastro-irritant and also mildly irritant to skin, eye and mucous membranes [6-7]. Sensitivity is generally considered to be mild in nature [7]. Sorbic acid sensitization is considered relatively uncommon [3]. Similarly, allergic dermatitis and allergic conjunctivitis are considered to be relatively infrequent in nature. Systemic toxicity has not been reported [3].

The parabens are considered unsuitable for parenteral and ophthalmic use due to irritancy [8-10]. There are numerous reports of delayed hypersensitivity to their topical use [3]. However these reactions are fairly uncommon[11].

Adverse reactions attributable to phenol are fairly rare, probably reflecting the low inclusion levels that are typically utilized. The total should not exceed 50mg in any ten hour period [12]. Chlorocresol, whilst less toxic than phenol can be irritant to skin, eyes and mucous membranes. It cannot be used in intrathecal, intra-cisternal or peridural injections [12-13]. Cross sensitivity to chloroxylenol has been reported [14-15]. However, chloroxylenol is generally considered to be less irritating than chlorocresol [3]. Hexachlorophene usage has declined because of concerns over neurotoxicity [3].

Sensitivity to the quaternary ammonium compounds (QACs) e.g. benzalkonium chloride (BKC) is infrequent, but it is a contact irritant and can exacerbate pre-existing dermatoses [3]. BKC should not be used in contact lens solutions where it can bind to soft lenses and then induce ocular irritation [16]. A maximum concentration of 0.02% of benzethonium chloride is recommended for ophthalmic and parenteral products [17]. BKC can cause broncho-constriction in some asthmatics [18]. Overdosing with solutions that exceed 0.03% may require urgent medical attention [19].

Organo mercurial preservatives were widely used in topical and parenteral formulations. However, concerns about the toxicity of mercurial compounds are regularly expressed [20] leading to usage constraints, e.g. intra-vaginal use [20]. Phenylmercuric salts have the potential to cause mercurialetis when used in eye drops. Incidence is fairly low (ca. 6%) and does not lead to visual impairment, but is not suitable for extended use [21]. Phenylmercuric salts are skin irritants at concentrations of 0.1% or more [22].

Thimerosal is a potent sensitizer, particularly in topical products [3]. Reactions occur in soft contact lens users and up to 10% of wearers may be impacted [23]. Hypersensitivity has been reported, hence it should not be used in either eye drops [24] or vaccines [25]. Indeed, EMA and FDA have recommended the general phasing-out of thimerosal in vaccines [26-27]. However, despite a concerted media campaign (see Public Relations Challenges) regulatory bodies found no link between thimerosal and incidences of autism [28].

Biguanides, e.g. chlorhexidine are used primarily as topical disinfectants. Levels in ophthalmic preparations must not exceed 0.05% [29].

The formaldehyde donators, e.g. imidurea [30], bronopol [31] are widely used in cosmetic and topical products, being generally considered non-irritant (up to 0.1%) and non-toxic [32]. However, there is a growing body of evidence linking both with sensitization (caused by the release / action of formaldehyde), particularly in vulnerable populations [3, 33].

EDTA is sometimes used as a preservative enhancer. Dose-related broncho-constriction has been observed when it is used in nebuliser solutions, and it use is now discouraged in such products [18]. EDTA salts can cause nephrotoxicity and should not be used in patients with renal impairment. EDTA can readily chelate calcium, leading to calcium depletion [34]. Despite such reports EDTA is generally considered as having low toxicity.

The potential for undesirable side effects, needs to be carefully considered when selecting a preservative system. At the same time these concerns need to be balanced with the low concentrations and the low probability of adverse effects, particularly if the medication is for short term use.

Public Relations Challenges

The popular press, at times seem to seek to engender a chemophobic mindset in its readers by publishing articles based on questionable, hypothetical, or even non-existent scientific evidence [35].

Such sentiments are particularly germane to many so-called “Healthscare” reports. Public reaction can, in extreme cases be irrational and lead to unforeseen consequences. Muslim clerics in Kano, Nigeria claimed (without any material evidence) that multi-use preserved Polio vaccine provided by the World Health Organisation contained oestrogens to ‘sterilize’ the local male population. The vaccine was banned from this region for 12 months. A subsequent polio epidemic claimed 792 victims [36]

The organo mercurial preservative, thiomersal has been used in vaccines since the 1930’s following the deaths of 28 children from staphylococcal infection following immunisation with an unpreserved diphtheria vaccine. During the past two decades, reports claiming a causal link between pediatric vaccines and autism have claimed that thiomersal is the culprit. Regulatory agencies in some countries have accordingly moved to eliminate it or to reducing levels in pediatric vaccines [26-27]. However, it is still commonly utilized in influenza vaccines.

The claim that autism was associated with a special form of mercury poisoning [37] was subsequently refuted by the American Academy for Pediatrics [38]. An independent committee of the US Institute of Medicine and the US National Institute of Health (NIH) reported that “neither thimerosal-containing vaccines nor MMR vaccine are associated with autism. The hypothesis regarding a link between autism and MMR vaccine and thimerosal-containing vaccines lack supporting evidence and are only theoretical. Future research to find the cause of autism should be directed toward other promising lines of inquiry that are supported by current knowledge and evidence and offer more promise for finding an answer.” [38]. A recent study in Italy backed up this position. No link was found between the presence of thiomersal in whooping cough vaccines and autism in 1403 children, 10 years after immunisation [39].

Media reports have linked parabens in under-arm deodorants with breast cancer [40]. Cancer specialists consider that there is no plausible biological mechanism to explain such a link. Furthermore, the sample size in the reported study was very small [41-42]. The dermal enzyme, Esterase III in human keratinocytes is sufficient to hydrolyze any paraben residues on skin from applied ointments and other dermal products [43].

Regulatory Challenges

The German Federal Institute for Drugs and Medicinal Devices (BfArM) have proposed that benzalkonium chloride (BKC) is removed from intranasal products in Germany because of concerns about mucociliary effects. The claim is based on in vitro and in vivo investigations in rats [44-46]. Such claims appear to contrast with clinical experience. The intranasal corticosteroid product, Flixonase (fluticasone propionate) which is preserved with benzalkonium chloride, improved nasal mucociliary clearance following twice daily use, over one year [47]. Another controlled six-week study in patients with perennial rhinitis evaluated the following formulations using a crossover design:

  • fluticasone propionate nasal spray preserved with BKC.
  • placebo solution containing BKC.
  • placebo solution without BKC.

The study showed no deleterious effects following intranasal application of BKC. There was no evidence of disturbance of mucociliary clearance that could be attributed to BKC [48].

A recent review of 18 studies (14 in vivo and 4 in vitro) explored the effects of long and short term exposure to solutions of BKC, dosed at concentrations ranging from 0.00045% to 0.1% [49]. There were conflicting data and opinions across the various reports but 8 studies (including one 6-month and one 12-month study) showed no deleterious effects attributable to BKC. Of the remaining 10 studies, 2 showed statistically significant differences between BKC and placebo [50-51]. In these cases the active ingredient was oxymetazoline, an alpha-2 agonist used to treat nasal congestion, but associated with clinical reports of rhinitis medicamentosa. Hence any effect cannot be categorically attributed to the BKC. In the light of such findings the rodent studies, underpinning BfARM’s stance may not be relevant to clinical experience.

Marple et al [49] indicated that; ‘Given the limited number of materials that are available to serve as preservatives, as well as the years of clinical safety exhibited by BKC, careful risk-to-benefit analysis is warranted before dismissing it as a preservative option’. The same authors go on to say ‘removal of antimicrobial preservatives in cosmetic or pharmaceutical products leads to increased risk of colonization by fungal, bacterial and viral pathogens and potential health and/or life threatening consequences’.

The cost and time associated with the introduction of new excipients e.g. preservative(s) can be extremely high, as a full safety package needs to be submitted covering the proposed route of administration [52]. This has ensured limited numbers of new preservatives available to replace the existing portfolio.

The EMEA [53] recently re-iterated that the levels of preservatives within a formulation should be maintained at the minimum concentration consistent with antimicrobial effectiveness in each individual preparation. They also were also keen to promote the use of novel preservatives which did not contain mercury, in any new ophthalmic preparations. Tellingly, they did not give any general recommendations not to use preservatives in eye drops.

Preservation Challenges: Case Histories

The following case studies involving programs aimed at developing (i) multi-use intra-nasal formulations and (ii) multi-use oral products, highlight some of the issues facing pharmaceutical scientists, with respect to the development of preservation systems.

Case Study 1: Preservation of a Multi-use Intra-nasal Formulation

Preclinical studies showed that the potassium salt of compound A, was a potent and selective inhibitor of the α-4 chain of the VLA-4 integrin (very late antigen) and potentially useful for the treatment of seasonal rhinitis. A is highly soluble in water at pH 7 (200mg/ml), but solubility decreases markedly with decreasing pH (e.g. < 2mg/ml at pH 5). The optimal dose was defined as 15 mg/ml for nasal application. Attempts to preserve this aqueous solution highlighted several confounding issues. The proposed high dose, together with product pH (pH 7) restricted the use of many of the common preservatives with optimal preservative efficacy at acidic pHs e.g. benzoic acid, benzyl alcohol, sorbic acid.

Incompatibility was observed between the drug substance and cationic preservatives. Inclusion of BKC or benzethonium chloride resulted in product cloudiness, and the addition of co-solvents did not resolve the problem. Synergistic mixtures of parabens and 2-phenylethanol were evaluated and whilst meeting USP AET (antimicrobial effectiveness test) criteria [54], failed the more stringent Ph.Eur requirements [55]. The parabens also imparted a metallic taste.

Organomercurials were not considered, from a safety and hypersensitivity perspective [22, 25] as were the phenolic preservatives, phenol and chlorocresol [12, 13].

EDTA [34] was compatible with the drug substance, but there were concerns that EDTA could affect the binding of the drug to α-4 side chain (of the VLA-4 integrin), at the local site of action within the nose, making its inclusion in the formulation undesirable.

Thus, a combination of the pH-solubility profile, incompatibility, palatability and potential for pharmacological interactions depleted the entire anti-microbial preservative palette considered acceptable for intra-nasal administration.

Preservatives, not previously used in intra-nasal formulations and thus likely to incur additional regulatory scrutiny together with additional safety testing were assessed. However, there were still some intractable issues.

  • 2-Phenoxyethanol was compatible with the formulation constituents, but it has a slight odor, which was considered unacceptable by the commercial groups. In addition it has a very narrow spectrum of anti-microbial activity, so is unlikely to be efficacious on its own [56].
  • 4-Chloroxylenol, which is the least sensitizing of the common phenols [14] and piroctone olamine [57] were insoluble in the formulation constituents and therefore inappropriate.
  • Phospholipid PTC (an alkyl polyglucoside preservative) [58] was physically incompatible with the formulation constituents.
  • Sodium Iodate [59] was physically and chemically compatible, but it is only utilized in oral mouthwashes. In addition, it has been linked with retinal degeneration and was therefore considered inappropriate for administration to the nasal mucosa.

Finally, purite [60], a stabilized oxychloro-complex was evaluated. It was physically and chemically compatible with the formulation constituents and effective at neutral pH’s. It has similar preservative capability to parabens for fungal organisms, but was less effective against yeasts and molds. However, it would still require combination with other preservatives to meet the requirements of the Ph.Eur, as well as comprehensive safety studies for the intranasal route of administration. Development of the compound was terminated for reasons other than preservation, but the summary highlights the barriers to dosage form development (and significant resources required), even for simple formulations for use in early clinical studies.

Case Study 2: Preservation of a Multi-use Oral Formulation

The need for pediatric / geriatric dosage forms for patients who have difficulties swallowing oral medications, e.g. tablets and capsules, is often the stimulus to develop an oral liquid multi-use product. Often the greatest formulation challenge (in addition to adequate preservation) is the development of a palatable dosage form that children will accept. Indeed, poor compliance caused by unacceptable taste is a major challenge in pediatric (and geriatric) medicines.

Compound B was approved for use in depression and anxiety but non-compliance, particularly in pediatric and institutionalized geriatric patients was a significant clinical issue. Due to relatively high solubility (4.9mg/ml) and extremely bitter taste most common taste-masking approaches were inappropriate. A promising strategy was the use of ion exchange resins to mask the bitter taste [61]. This approach relied on the formation of an insoluble tasteless drug-resin complex between the cationic drug and a cation exchange resin (CER) at buccal pHs (ca. pH 6), which can then rapidly dissociate at gastric pHs (ca. pH 1.2) to release the drug for rapid and complete absorption.

A literature review identified the most promising resin to be the strong CER, Amberlite IRP-69 [62], which is a compendial material (polacrilin potassium), available as a pharmaceutical grade material. IRP-69 readily formed palatable complexes with compound B and as the intrinsic pH of the complex was about pH 4, the formulation was preserved with sodium benzoate, which has a pH of optimal anti-microbial efficacy at the same pH [5]. A prototype formulation showed compliance with USP [54] and Ph. Eur. [55] AET criteria and on that basis this product was progressed to a comparative bioavailability study versus the commercial tablet. However, the resin-based formulation was shown to be bio-inequivalent. This was attributable to the very strong binding efficiency of the strong CER with the cationic drug. The complex did not adequately dissociate in fasting conditions (gastric residence time about 30-minutes) resulting in incomplete absorption.

The product was re-formulated using the weak CER, Amberlite IRP-88 [62]. The intrinsic pH of this formulation was higher (ca. 6) than the previous Amberlite IRP-69 formulation and consequently failed the USP [54] and Ph. Eur. [55] AET criteria. Attempts to re-adjust the pH to pH 4 (optimal for sodium benzoate efficacy) improved the preservation efficacy but adversely impacted palatability.

The parabens [8-10] have a wider effective pH range (pH 4-10) than sodium benzoate and therefore a design of experiments utilizing various ratios of these parabens was evaluated. Butyl paraben was found to be incompatible and formulations discolored rapidly. Hence mixtures of methyl and propyl paraben were evaluated. Combinations of methyl and propyl paraben utilizing lower concentrations of methyl paraben (0.060-0.015% w/v) and higher concentrations of propyl paraben (0.20-0.050% w/v) were ineffective at adequately killing fungal organisms (in particular Aspergillus niger) in AET studies; which is surprising as anti-microbial efficacy of the parabens is reported to increase with increasing alkyl chain length [8-10]. However, the inverse combinations utilizing higher concentrations of methyl paraben (0.20-0.050% w/v) and lower concentrations of propyl paraben (0.060-0.015% w/v) had adequate preservative efficacy.

Additional investigations demonstrated that both parabens were adsorbed to the weak CER, but they had very different binding efficiencies. The less hydrophobic methyl paraben bound less effectively to the weak CER (16-20%), than the more hydrophobic propyl paraben (45-56%) over the same concentration range. As bound preservatives are ineffective at killing micro-organisms this explained why higher concentrations of propyl paraben were less effective than corresponding concentrations of methyl paraben.

Thus, a combination of pH-preservative effectiveness, palatability, bio-inequivalence, chemical incompatibility, and binding of preservatives to a key excipient, required extensive formulation activities to develop an adequately preserved, palatable and bioequivalence formulation that was subsequently commercialized.

Conclusions

This article demonstrates the many challenges facing the formulation scientist. In addition to safety and regulatory concerns, there is a greater public debate about the perceived safety of preserved medicinal products. The lay public often questions the need for preserved formulations, perhaps forgetting the significant health issues that were caused by non-preserved or poorly preserved medicines in earlier times. The two case studies demonstrate how the requirements of the preservative to show maximal efficacy are often conflicted with other needs of the formulation (palatability, chemical or physical incompatibilities, efficacy or bioavailability).

Hence, although preservatives may face an uncertain future, it is vital that they remain available for incorporation into the many multi-use pharmaceutical products, to assure quality and patient safety. The evidence used to highlight concerns and calls for constraints [63] can be contrasted with the well-established record of preservatives with respect to performance and safety. The limited number now available should not be constrained further.

In summary and, to paraphrase Marple et al [49]; given the limited choices that are currently available to serve as anti-microbial preservatives, as well as the years of clinical safety that have been demonstrated, a careful risk-to-benefit analysis should be considered before discounting preserved multi-dose formulations. Multi-use oral, topical and parenteral products need to be preserved if patient safety is to be assured.

Acknowledgements

Dr. Paul Newby and Dr. Don Singer, GSK for their review and comments of this manuscript. Anna Slater, Trevor Shreeves and Gary Cannon (all from GSK) for their input into case study 1; Tracey Wood, Nicola Marzolini, Liz Jarvis and Padma Patel (all either from or former GSK) for their input into case study 2.

References

  1. E.Cahill, Benzyl Alcohol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 69-71.
  2. Anon., Benzyl Alcohol May be Toxic to Newborns, FDA Drug Bull., 12: 10-11 (1982).
  3. A.F. Fransway, The Problem of Preservation in the 1990s: III Agents with Preservation Function Independent of Formaldehyde Release, Am. J. Cont. Derm., 2: 145-174 (1991).
  4. Y. Boer, Irritation by Eye drops Containing 2-Phenylethyl Alcohol, Pharm. Weekbl (Sci)., 3: 826-827 (1981).
  5. R.M.E. Richards, R.J. McBride, Phenylethanol Enhancement of Preservatives Used in Ophthalmic Preparations, J. Pharm. Pharmac., 23: 141S-146S (1971).
  6. C.E. Downward, L.I. Roberts, L.IJ.D. Morrow, Topical Benzoic Acid Induces the Increased Biosynthesis of PGD2 in Human Skin In Vivo, Clin. Pharmacol. Therap., 57: 441-445 (1995).
  7. P.J.Weller, Benzoic Acid Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 66-68.
  8. R.Johnson, R.Steer, Methylparaben Monograph in: R.C. Rowe,P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 466-470.
  9. R.Johnson, R.Steer, Propylparaben Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 629-632.
  10. R.Johnson, R.Steer, Butylparaben Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 83-85.
  11. M. Weiner, M.L. Bernstein, Adverse Reactions to Drug Formulation Agents: A Handbook of Excipients, New York, Marcel-Deckker, pp. 298-300 (1989).
  12. D.J. Brancato, Recognising Potential Toxicity of Phenol, Vet. Hum. Toxicol., 24: 29-30 (1982).
  13. S.Nema, Chlorocresol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 171-173.
  14. L.M.E.McIndoe, Chloroxylenol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 180-181.
  15. B.W. Hancock, A. Naysmith, Hypersensitivity to Chlorocresol Preserved Heparin, Br. Med. J., 3: 746-747 (1975).
  16. A.R. Gasset, Benzalkonium chloride Toxicity to the Human Cornea, Am. J. Ophthamol., 84: 169-171 (1977).
  17. The Expert Panel of the American College of Toxicology. Final Report on the Safety Assessment of Benzethonium Chloride and Methylbenzethonium Chloride, J.Am. Coll. Toxicol., 4: 65-106 (1985).
  18. C.R.W. Beasley, P. Rafferty, S.T. Holgate, Bronchoconstrictor Properties of Preservatives in Ipratropium Bromide (Atrovent) Nebuliser Solution, Br. Med. J., 294: 1197-1198 (1987).
  19. M.S. Parker, The Preservation of Pharmaceuticals and Cosmetic Products, in Principles and Practices of Disinfection, Preservation and Sterilization, Editors: A.D.Russell, W.B. Hugo, W.B. G.A.J. Ayliffe, Oxford, Blackwell Scientific, (1982) pp. 287-305.
  20. L.Lohr, Mercury Controvesy Heats Up, Am. Pharm., 18:23 (1978).
  21. A.F. Winder, N.J. Astbury, G.A.K. Sheraidah, M. Ruben, Penetration of Mercury from Ophthalmic Preservatives into the Human Eye, Lancet, ii: 237-239(1980).
  22. G.A. Koby, A.A. Fisher, A.A., Phenylmercuric Acetate as a Primary Irritant, Arch. Dermatol., 106: 129 (1972).
  23. J.R.Miller, Sensitivity to Contact Lens Solutions, West J. Med., 140:791 (1984).
  24. J.L. Ford, M.W. Brown, P.B. Hunt, A Note on the Contamination of Eye-Drops Following Use by Hospital Out-Patients, J. Clin. Hosp. Pharm., 10:203-209 (1985).
  25. N.H. Cox, A. Forsyth, Thiomersal Allergy and Vaccination Reactions, Contact Derm., 18: 229-233 (1988).
  26. American Academy of Pediatrics, United States Public Health Service. Thimerosal in vaccines: a joint statement of the American Academy of Pediatrics and the Public Health Service, MMWR, 49: 563-565 (1999).
  27. European Agency for the Evaluation of Medicinal Products (EMEA) EMEA public statement on thiomersal containing medicinal products, 8th July (1999), EMEA publication 20962/99.
  28. Anon. Department of Health. Public Letter from the Chief Medical Officer: Current Vaccine and Immunisation Issues, 15th October 2001, Pl/CMO/2001/5.
  29. S.C. Owen, Chlorhexidine Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 163-167.
  30. R.T.Guest, Imidurea Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 359-361.
  31. S.P.Denyer, N.A.Hodges, Bronopol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 76-78.
  32. W.E. Rosen, P.A. Berke, Germall 115: A Safe and Effective Modern Preservative, Cosmet. Toilet. 92: 88-89 (1977).
  33. H.I. Maibach, Dermal Sensitization Potential of 2-Bromo-2-nitropropane-1,3-diol (bronopol), Contact Derm., 3:99 (1977).
  34. S.C. Owen, Edetic Acid Monograph, in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 260-263.
  35. R. Kava, A.N. Stimola, R. Weiser, The Top Ten Unfounded Health Scares of 2004, American Council on Science and Health, http://www.acsh.org/healthissues/newsID.1007/healthissue_detail.asp, accessed 15th December 2010.
  36. F. Walsh, Muslim fears hamper drive to eradicate polio, The Observer, Sunday May 29th (2005).
  37. M.F. Blaxhill, L. Redwood, S. Bernard, Autism: A Novel Form of Mercury Poisoning, Medical Hypothesis, 56: 462-471 (2001).
  38. Vaccines and Autism: An Institute of Medicine Report, 18th May (2004), www.iom.edu/Reports/2004/Immunization, accessed on 15th December 2010.
  39. A.E. Tozzi, P. Bisiacchi, V. Tarantino, B. De Mei, L. DeElial, F. Chiarotti, S. Salmaso, Neuropsychological Performance 10 Years after Immunization in Infancy with Thimerosal-Containing Vaccines, Pediatrics, 123: 475-482 (2009).
  40. P.D. Darbre, A. Alijarrah, W.R. Miller, N.G. Coldham, M.J. Sauer, G.S. Pope, Concentrations of Parabens in Human Breast Tumours, J. Appl. Toxicol., 24: 5-13 (2004).
  41. R. Sullivan, Report in The Independent, 12th January, (2004).
  42. D. Morgan, Report in the Daily Mail, 12th January, (2004).
  43. C. Lobermeier, C. Tschoetschel, S. Westie, E. Heymann, Hydrolysis of Parabens by Extracts from Differing Layers of Human Skin, Biol. Chem., 377: 647-651 (1996).
  44. Ø.H. Berg, K. Lie, S.K. Steinsvag, The Effect of Topical Nasal Steroids on Rat Respiratory Mucosa with Special Reference to Benzalkonium Chloride, Allergy, 52: 627-632 (1997).
  45. Y. Kuboyama, K. Suzuki, K. T. Hara, Nasal Lesions induced by Intranasal Administration of Benzalkonium Chloride in Rats, J. Tox. Sci., 22: 153-160 (1997)
  46. J-H. Cho, Y.S. Kwun, H.S. Jang, Long Term Use of Preservatives on Rat Nasal Respiratory mucosa: Effects of Benzalkonium Chloride and Potassium Sorbate, Laryngoscope, 110: 312-317 (2000).
  47. G.K. Scadding, V.J. Lund, M. Holmstrom, M. Y. Darby, Clinical and Physiological Effects of Fluticasone Propionate Aqueous Nasal Spray in the Treatment of Perennial Rhinitis, Rhinology, 11: 37-43 (1991).
  48. J.P.M. Braat, G. Ainge, J.A.K. Bowles, D.H. Richards, D. van Riessen, W.J. Visser, E. Rijntjes, The Lack of effect of Benzalkonium Chloride on the Cilia of the Nasal Mucosa in Patients with Perennial Allergic Rhinitis: A Combined Functional Light, Scanning and Transmission Electron Microscopy Study, Clinical and Expt. Allergy, 25: 957-965 (1995).
  49. B. Marple, P. Roland, M. Benninger, Safety Review of Benzalkonium Chloride used as a Preservative in Intranasal Solutions: An Overview of Conflicting Data and Opinions, Otol. Head and Neck Surgery, 130: 131-141 (2004).
  50. Graf, P., Hallen, H. and Juto, J.E., Benzalkonium Chloride in a Decongestant Nasal Spray Aggravates Rhinitis Medicamentosa in Healthy Human Volunteers, Clin. Exp. Allergy, 25 (1995) 395-400.
  51. Hallen, H. and Graf, P., Benzalkonium Chloride in Nasal Decongestant Sprays has a Long Lasting Effect on the Nasal Mucosa of Healthy Human Volunteers, Clin. Exp. Allergy, 25 (1995) 401-405.
  52. B.R. Matthews, Preservation and Preservative Efficacy Testing: European Perspective, Eur. J. Parent. Pharm. Sci., 8: 99-107 (2003).
  53. Anon, EMEA Public Statement on Antimicrobial Preservatives in Ophthalmic Preparations for Human Use. EMEA/622721/2009, London, 08 December, 2009.
  54. United States Pharmacopeia General ChapterAntimicrobial Effectiveness Testing, USP 34-NF29, US Pharmacopeia, Rockville, Maryland, USA, 2010.
  55. European Pharmacopoeia 5.1.3 Efficacy of Antimicrobial Preservation, EP 6.4, European Directorate for Quality of Medicines, Strasbourg, France, 2010.
  56. S.C.Owen, 2-Phenoxyethanol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 517-518.
  57. H.C. Sigle, M. Schäfer-Kortning, H.C. Korting, B. Hube, M. Niewerth, In Vitro Investigations on the Mode of Action of the Hydroxypyridone Antimycotics Rilopirox and Piroctone on Candida albicans, Mycoses, 49: 159-158 (2006).
  58. E.J. Fendler, R.A. Williams, M.J. Dolan, Antimicrobial Cleansing Composition Containing Chlorhexidine, an Amphoteric Surfactant and an Alkyl Polyglucoside, United States Patent 5, 719,113, 17th February 1998.
  59. D.J. Picciano, Iodine Containing Nasal Moisturizing Saline and Mouthwash Solutions, United States Patent 6,171,611, 09th January 2001.
  60. R. Noecker, Effect of Common Occular Preservatives on Occular Health, Advances in Therapy 18: 205-215 (2001).
  61. T.B. Ernest, D.P. Elder, L.G. Martini, M. Roberts, J.L. Ford, Developing Paediatric Medicines: Identifying the Needs and Recognizing the Challenges, J. Pharm. Pharmac., 59: 1043-1055 (2006).
  62. A. Palmieri, Polacrillin Potassium Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp 532-534.
  63. C. Baudouin, A. Labbè, H. Liang, F. Brignold-Baudouin, ‘Preservatives in Eyedrops: The Good, the Bad and the Ugly,’ Progress in Retinol and Eye Research, 29: 312-334 (2010).

Author Biographies

David P. Elder has 34-years experience in the pharmaceutical industry. He is a director in the pre-clinical SCINOVO group at GSK. He has a PhD from Edinburgh University, UK. He is a member of the British Pharmacopoeia Commission and an FRSC. He has written and lectured widely on the theme of product development and the challenges of preservation.

Patrick Crowley is a pharmacist by training (FRPhSGB). He worked in the Pharmaceutical Industry for over 40 years and was a VP of product development at GSK. He currently operates as a consultant and teaches Pharmaceutical Sciences at a number of Institutions. Has authored / presented on over 40 topics related to pharmaceutical sciences.

  • <<
  • >>

Join the Discussion