BioScience Laboratories, Inc., personnel participate in the activities of numerous national and international professional associations that focus on microbiology and infection control in the healthcare and food service industries.  As our website indicates, our interests relate to disinfectant and topical antimicrobial formulations, their importance in reducing the risk of disease transmission, and fair assessments of their antimicrobial efficacy.  Because such assessments require methods of testing that provide reliably reproducible data meaningful in the context of infection control, our personnel have, for many years, been deeply involved in method development through the American Association for Testing and Materials (ASTM), specifically, Subcommittee E 35.15 on Antimicrobials.  Four members of our staff, including myself as Subcommittee Co-Chair, serve on E 35.15.

As of the conclusion of our semiannual meeting last week, our Subcommittee has 96 members and is responsible for 45 approved methods, plus another 13 currently in the process of development.  In the interest of brevity, I will describe only two examples of the latter.

The first of special note is a modification of E 1174, the ASTM version of the FDA method specified for testing of handwash products intended for use in healthcare. The modification involves the procedure for contaminating the hands with Serratia marcescens, the indicator bacterium used to challenge product antimicrobial efficacy, and is particularly important in that the new method will be much more appropriate for testing leave-on (non-water-aided) hand sanitizers.

Another method-in-the-making is one for testing liquid microbicides versus bacterial biofilms, organized assemblages that are considerably more resistant to antibiotics, topical antimicrobials, and disinfectants than are planktonic (free-floating) bacteria.  Only in the last decade, or so, has the important role that biofilms play in disease causation and environmental fouling been understood, and colleagues from the Center for Biofilm Engineering at Montana State University here in Bozeman have been in the forefront of methods development in E 35.15.

Although I have selected for comment only these from among our many methods, I would welcome any questions you may have about testing of antimicrobial formulations and how the testing methods are created collaboratively by volunteers from industry, regulatory agencies, and CROs such as BioScience Laboratories.

– John Mitchell, Director of Quality Assurance and Chief Medical Officer

As required by <USP 1072> clean-room disinfectant validation is required “to demonstrate the efficacy of a disinfectant within a pharmaceutical manufacturing environment”.

What you must do:  Take the time to think through all the parts and pieces that make up your overall cleaning program to ensure the program is effective, practical for every day activities.

Product Selection:  

Alcohols:  Broad-spectrum efficacy against vegetative bacteria.  Typical concentration of 70% used.  Not effective against molds or spores. 

Aldehydes: Powerful and aggressive disinfectants.  However, are highly toxic to personnel and require long contact times for sporicidal claims.  

Sodium hypochlorite (NaOCl) and other chlorine compounds:  Broad-spectrum biocidal activity.  Chlorine solutions are corrosive, unstable over time, and rapidly lose activity.  Typically concentrations for sodium hypochlorite are 500 to 50,000ppm.  A low ppm will be effective against most vegetative bacteria within 10 minutes.  Unfortunately, to kill spores/molds the concentration must be greater.  Good disinfectant – Poor cleaner.  Will not remove soil load.   

Hydrogen peroxide: A potent biocide and environmentally friendly.  Peroxides are deactivated in the presence of soil loads, so pre-cleaning is required to achieve the desired reduction in the microbial population.  Typical concentrations as low as 0.5 percent.  Hydrogen peroxide can be combined with other ingredients to dramatically increase its germicidal potency and cleaning performance.

Phenolics: Broad range of disinfectants that are used on environmental surfaces. Typical concentrations are 2 to 5 percent with contact times of 5 to 10 minutes.  Added detergents are effective in removal of soil loads. 

Quaternary ammonium compounds: Non-irritating and non-corrosive to surfaces.  Typical concentrations of 0.1 to 2 percent and require 10 minutes of contact time to kill microorganisms.  However most are not effective in removing biofilms and leave surfaces with a residue that must be removed after disinfection.

Application Procedure:

A spray procedure is a quick way to effectively treat a surface.  However, with many of the disinfectants a pre-clean may be required to remove any soil load.  A wipe procedure is also very effective in mechanically removing microorganisms and is a great addition to any disinfectant cleaning program.

How can BioScience help?

We can perform the following testing (1) use-dilution tests (screening disinfectants for their efficacy at various concentrations and contact times against a wide range of standard test organisms and environmental isolates); and (2) surface challenge tests (using standard test microorganisms and microorganisms that are typical environmental isolates, applying disinfectants according to your cleaning procedures. 

– Liv Graving, Microbiologist and In-Vitro Study Director 

We have been very fortunate in the past regarding results from FDA, EPA, and Sponsor audits.  We take each one seriously, as a learning experience, and implement appropriate changes to our processes in a timely fashion.  The audits are a constant reminder for BioScience to strive to be the best that we can be.  Although we are not ISO 17025 certified, we have been audited using those guidelines, as well as Good Laboratory Practice Regulations (GLPs) and Good Clinical Practice Regulations (GCPs).  Some outcomes of the audits have included: adding “Controlled” and “Uncontrolled” stamps on copies given to auditors; adding “Obsolete” on documents once a revision has been put into place; and being more diligent in referencing other SOPs in our SOP documents.  We are accommodating in sending, prior to an auditor’s visit, such information as our current organizational chart, corporate resumes for key employees, and the index of our Standard Operating Procedures.  We have received praise on the ease with which we are able to access documentation requested by an auditor.  All in all, each audit has been a positive experience for us, and I believe for the auditor as well.  Come visit us and see for yourself.  Scientific Expertise with Montana Hospitality is a self-appraisal we take very seriously.

Amy L. Juhnke, Manager of Quality Assurance/Document Control

At BioScience Laboratories, Inc., we currently use EpiOcular and EpiDerm tissue models in ocular and dermal irritation studies, respectively, performed according to the MTT Effective Time-50 viable cell assay.  The EpiOcular viable cell assay is designed to provide both a Draize and a potential ocular irritation score.  The EpiDerm viable cell assay is designed to provide a potential dermal irritation score only.  These testing methods are becoming increasingly popular as a move is made toward replacing animal testing with an in-vitro means of evaluation.  We have successfully used these models to test a wide range of products: cosmetics (liquids, powders, gels, mascaras or creams at multiple concentrations), personal care products, household products (cleaners, inks, shaver heads, etc.), pharmaceutical products, and surfactant-based products tested at a concentration of 10% to simulate “rinse-off” exposures (general use hand soaps and detergents) utilizing both of these methods.

Additionally, other testing methods have also been developed using the EpiDerm tissue model.  These other testing methods are conducted in ways very similar to the dermal irritation method and are also performed at BioScience Laboratories:

Dermal corrosion testing measures the production of scarring usually as a result of corrosive tissue destruction (necrosis) following the application of a substance (irreversible).  The potential for chemically-induced dermal corrosion is an important consideration in establishing procedures for the safe handling, packing, and transport of chemicals.

Dermal phototoxicity testing identifies the existence or absence of possible hazards likely to arise from a test substance in association with exposure to UV and visible light.  A typical application is testing the phototoxicity of new sunscreen product formulations while in development to identify chemicals that might induce adverse skin reactions.

Percutaneous absorption testing uses various EpiDerm kits to calculate the permeability coefficient of a test article.  Also, this is the underlying technology used in transdermal drug delivery studies.

Looking towards the future, BioScience Laboratories is developing test methods to make use of the other tissue models available from the manufacturer of the EpiOcular and EpiDerm models. 

MelanoDerm is the model of choice in tissue testing procedures useful as an in-vitro means to evaluate cosmetic and pharmaceutical agents designed to modulate skin pigmentation and usually involves topical application of skin lighteners or self-tanning agents.

EpiAirway is the model of choice in tissue testing procedures for gas phase exposure of volatile articles for airway inflammation and irritancy studies.  These methods also allow the measurement of transepithelial permeability for inhaled drug delivery studies.

EpiOral is the model of choice in tissue testing procedures which enable the in-vitro study of irritation, oral pathologies, and basic oral cavity phenomena.  Some companies have also used this model to grow commensal and pathogenic bacteria in order to study their effects on the oral tissues.

Dendritic/Langerhans cells can be used to develop in-vitro assays for contact sensitization and other immunological reactions of the body.

As you look to develop new products and differentiate them within the marketplace, which of these tissue models are most attractive to your company to use in product testing?  How important is it to your customers that products are tested without harming animals?

Jessica McDonnell-Philipp, In-Vitro Laboratory Study Director

We work in a customer service environment dealing with the in-vitro efficacy evaluation of many and varied antimicrobial products. One of the most important considerations in almost every study that we perform is proving effective neutralization/inactivation of the antimicrobial activity of the product(s) to be tested. In brief, proof of neutralization is necessary to ensure the validity of the test method. Without verification of neutralization, differentiation between –cidal (kill) or inhibitory activity is difficult, at best. And since most competitive marketing of products is based upon claims of “kills 99.9% of germs in 15 seconds” or “reduces bacteria by 99.99% in 10 minutes,” proof of neutralization is as important as the results of the bactericidal test itself.

Depending upon the active ingredient(s) of a test product, neutralization is accomplished chemically through the addition of neutralizing agents (i.e., lecithin, Tween 80, catalase, etc.), by dilution of the active ingredient to a sub-inhibitory or sub-lethal level, or by a combination of the two. One of the first questions asked of a potential customer is “what is the active ingredient of your product?” Without an accurate answer, the study design is a “best guess” – which neutralizing formula will work best to immediately inactivate a given product?

ASTM E 1054-08, Standard Test Methods for Evaluation of Inactivators of Antimicrobial Agents, provides excellent guidance on methodologies for demonstration of adequate neutralization. All neutralization verification procedures should, at minimum, include the following:

1) An arm/phase that provides data demonstrating that the neutralizing fluid immediately and completely neutralizes the product. If neutralization is not complete or immediate, residual “killing” activity may occur – a bacterial reduction reported for a 15 second exposure time may actually be a reduction for 30 seconds, 1 minute, or longer. If this happens, the danger is that a product may be reported as being more effective than it actually is.

2) An arm/phase demonstrating that the neutralizing fluid itself is not toxic to the organism(s) tested. If the neutralizer exhibits some antibacterial activity, it may actually “add to” the apparent antimicrobial activity of a product. Again, the danger is that a product may be reported as being more effective than it actually is.

3) An arm/phase providing data showing the starting bacterial challenge population. The data from this phase/arm are the basis of comparison for the previous phases – in general, neutralization procedures should employ a LOW population of bacteria, in terms of CFU/mL. If a challenge population is too high, a neutralizing system may appear to be effective, when in fact it is not.

4) REPLICATION – this allows for a statistical comparison of the data – PROOF that the neutralizing system is effective.

Verification of neutralization must be performed for all in-vitro efficacy evaluations of antimicrobial products to ensure the validity of the data; depending upon the type of evaluation and the familiarity with the active ingredient(s), this verification may be conducted in advance of a efficacy evaluation, or concurrent with the efficacy evaluation — as long as it is conducted using the same test formulation.

– Terri Eastman, Manager of the In Vitro Laboratory

To be successful in the marketplace eye care cosmetics must be non-irritating to the consumer.  Traditionally, a Draize rabbit eye test has been used to determine the safety of cosmetic products before sale to the general public.  The Draize Test involves applying the products directly to an animal’s eye.  The animals are observed for up to 14 days for signs of irritation (redness, swelling, discharge, cloudiness, or blindness in the tested eye).  Animal rights concerns, the cost of testing, and current European legislation banning cosmetics that have been tested using animals begs for an alternative model.  The MatTek  EpiOcular™ Tissue Model is a highly reproducible human cell-based in vitro tissue model that can be used to replace the traditional Draize ocular irritation testing.  The EpiOcular™ tissue model consists of normal, human-derived cells similar to cells found in the cornea (eye).  These cells are cultured on specially prepared cell culture forms creating multi-layered structures that are mitotically and metabolically active and induce the same inflammatory response as the human eye and can be used for detection of ocular irritation.  

We performed a collaborative study with MatTek to determine if the EpiOcular™ tissue model could also be used to differentiate between ultra-mild eye care cosmetic formulations.  Ultra-mild classifications can not be determined using the standard Draize rabbit eye test due to the insensitivity to the low levels of irritation induced by these products.  For the mildness testing, 10 commercially available mascara products, all with non-irritating or hypoallergenic claims, were purchased representing a broad range of manufacturers including Almay, Revlon, L’Oreal, Maybelline, and Covergirl.  The mascara products were tested using the EpiOcular™ model with an extended time exposure protocol (up to 24 hours).  The mascaras were applied to the surface of the ocular cells and remained in direct contact with the tissues for 8, 16, or 24 hours.  Following the exposure times, the mascaras were removed and the tissues and exposed to MTT for 3 hours.  Following the MTT exposure the tissues were removed from the MTT and rinsed with a Phosphate Buffered Saline solution. An alcohol extractant was added to the tissues following the MTT exposure.  The extraction process was allowed to proceed overnight.  The optical density (color change) of the extract was read the following day using a spectrophotometer.  If the mascara was non-irritating to the cells, the cells remained viable and were able to metabolize the MTT, thereby reducing the MTT and creating a visible color change from red/orange to blue/purple.  The absence of this darkening indicated that the mascara was irritating to the cells.  These optical density readings were used to determine the percent (%) viability of each tissue following exposure to each of the 10 mascara products.  An ET-50 (the time it takes for the viability of the tissues to decrease to 50%) irritation score was then assigned to each mascara.   The commercially available mascaras showed ET-50 scores ranging from 8.7 hours to > 20 hours.  These results relate to very mild products whose irritation potential would not have been detected with the standard animal model.  As such, the extended time exposure protocol appeared to be a facile, cost-effective means to screen ultra-mild eye care cosmetics. 

LIV GRAVING AND JESSICA MCDONNELL