BEFORE THE ILLINOIS POLLUTION CONTROL BOARD

BIOMEDICAL TECHNOLOGY SOLUTIONS,

INC., a Colorado Corporation,

Petitioner,

v.

RECEIVED

CLERK'S OFFICE

NOV 2 8 2007

STATE

OF ILLINOIS

Pollution Control Board

(Adjusted Standard Petition)

ILLINOIS ENVIRONMENTAL PROTECTION

AGENCY,

?

HEARING WAIVED

Respondent.

NOTICE OF FILING

To:

?

Division of Legal Counsel

Illinois Environmental Protection Agency

1021 North Grand Avenue East

P.O. Box 19276

Springfield, Illinois 62794-9276

PLEASE TAKE NOTICE that I have filed with the Office of the Clerk of the

Pollution Control Board the Petition for Adjusted Standard of BioMedical Technology

Solutions, Inc., a copy of which is herewith served upon you.

Dated: November 28, 2007

Neal H. Weinfield

Jason B. Elster

GREENBERG TRAURIG, LLP

Firm No. 36511

77 West Wacker Drive, Suite 2500

Chicago, Illinois 60601

312-456-8400 (Telephone)

312-456-8435 (Facsimile)

weinfieldn@gtlaw.com

elsterj@gtlaw.com

Neal

iteetteafra"

H. Weinfield

---

BEFORE THE ILLINOIS POLLUTION CONTROL BOARD

BIOMEDICAL TECHNOLOGY SOLUTIONS,

INC., a Colorado Corporation,

RECEIVEDC

LERK'S

OFFICE

NOV 2 8 2007

STATE OF ILLINOIS

Pollution Control Board

v.

Petitioner,

OD

PCBe-

ILLINOIS ENVIRONMENTAL PROTECTION

AGENCY,

(Adjusted Standard Petition)

HEARING WAIVED

Respondent.

PETITION FOR ADJUSTED STANDARD

Petitioner BioMedical Technology Solutions, Inc. ("BMTS"), by and through its

undersigned attorneys, hereby petitions the Illinois Pollution Control Board (the "Board")

for an Adjusted Standard from a provision of 35 IAC 1422.

1

BMTS, which manufactures

a countertop medical waste treatment device, the Demolizer® technology, seeks a

technology-specific Adjusted Standard from 35 IAC 1422, which requires the use of a

particular microorganism,

Bacillus subtilis

(ATCC 19659), to determine the initial

efficacy of the technology. In conducting the initial efficacy test required under the

Board's regulations, BMTS seeks permission to use a subspecies of

Bacillus subtilis

commonly referred to as

Bacillus subtilis

var.

niger

(recently reclassified as

Bacillus

atrophaeus)

that is the preferred and most appropriate biological indicator organism for

the validation of dry heat sterilization processes.

The proposed Adjusted Standard exhibits superior dry heat resistance and can be

distinguished from the generic

Bacillus subtilis

primarily through differences in color or

pigmentation response to certain media. Importantly, the proposed Adjusted Standard is

nationally and internationally recognized by microbiologists and governing standards

BMTS and the Illinois Environmental Protection Agency have agreed to waive a hearing for this petition.

---

organizations as the preferred and most appropriate biological indicator organism for the

validation of dry heat sterilization technologies, the underlying technology of the

Demolizer® system. Further, it is the only

Bacillus subtilis

organism available in a

tested, certified carrier form. This petition for an Adjusted Standard (the "Petition") is

brought pursuant to Section

35

of the Illinois Environmental Protection Act (the "Act"),

415 ILL.

COMP. STAT.

5/35,

and Part

104

of Chapter

35

of the Illinois Administrative

Code,

35 IAC 104.

In support of its Petition, BMTS states as follows:

I.

Introduction

BMTS manufactures medical waste treatment devices that, employing

Demolizer® technology, destroy potentially infectious microorganisms through the use

of dry-heat. Prior to conducting a treatment cycle, medical wastes, including "sharps,"

are placed into the device, which is approximately the size of the common microwave.

Through the course of a treatment cycle, the waste is sterilized and rendered into a non-

recognizable solid waste that can then be disposed of as any other refuse. Businesses that

generate relatively low volumes of medical waste such as nursing homes, medical, dental

and veterinary offices, and pharmacies can use BMTS devices on-site as a safe and

efficient method of treating and disposing these materials. It also avoids having to ship

medical waste off-site for treatment and disposal. In fact, BMTS devices can be found

throughout the United States and BMTS has begun marketing the technology world-wide.

The technology is formally approved or meets statutory requirements in

46

states.

In order to sell its devices in Illinois, the Board's regulations require that BMTS

demonstrate that its Demolizer® technology is effective in eliminating potentially

harmful microorganisms by performing an Initial Efficacy Test ("IET"). The purpose of

2

---

an IET is to validate the sterilization efficacy of a treatment device. Currently, the

Board's regulations specify that a particular microorganism, ATCC 19659

Bacillus

subtilis

("Chemical Indicator"), must be used in the IET. However, ATCC 19659

2 is not

commercially available in a certified form, and the procedure for growing and certifying

ATCC 19659 to the same standards achieved using the most appropriate

Bacillus subtilis

certified microorganism could take close to two and a half years and cost upwards of

$320,000 - which would require that BMTS sell numerous additional Demolizer® units

just to cover these costs.

The alternative to ATCC 19659 is a variant of the same species, ATCC 9372

Bacillus subtilis

var.

niger,

also known as

Bacillus atrophaeus

("Certified Indicator" or

"Dry Heat Indicator"), which is commercially available in a certified form and is the

scientifically-recognized standard in 46 states as well as the international community for

the validation of dry heat sterilization processes due to its superior growth and heat

resistance properties.

The Certified and Chemical Indicator organisms are very similar organisms. The

Chemical Indicator,

Bacillus subtilis,

is commonly used for the validation of chemical

disinfectants and is, therefore, most appropriate for the validation of alternative

technologies employing a chemical sterilization agent. The Chemical Indicator is not

recognized by international standards organizations or in the scientific literature for the

validation of dry heat sterilization technologies.

2

The American Type Culture Collection, commonly known as the ATCC, is an international nonprofit

organization that provides biological products and technical services to the scientific community. The

biological samples deposited with the ATCC are used internationally as the reference standard for

biological materials.

See

ATCC,

http://www.atcc.org/About/AboutATCC.cfm (last visited June 20, 2007).

3

---

The Certified Indicator,

Bacillus subtilis

var.

niger

(reclassified as

Bacillus

atrophaeus

in 2004), exhibits enhanced resistance in dry heat applications compared to a

generic

Bacillus subtilis

organism, typical of the Chemical Indicator. In a definitive

study conducted by Gurney and Quesnel, the dry heat resistance performance of a generic

Bacillus subtilis

and

Bacillus subtilis

var.

niger

were compared at dry heat treatment

temperatures ranging form 140 to 170°C. At all temperatures,

Bacillus subtilis

var

niger

demonstrated superior dry heat resistance. The study definitively found that "the var.

niger

strain is clearly the organism of choice as an indicator of dry heat sterilization..."

See

Group Exhibit J,3

Gurney, T.R. & Quesnel, L.B.,

Thermal Activation and Dry-heat

Inactivation of Spores of Bacilus subtilis MD2 and Bacillus subtilis var. niger, J.

APPLIED

BACTERIOLOGY, 48, 231-247 (1980).

Based on these findings and the preponderance of evidence in the scientific

community, the Certified Indicator has been universally adopted as the preferred and

most appropriate biological indicator organism for the validation of dry heat sterilization.

The following international standards organizations specify the proposed Adjusted

Standard,

Bacillus subtilis

var.

niger

(ATCC 9372) as the preferred biological indicator

organism for dry-heat processes. Each standards organization convenes an expert panel

of microbiologists and specialists in sterilization assurance that review the body of

scientific evidence to substantiate their recommendations and published standards.

Manufacturers of certified biological indicators must then test each production lot against

3

True and correct copies of relevant portions of the scientific authorities cited in this Petition are attached

collectively hereto as Group Exhibit J.

4

---

these standards meeting stringent performance requirements for resistance as measured in

D-values and z-values.4

1. US

Pharmacopoeia.

USP28-NF23 USP. Monographs: Biological Indicator

for Dry-Heat Sterilization, Paper Carrier; Rockville, MD; 2005.

2.

FDA.

Guidance on Premarket Notification [510(k)] Submissions for

Sterilizers Intended for Use in Health Care Facilities. Infection Control

Devices Branch, Division of General and Restorative Devices (March 1993).

3. FDA.

Premarket Notifications [510(k)] for Biological Indicators Intended to

Monitor Sterilizers Used in Health Care Facilities; Draft Guidance for

Industry and FDA Reviewers; U.S. Department of Health and Human

Services, Food and Drug Administration, Center for Devices and Radiological

Health, Infection Control Devices Branch (March 2001).

4.

British Pharmacopoeia Commission.

Methods of sterilization. London,

UK: British Pharmacopoeia Commission; British Pharmacopoeia Appendix

XVIII (2003).

5. European Pharmacopoeia Commission.

Biological indicators of

sterilization. Strasbourg, France: European Pharmacopoeia Commission;

European Pharmacopoeia EP 5.1.2 (1997).

6.

Japanese Pharmacopoeia.

JP14e.partII.15 JP. Terminal Sterilization and

Sterilization Indicators.

7.

ISO and ANSI.

Sterilization of health care products — Biological indicators;

Part 4: Biological indicators for dry heat processes. Geneva (Switzerland):

International Organization for Standardization/ANSI; ISO 11138-4:2006.

BMTS is requesting relief from the Board's requirement of using the Chemical

Indicator in the IET and seeks permission to demonstrate the effectiveness of its devices

by conducting the IET using the Certified Indicator. Currently, out of the 46 states that

have approved the Demolizer® or for which the Demolizer® meets statutory

requirements, Illinois is the only state that has required use of the Chemical Indicator in

the IET for the Demolizer® technology rather than the Certified or Dry Heat Indicator for

the validation of the dry heat sterilization technology.

4

The D-value is the time required to destroy 90% (1 log10 reduction) of cells under specified conditions

while the z-value is the increase in temperature required to reduce the thermal death time by a factor of 10.

5

---

II. Regulatory

Requirements For Conducting An Initial Efficacy Test

35 IAC 104.406(a) requires that the Petition contain a statement describing the

regulation from which an Adjusted Standard is sought. Pursuant to 35 IAC 1422.124,

"[t]he manufacturer, owner or operator of a treatment unit shall conduct an Initial

Efficacy Test, pursuant to Appendix A of this Part, for each model prior to its operation."

35 IAC 1422.124(a). The IET is a scientifically-controlled demonstration that the

treatment unit does in fact eliminate the infectious potential from potentially infectious

medical waste. Section 1422.Appendix A ("Appendix A"), titled Initial Efficacy Test

Procedures, sets forth the procedures for conducting an IET for three classes of treatment

units.

See

35 IAC 1422.Appendix A.

The IET procedure that applies to BMTS involves placing carriers of indicator

microorganisms inside the device, conducting a treatment cycle, and then measuring the

number of indicator microorganisms that remain viable.

See id.

Appendix A identifies

three indicator microorganisms to be used in an IET for treatment units that use thermal

treatment and maintain the integrity of the container of indicator microorganisms (e.g.,

incinerators, autoclaves, and radiation-based processes): 1)

Bacillus subtilis

(ATCC

19659); 2)

Bacillus stearothermophilus

(ATCC 7953); and 3)

Bacillus pumilus

(ATCC

27142).

See

35 IAC 1422.Table B ("Table B"). The Agency has agreed that the second

and third indicator microorganisms are not scientifically appropriate for verifying the

efficacy of the Demolizer® system because they are not recognized for the validation of

dry heat systems. The effective date of the regulation is March 1993.

6

---

III.

Statement of Applicability

As required by 35 IAC 104.406(b), the regulation of general applicability was not

promulgated to implement, in whole or in part, the requirements of the CWA (33 USC

1251 et seq.), Safe Drinking Water Act (42 USC 7401 et seq.), or the State programs

concerning RCRA, UIC, or NPDES [415 ILCS 5/28.1].

IV.

Level of Justification

35 IAC 104.406(c) requires the Petitioner to state whether a specific level of

justification is provided in the regulation of general applicability. 35 IAC 1422 does not

specify a level of justification or other requirements.

V.

Description of the Nature of the Petitioner's Activity

35 IAC 104.406(d) requires a complete and concise description of the nature of

BMTS' activity that is the subject of the proposed Adjusted Standard. BMTS was

incorporated in 2005 as a Colorado corporation. BMTS produces medical waste

treatment devices that employ Demolizer® technology, which is based on a dry-heat

treatment process that was developed and broadly approved throughout the United States

in the mid-1990s. The technology heats one gallon of medical waste to a minimum

treatment temperature of 350°F for a minimum of 90 minutes. The Demolizer®

technology has demonstrated broad-scale efficacy under these treatment conditions

through studies at Stanford University, Kansas State University, and various private

laboratories. BMTS has customers in almost every state and has begun marketing the

technology world-wide. Further, the temperature profile completely destroys sharps

waste through a slow-melting of the plastic components of used syringes. The resulting

7

---

melted mass is further contained in the bottom of the metal collector for final disposal as

ordinary solid waste.

A.

BMTS' Initial Efficacy Test Using the Certified Indicator

In 2006, BMTS commissioned Dr. James Marsden, Regent's Distinguished

Professor at Kansas State University, to conduct an initial efficacy test for its updated

Demolizer® technology that could be used to secure regulatory approval both in the

United States and internationally (the "KSU Efficacy Test"). In selecting an appropriate

indicator microorganism, Dr. Marsden conducted a comprehensive review of the

scientific literature prior to initiating the efficacy trial.

In his preparations for the KSU Efficacy Test, Dr. Marsden discovered that the

Chemical Indicator was not commercially available in a certified spore carrier form.

However, the scientifically similar Certified Indicator, which is the industry standard for

validating dry-heat sterilization technologies due to superior heat resistance, was readily

available from multiple certified manufacturers including STERIS Corporation, NAMSA,

Raven Laboratories, STS, and Charles River Laboratories, to name a few. Through his

literature review, Dr. Marsden concluded that the Chemical and Certified Indicators are

essentially equivalent with primary differentiation based on pigmentation response to

certain media. In fact, over 99.8% of their genetic material is

identical -

meaning that,

but for their color, the Chemical and Certified Indicators are indistinguishable.5

Most importantly, Dr. Marsden determined that the international scientific

community, including many of the world's most prestigious standards organizations,

recognizes the Certified Indicator as the preferred and most appropriate biological

5

See

Group Exhibit J

infra,

K.S. Blackwood, C.Y. Tureene, D. Harmsen, and A.M. Kabini„

Reassessment

of Sequence-Based Targets for the Identification Bacillus Species, J.

CLINICAL MICROBIOLOGY,

42, No. 2

(2004).

8

---

indicator for the validation of dry heat processes. As cited in the previously,

Bacillus

subtilis

var.

niger,

the Certified Indicator, exhibits enhanced resistance in dry heat

applications compared to a generic

Bacillus subtilis

organism, typical of the Chemical

Indicator.

Therefore, it was the recommendation of Dr. Marsden, consistent with the

overwhelming body of scientific literature, to use the commercially available Certified

Indicator in the KSU Efficacy Test. This approach poses the most rigorous challenge for

the Demolizer® technology and relies on the use of tested and standardized indicator

spore carriers.

The results from the KSU Efficacy Test conclusively established that the

Demolizer® technology is an effective sterilization treatment for potential infectious

medical waste. Since complete elimination or destruction of all forms of microbial life is

difficult to prove, sterilization is usually expressed as a probability function in terms of

the number of microorganisms surviving a particular treatment process. Under the

Board's regulations, a valid sterilization process must demonstrate a one-millionth

survival probability in the indicator microorganism population.

6

The Demolizer®

devices used in the KSU Efficacy Test unequivocally demonstrated their ability to meet

Illinois' requirements for sterilization devices.

B.

?

Historical Classification and Subsequent Sub-Classification of

the

Bacillus Subtilis

Species

The following provides a discussion of the subspecies reclassification of the

Bacillus

genus that affects

Bacillus subtilis

organisms.

6

The Board's regulations express this probability function is a 6 Log

i() reduction, i.e.,

a

99.9999%

reduction in microbial life.

9

---

Until 1989, the scientific community recognized the Chemical and Certified

Indicators as members of the

Bacillus

family commonly referred to as

Bacillus subtilis.

Migula first described the species now know as

Bacillus subtilis

in 1900.

See

Migula,

W.,

System der Bakterien,

vol. 2.

JENA: GUSTAV FISCHER

(1990). In 1952, Smith

et at

noted that certain strains of

Bacillus subtilis

produced different colored pigments when

exposed to varying culture conditions, but otherwise found no other discriminatory

property between the strains other than pigmentation.

See

Smith, N.R., Gordon, R. E. &

Clark, F.E.,

Aerobic Spore-forming Bacteria,

AGRICULTURE MONOGRAPH

NO. 16,

Washington, DC: United States Department of Agriculture (1952). In that same work,

Smith

et at

allocated certain strains into a subspecies variety called

Bacillus subtilis

var.

niger. See id.

However, in 1973, these different varieties were once again subsumed into the

broader species designation

Bacillus subtilis

through the work of Gordon

et al.

due to the

lack of differentiation between varieties.

See

Gordon, R.E., Haynes, W.C. & Pang, C.

H.-N.,

The Genus Bacillus,

AGRICULTURE HANDBOOK

No. 427, Washington, DC: United

States Department of Agriculture (1973). In 1989, Nakamura re-examined the pigment-

producing strains of

Bacillus subtilis

and, just like Smith

et at,

once again differentiated

certain subspecies based on pigmentation.

See

Group Exhibit J,

infra,

Nakamura, L.K.,

Taxonomic Relationship of Black-Pigmented

Bacillus Subtilis

Strains and a Proposal for

Bacillus Atrophaeus

sp. nov.,

NT.

J.

SYST.

BACTERIOLOGY

39, 295-300 (1989).

This time, Nakamura created a new subspecies designation,

Bacillus atrophaeus,

which included 21 of the 25 strains that had previously been designated as

Bacillus

subtilis

var.

niger. See id.

Henceforth, the Certified Indicator belonged to the subspecies

10

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atrophaeus

while the Chemical Indicator remained part of the subspecies

subtilis.

In

making this distinction between strains, Nakamura noted that the species descriptions of

Bacillus subtilis

and

Bacillus atrophaeus are

not affected by the re-classification because,

"except for the colour of the soluble pigment, all of the strains were indistinguishable by

the standard characterization method;

i.e.

they exhibited the traits typical of

B. subtilis."

Id.; see also

Fritze, D. and Pukall, R.,

Reclassification of Bioindicator Strains

Bacillus

Subtilis

DSM 675 and

Bacillus Subtilis

DSM 2277 as

Bacillus Atrophaeus, INT'L. J.

SYSTEMATIC EVOLUTIONARY MICROBIOLOGY,

51, 35-37 (2001).

Since Nakamura's 1989 re-classification of

Bacillus subtilis

strains, the scientific

community has consistently and unanimously found that members of the

Bacillus subtilis

and

Bacillus atrophaeus

are phenotypically identical except for color.

See generally,

Group Exhibit J,

infra.

C.

?

BMTS' Regulatory Approval Efforts

As part of the KSU Efficacy Test, extensive trials were conducted on the updated

Demolizer® technology utilizing an array of organisms under varying conditions as

required by the Illinois statutes and other state agencies across the United States. These

results have been exhaustively reviewed by many of the states that formally approve such

technologies and resulted in the issuance of technology approval letters. Only three states

specifically identify the Chemical Indicator in their regulations for use in validation

procedures: Arizona, Illinois, and Delaware. In fact, both Arizona and Delaware have

reviewed the KSU Efficacy Test that used the Certified Indicator and issued approval for

the technology based on its findings. To date, BMTS' Demolizer® technology is either

approved or meets statutory requirements in 46 states. Historically, the technology has

11

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been reviewed favorably by over 75 federal, state, and local agencies, and it meets

statutory requirements for treatment across the United States and throughout the

international community. Exhibit A, attached hereto, contains regulatory approval

documentation from select states, including the States of Arizona and Delaware, which

have accepted the Certified Indicator as equivalent to the Chemical Indicator. This

information has been previously provided to the Illinois Bureau of Land in September

2007 in support of the Agency's review of this petition.

In mid-October 2006, BMTS contacted the Illinois Environmental Protection

Agency (the "Agency") to request that the Agency consider a continuous monitoring

system as an alternative to biological testing consistent with the provisions of 35 IAC

1422.125(a)(3).

7

After speaking with an Agency representative, BMTS submitted a

formal request that included the KSU Efficacy Test results on October 19, 2006. Over

the next few months, BMTS periodically contacted the Agency to check on the status of

its request and was told that a response would be issuing shortly. In January 2007,

BMTS received a formal response from the Agency stating that, in the Agency's opinion,

the KSU Efficacy Test did not conform with the IET requirements. A true and correct

copy of the Agency's January 5, 2007 Letter is attached hereto as Exhibit B.

After receiving the Agency's January 5, 2007 letter, BMTS agreed to provide the

Agency with additional information to resolve the issue regarding the IET, which was

transmitted on January 10, 2007. A true and correct copy of BMTS' January 10, 2007

Correspondence is attached hereto as Exhibit C. Over the next four months, BMTS

periodically contacted the Agency to inquire as to its review of the additional information

7

Formal approval from the Agency is required in order for a manufacture like BMTS to use a continuous

monitoring approach to periodic verification initiatives.

12

---

BMTS provided. On May 7, 2007, BMTS received a response from the Agency that

reiterated its prior position.

8

A true and correct copy of the Agency's April 4, 2007

Letter is attached hereto as Exhibit D. The Agency's representative referred BMTS to

Agency attorney Bill Ingersoll, who in turn referred BMTS to the Agency Attorney, Kyle

Davis.

From May 8, 2007 through early June 2007, BMTS exchanged correspondence

with Mr. Ingersoll regarding the IET. A true and correct copy of the e-mail

correspondence between BMTS and Mr. Ingersoll is attached hereto as Exhibit E. Mr.

Ingersoll recognized that the Chemical Indicator was not commercially available. 9 Even

so, Mr. Ingersoll stated that "it seems that we are unable to help you . . ."

See

Exhibit E.

Pursuant to the suggestion of Mr. Ingersoll, BMTS filed a Variance Petition on or about

June 24, 2007. (The Variance Petition was subsequently dismissed on July 26, 2007).

On August 24, 2007, BMTS, IEPA, and Agency attorney Kyle Davis, discussed

concerns related to the Variance Petition. Dr. Marsden participated in this teleconference

to try to answer specific technical questions on the appropriateness of the use of the

Certified Indicator in the KSU Efficacy Study. As an outcome of this conference, BMTS

agreed to provide additional information supporting the assertion that the Certified

Indicator is the preferred and most appropriate biological indicator organism for the

validation of dry heat sterilization processes. As part of this effort, BMTS provided the

8

Although the Agency's letter was dated April 4, 2007, which appears in a different type-font than the rest

of the letter, BMTS received the letter on May 7, 2007.

9

The Chemical Indicator cannot be purchased in a certified form. However, it is available in freeze-dried

form, which would require the purchaser to grow a viable population. However, this method necessitates

that the purchaser conduct rigorous testing to certify that the custom-grown population has the proper

resistance properties to validate a treatment process. In most cases, the purchaser will have to grow and

test several populations in order to certify a custom-grown population.

13

---

Agency with additional information regarding the acceptance of the Certified Indicator

by other states.

See

Exhibit A.

Dr. Daniel Y.C. Fung, an internationally known food, environmental and public

health microbiologist, and authority in the field of sterility control, reviewed the body of

scientific literature and provided an assessment on the appropriateness on the use of the

proposed Adjusted Standard for the validation of the Demolizer® technology.

Specifically, Dr. Fung concludes:

Based on the overwhelming evidence, it is my expert opinion that

Bacillus

subtilis var. niger

(ATCC 9372, also known as

Bacillus atrophaeus)

is the

most appropriate

biological indicator organism for the validation of dry

heat sterilization technologies. This specific subspecies of

Bacillus

subtilis

demonstrates excellent growth and dry heat resistance

characteristics. Standards for performance have been established by USP,

ISO, and others to ensure that certified biological indicators for dry heat

sterilization deliver predictable and standardized resistance.

The Demolizer® technology is an alternative infectious waste treatment

system that employs dry heat as the sterilization agent. As such, the most

appropriate biological indicator organism for the validation of the efficacy

of the Demolizer® technology is the ISO and USP recognized standard,

Bacillus subtilis

var.

niger

(also known as

Bacillus atrophaeus).

Further,

certified carriers manufactured under rigorous quality standards should be

used, wherever possible, since such carriers are tested for purity and

performance meeting defined D-value and z-value performance criteria.

Letter from Dr. Daniel Y. C. Fung to Diane Gorder, August 27, 2007, a true and correct

copy of which is attached hereto as Exhibit F.

Dr. Fung has published extensively in Food Microbiology, Applied Microbiology

and Rapid Methods with more than 700 Journal articles, meeting abstracts, proceeding

papers, book chapters and books in his career. He has served

as

the major professor for

more than 90 M.S. and Ph.D. graduate students. The Kansas State University Rapid

Methods and Automation in Microbiology Workshop, directed by Dr. Fung, has attracted

14

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more than 3,500 participants from 60 countries and 46 states to the program in the past 27

years. Dr. Fung is a Fellow in the American Academy of Microbiology, Institute of Food

Technologists (IFT), International Academy of Food Science and Technology and

Institute for Food Science and Technology (UK). He has won more than 30 professional

awards which included the International Award from IFT (1997), Waksman Outstanding

Educator Award from The Society of Industrial Microbiology (2001), KSU College of

Agriculture Excellence in Graduate Teaching Award (2005), and the Exceptional

Achievement and Founder of the KSU International Workshop on Rapid Methods and

Automation in Microbiology Award given by the Director of the Center for Food Safety

and Applied Nutrition, U.S. Food and Drug Administration, 2005. Dr. Fung received the

B.A. degree from International Christian University, Tokyo, Japan in 1965, M.S.P.H. at

University of North Carolina-Chapel Hill in 1967, and the Ph.D. in Food Technology

from Iowa State University in 1969. He is currently a Professor of Food Science,

Professor of Animal Sciences and Industry and Ancillary Professor of Biology at Kansas

State University and Distinguished Professor Universitat Autonama de Barcelona, Spain.

Based on all of this information, the Agency has agreed to recommend to the

Board that it grant this Petition for an Adjusted Standard.

VI. Difficulties Meeting 35 IAC

§

1422.Table B

In developing the specific protocol used for demonstrating treatment efficacy,

BMTS attempted to acquire the Chemical Indicator in a certified carrier form.

Unfortunately, this subspecies is not available commercially in a certified carrier form.

With the help of researchers at Kansas State University, BMTS reviewed a

comprehensive scientific literature survey and identified an equivalent subspecies, the

15

---

Certified Indicator, as the industry standard for the validation of dry-heat sterilization

processes. The overwhelming use of the Certified Indicator as the preferred and most

appropriate indicator organism for dry-heat processes stems from its demonstrated

excellent dry heat resistance compared to dry heat sterilization compared to other

B.

subtilis

organisms.

See

Exhibit F, Letter from Dr. Daniel Fung; Group Exhibit J Gurney,

et al,

for expanded discussion on the appropriateness of the Certified Indicator for the

validation of dry heat sterilization processes. The Certified Indicator is cited in numerous

national and international standards including the U.S. Pharmacopoeia, the International

Standards Organization, and over three dozen scientific papers related to the validation of

sterilization processes.

See

Group Exhibit J,

infra.

BMTS made the decision to use the Certified Indicator because: 1) the indicators

are phenotypically identical with the exception of pigmentation response; 2) the Certified

Indicator is nearly universally recognized as the appropriate indicator microorganism to

demonstrate the effectiveness of dry-heat treatment processes, the underlying treatment

technology of the Demolizer® system; and 3) use of a Certified Indicator comports with

the best practices of the scientific community since Custom Indicator populations must be

grown in more non-controlled laboratory environments where it is possible to

inadvertently compromise the resistance and growth properties. Each manufacture must

test all production lots against stringent dry heat resistance performance standards as

expressed in D-values and z-values. Since the Certified Indicator is indisputably

recognized as the most appropriate

Bacillus

indicator organism for dry heat sterilization

processes and considered superior, from a heat resistance perspective, to the Chemical

Indicator and, unlike the Chemical Indicator, is available in a certified form that comports

16

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with the industry's best practices, BMTS used the Certified Indicator in the KSU Efficacy

Test.

VII.

Description of Efforts Necessary for BMTS to Achieve Immediate

Compliance

35 IAC 104.406(e) requires that the Petition contain a description of the efforts

required to come into immediate compliance. Under the Agency's current interpretation

of the Board's regulations, it is impossible for BMTS to achieve immediate compliance,

which could take as long as two and a half years due to the time and resources required to

grow and certify a Chemical Indicator to the same standards already demonstrated in the

KSU Efficacy Test. However, BMTS has already conducted a successful IET using the

preferred and most appropriate

Bacillus subtilis

indicator microorganism with a dry heat

resistance, understood in the scientific community, to be superior to that of the specific

species identified in the regulations. Therefore, if the Board were to accept the proposed

Adjusted Standard recognizing the overwhelming evidence in the scientific community,

BMTS would be in immediate compliance with the Board's regulations.

VIII.

Immediate Compliance Would Impose an Arbitrary and Unreasonable

Hardship

35 IAC 104.406(e) requires that BMTS set forth reasons why immediate

compliance with the regulation would impose arbitrary and unreasonable hardship. Table

B's requirement of using a Chemical Indicator over a Certified or Dry Heat Indicator is

inappropriate and would impose an arbitrary and unreasonable hardship because it does

not take into consideration the body of scientific evidence that unequivocally supports the

claim that the Certified Indicator is most appropriate due to enhanced heat resistance

under dry heat conditions.

17

---

Further, 35 IAC 1422 has not been updated to include the Certified Indicator as an

equivalent alternative

B. subtilis

organism for the validation of dry heat and gas

sterilization technologies consistent with the market availability of such sterilization

technologies and the consensus within the standards and scientific community. At the

time of the adoption of 35 IAC 1422, prevalent sterilization technologies included

incineration, steam sterilization, chemical disinfection and radiation. The selection of the

specific subspecies in Table B are appropriate and consistent with scientifically

recognized indicator organisms for these traditional sterilization processes but are

inconsistent with domestic and international standards for the qualification of dry heat

treatment processes. These international standards promulgated by the US

Pharmacopoeia, International Standards Organization, the U.S. Food and Drug

Administration, the European Pharmacopoeia Commission, and others are the primary

reason why

B. subtilis

is only available commercially both domestically and

internationally as the Certified Indicator used in the KSU Efficacy Study.

Most states modeled their statutes and regulations off of a report titled

Technical

Assistance Manual: State Regulatory Oversight of Medical Waste Treatment

Technologies

that was prepared by the State and Territorial Association on Alternate

Treatment Technologies (the "STAATT Report").

10

True and correct portions of the

STAATT Report are attached hereto as Exhibit G. The STAATT Report identified the

Chemical Indicator strain as a representative example of

Bacillus subtilis.

However, the

STAATT Report stressed that the Chemical Indicator spore was only a representative

strain of the species and was not selected based on any special resistance properties.

I°

The STAATT Report was a culmination of conferences and debates beginning in 1992, the conclusions

of which were widely disseminated prior the publication of the final STAATT Report in April 1994.

18

---

Further, the STAATT Report stated that "the guidelines developed through this series of

meetings should serve only to provide guidance to states in the development of a review

and approval process for medical waste treatment technologies." Exhibit G, STAATT

Report at p. 3.

As explained by Dr. Nelson S. Slavik, the primary author of the STAATT Report,

BMTS' "selection of

B. subtilis

ATCC 9372 spores is consistent with the criteria

provided by STAATT in their publication. This strain [the Certified Indicator] provides

the dry-heat resistance which is appropriate for your treatment process." Letter from

Nelson S. Slavik to Diane Gorder, June 11, 2007, a true and correct copy of which is

attached hereto as Exhibit H.

This opinion is supported by Dr. Daniel Y. C. Fung, internationally renowned

microbiologist.

See

Section V-C of this Petition and Exhibit F for a review of Dr. Fung's

analysis on the appropriateness of the Certified Indicator. The 35 IAC 1422 requirements

that dry-heat based sterilization processes use the Chemical Indicator as opposed to the

Certified or Dry Heat Indicator in the IET is clearly arbitrary.

Moreover, BMTS will incur significant and unreasonable costs if it is required to

repeat the KSU Efficacy Test using a different colored indicator microorganism that is

likely to exhibit inferior heat resistance in a dry heat sterilization process. After learning

of the Agency's position, BMTS requested that KSU prepare an estimate to repeat the

KSU Efficacy Test using the Chemical Indicator to the same quality standards as attained

in the original KSU Efficacy Study. In preparation of this estimate, BMTS again

contacted Dr. Marsden, who would be responsible for repeating the study. Dr. Marsden

informed BMTS that, in order to grow a custom indicator and ensure comparable quality

19

---

standards to the previously conducted study using a certified carrier, the study would

require two major phases.

The first phase would involve growing a culture population of the Custom

Indicator and certifying its resistance properties through exhaustive D-value studies."

Dr. Marsden would use standard protocols for validating the resistance of the culture

similar to those used throughout the industry. This study will likely need to be repeated

several times until a population is grown to the standards comparable to a Certified

Indicator like those obtained from certified manufactures.

Dr. Marsden provided an estimate of a minimum of $60,000 for a single D-value

evaluation of a population. It is very possible that repeated trials could result in a

total

cost approaching $250,000

to properly certify the population with a

total time frame of

up to two years.

These estimates are phase-one costs only.

Once a Custom Indicator population has been grown and certified, Dr. Marsden

would begin the second phase, which involves repeating the Demolizer® efficacy study

using appropriate replicates, load conditions, etc. This requires a

minimum of 2-4

months

to coordinate and report the study. Upon completion of both phases, validation

results comparable to those already reported could be obtained. The estimate provided by

Dr. Marsden for phase two of the validation study using ATCC 19659 is

$40,000.

In

addition to these costs, BMTS would incur

direct costs totaling more than $30,000,

which includes the cost of three dedicated systems and the cost of BMTS staff time to be

on-site at Kansas State University to facilitate the trial.

II

An organism's D-value is the treatment time required for 90% deactivation (sterilization), i.e.,

a

measure

of an organism's resistance to a particular treatment method - here, dry-heat.

20

---

Therefore, the total cost for repeating the efficacy study using a Custom

Indicator is estimated to be between $130,000 and $320,000 dollars and could take

up to two and

a

half years to complete.

A true and current copy of the estimate is

found in Exhibit I. This information was also provided to the Illinois Bureau of Land in

September 2007 in support of the agency's review of this petition. BMTS would have to

sell numerous additional Demolizer® units to make up for the cost of repeating the IET

with the Chemical Indicator. Given that the Certified Indicator is reported to demonstrate

greater heat resistance than other

Bacilus subtilis

isolates, requiring BMTS to repeat the

same efficacy test using a Chemical Indicator is an arbitrary and unreasonable hardship.

Further, BMTS envisions continuous improvements of the technology which may

necessitate future IET trials to validate such improvements have not adversely impacted

treatment efficacy. The Certified Indicator is the scientifically recognized and widely

accepted indicator organism for the validation of the Demolizer® technology. If the

Adjusted Standard is not granted, BMTS will continue to incur substantial ongoing costs

to conduct efficacy studies using two similar and likely equivalent organisms, the

Certified Indicator and the Chemical Indicator organisms. Such duplicate effort is not

scientifically justified and is an arbitrary and unreasonable hardship.

IX.

?

Narrative Description of the Proposed Adiusted Standard

35 IAC 104.406(1) requires that the Petition provide a narrative description of the

proposed adjusted standard as well as proposed language for a Board order that would

impose the standard. The Adjusted Standard would simply involve formally recognizing

the appropriateness of both the Certified and Chemical Indicators in Table B of 35 IAC

1422 for the validation of dry heat and chemical sterilization processes, respectively. The

21

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proposed language for a Board order would involve amending Item 1 of Table B from

"I. Bacillus subtilis (ATCC 19659),"

to

"1. Bacillus subtilis (ATCC 19659) or Bacillus

subtilis var. niger (ATCC 9372)." If the Board wishes to recognize the recent change in

species classification, the proposed language could read, "1. Bacillus subtilis (ATCC

19659) or Bacillus atrophaeus (ATCC 9372)."

35 IAC 104.406(f) further requires the Petition to describe efforts necessary to

achieve this proposed standard and the corresponding costs must also be presented.

BMTS has already completed an Initial Efficacy Test demonstrating a 6 log io reduction

of

B. subtilis

var.

niger

(ATCC 9372) under varying load conditions the Agency has been

acknowledged meets the requirements of 35 IAC 1422 with the exception of the use of

the Certified Indicator instead of the Chemical Indicator. Thus, no additional efforts are

required by the Petitioner if the proposed standard is adopted.

X.

No Environmental Impact

35 IAC 104.406(g) requires that the Petition describe the quantitative and

qualitative description of the impact of the petitioner's activity on the environment if the

Petitioner were to comply with the regulation of general applicability as compared to the

quantitative and qualitative impact on the environment if the Petitioner were to comply

only with the proposed Adjusted Standard. BMTS' activities, operating under either the

regulation of general applicability or the proposed Adjusted Standard, have no adverse

impact on human, plant, or animal life. This is established by the studies described

herein. There are no emissions, discharges or releases from the use of the Demolize®

technology. All infectious waste treated in a Demolizer® system meets the requirements

for sterilization and final disposal outlined in the regulations.

22

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XI.?

Justification for the Adjusted Standard

35 IAC 104.406(h) requires that the Petition explain how the Petitioner seeks to

justify, pursuant to the applicable level of justification, the proposed adjusted standard.

As presented in Section IV of this Petition, the regulation of general applicability does

not describe a specific level of justification therefore the level of justification outlined in

35 IAC 104.426 applies. The following outlines a statement of justification for each of

the four conditions outlined in 35 IAC 104.426.

A.

Change in Factors Relied Upon by the are Substantially

Different

35 104.426(a)(1) requires that the Petitioner demonstrate that factors relating to

that petitioner are substantially and significantly different from the factors relied upon by

the Board in adopting the general regulation applicable to that petitioner. At the time the

Illinois regulations were drafted (1992-1993), infectious waste treatment technologies

available both domestically and internationally primarily consisted of autoclave or steam

sterilization, chemical disinfection, and radiation. The Agency identified scientifically

recognized indicator organisms for these classes of sterilization technologies.

Bacillus

stearothermophilus

is the internationally recognized indicator organism for the validation

of steam sterilization technologies in the same manner that the Certified Indicator is the

USP and ISO recognized indicator organism for dry heat.

Bacillus subtilis

(ATCC

19659), the Chemical Indicator, is commonly used for the validation of chemical

disinfection processes, disinfectants and hand washing procedures.

Bacillus pumilis

is

generally recognized as the appropriate indicator organism for radiation sterilization

technologies. During the time period of the adoption of the Illinois regulation, the

STAATT committee, a group of state regulatory personnel and infection control

23

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scientists, strongly recommended that the specific subspecies

(Bacillus subtilis, Bacillus

stearothermophilus, and Bacillus pumilis)

are for example purposes only and should not

be integrated directly into regulations since future technologies may warrant the use of

better suited indicator organisms. Section VIII and Exhibit

H

hereto provide additional

supporting evidence to this effect.

In the late 1990s, the Demolizer® technology and other dry heat based systems

were formally introduced in the United States for the treatment of infectious wastes. The

regulations were, in fact, promulgated in 1993 before the Demolizer technology was

formally introduced. Further, published standards for the validation of dry heat

sterilization technologies both domestically and internationally converged on the

selection of the Certified Indicator in the mid to late 1990s as the most appropriate

indicator organism for the validation of such technologies. The specific selection of

biological indicators in Table B is consistent with chemical disinfection, steam

sterilization, and radiation-based technologies. Table B does not, however, include the

Certified Indicator which is specifically optimal for the validation of dry heat sterilization

processes.

The Agency acknowledged that an Adjusted Standard may be necessary to

address emerging technologies in the Second Notice for Rulemaking (R91-20) published

on March 25, 1993. On Pages 19 and 20 of this Notice, the Agency specifically cites the

following:

The record shows that the Study Group and the Agency proposed these

provisions to allow easy consideration for new technologies that do not fit

the definition of chemical, thermal or irradiation treatment. The Board

supports this concept. (Note, dry heat is not specifically listed in the

definition of thermal treatment provided in the regulation.)

24

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The Board emphasizes that it is sympathetic with the concerns of the

Agency regarding the administrative burden of adjusted standards. An

adjusted standard proceeding is resource consuming not only for the

Board, but for the Agency and the petitioning party as well. Accordingly,

reliance on the adjusted standard process must be contemplated with care

that an unnecessary and onerous administrative burden is not created.

By requiring the Board to grant adjusted standards consistent with Section

27(a), the statute requires the Board to consider the implications of certain

site-specific conditions when granting an adjusted standard. As long as

information requirements are met to the extent applicable, a technology-

specific adjusted standard may be granted.

Therefore, at the time of adoption of the general regulation, dry heat sterilization

had not been adapted for the treatment of infectious waste and was not included in the

definition of thermal treatment. In the late 1990s, the Demolizer® and other dry heat

treatment technologies became available in the U.S. and the international community.

The specific organisms listed in Table B of 35 IAC 1422 are consistent with technologies

available in the U.S. in the early 1990s. Table B, however, is not consistent with the

application of dry heat to treat infectious waste. The Agency and the Board envisioned

that adjusted standards may be warranted to include alternative biological indicators on a

technology-specific basis. Therefore, the absence of dry heat alternatives at the time of

drafting of the regulations is a factor that is substantially and significantly different than

factors existing today and warrant adoption of a technology-specific, adjusted standard.

B.

Existence of Those Factors Justifies an Adjusted Standard

35 104.406(a)(2) requires that the Petitioner demonstrate that existence of such

factors justifies an adjusted standard. As stated above, 35 IAC 1422 is not consistent

with the large body of scientific evidence for the selection of appropriate indicator

microorganisms for the validation of dry heat medical waste treatment technologies. The

scientific consensus in the domestic and international scientific community and the

25

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overwhelming body of evidence justify the use of the Certified Indicator for the

validation of dry heat sterilization technologies. Use of a different indicator organism,

such as

B. stearothermophilus

or

B. pumilis,

are not recognized in the scientific

community for the validation of dry heat technologies. The Chemical Indicator,

Bacillus

subtilis

(ATCC 19659) is recognized in the scientific literature for the verification of

chemical disinfectants, chemical disinfectant processes and hand washing procedures.

The Chemical Indicator is not recognized in the scientific community for the validation

of dry heat treatment processes. Similarly,

Bacillus subtilis

var.

niger

(ATCC 9372), the

Certified or Dry Heat Indicator, is the biological indicator of choice for dry heat

sterilization technologies due to its enhanced heat resistance under such conditions.

Further, the use of certified carriers for the validation of sterilization technologies

represents best practices in the scientific community since such certified carriers are

manufactured under strict international standards for quality and certification. The

Chemical Indicator is not commercially available in a certified form, thus insistence on

the use of a carrier that is not recognized for the validation of dry heat technologies and

must be grown under non-controlled conditions actually results in a lower quality result.

For these reasons, the factors presented hereto justify the proposed Adjusted Standard.

C.

No Environmental or Health Effects

35 104.406(a)(3) requires that the Petitioner demonstrate that the requested

standard will not result in environmental or health effects substantially and significantly

more adverse than the effects considered by the Board in adopting the rule of general

applicability. The extensive body of scientific evidence presented herein provides proof

that the proposed Adjusted Standard has no adverse environmental or health effect

26

---

compared to the standard stipulated in the regulation of general applicability. In fact, the

proposed Adjusted Standard is more beneficial. The proposed Adjusted Standard, the

Certified Indicator, poses a more difficult challenge for the Demolizer® technology than

the Chemical Indicator. BMTS has demonstrated that the Demolizer® technology

delivers a minimum 6 log i o reduction of the Certified Indicator consistent with the

regulatory disinfection standard.

D. Consistency with Applicable Federal Law

35 IAC 104.406(a)(4) requires that the Petitioner demonstrate that the adjusted

standard is consistent with any applicable federal law. The treatment of infectious waste

and the approval of alternative treatment technologies are not regulated at the federal

level. However, state, federal, and international authorities recognize the use of the

Certified Indicator as an appropriate indicator microorganism for dry heat sterilization

validation procedures.

BMTS' Demolizer® devices have been approved or meet statutory requirements

in 46 states based on the results of the KSU Efficacy Test. While some of the states that

have approved Demolizer® technology do not specify a particular strain of indicator

microorganism, e.g., California, New York, Michigan, Connecticut, North Carolina,

South Carolina, Georgia, and Louisiana, others such as Florida specify only that the

species

B. subtilis

be used to validate sterilization treatments. Of the three states that

particularly identify the Chemical Indicator in their regulations, Arizona, Delaware, and

Illinois, BMTS has already received approval from both Arizona and Delaware based on

the KSU Efficacy Test.

See

Exhibit A. The State of New Mexico regulations have

recently been updated effective August 2, 2007. The previous draft of the New Mexico

27

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Administrative Code, Solid Waste Regulations cited the

B. subtilis

ATCC 19659 (the

Chemical Indicator) as an indicator organism to demonstrate initial efficacy of alternative

treatment technologies. In the recently amended N.M.A.C. 20.9.8.13, the State of New

Mexico specifically recognizes

Geobacillus stearothermophilus

or

Bacillus atrophaeus

(the Certified Indicator) as appropriate and scientifically recognized indicator organisms

for the validation of alternative technologies consistent with the facts and the evidence of

scientific consensus described hereto.

The federal government recognizes the appropriateness of using the Certified

Indicator to validate sterilization procedures. The U.S. Food and Drug Administration

identifies the Certified Indicator as the appropriate test organism for dry-heat based

sterilization procedures.

See

Group Exhibit J,

Guidance on Premarket Notification

[510(k)] Submissions for Sterilizers Intended for Use in Health Care Facilities

(March

1993);

Guidance on Premarket Notification [510(k)] Submissions for Sterilizers Intended

for Use in Health Care Facilities; Draft Guidance for Industry and FDA Reviewers (May

2001),

supra.

In addition, the U.S. Pharmacopeia states that an appropriate biological

indicator for dry-heat sterilization should "compl[y] substantially with the morphological,

cultural, and biochemical characteristics of the strain of

Bacillus subtilis,

ATCC No.

9372 [the Certified Indicator], designated subspecies

niger . ."

Group Exhibit J, U.S.

Pharmacopeia, Monographs: Biological Indicator for Dry-Heat Sterilization, Paper

Carrier, USP28-NF23 USP (2005),

infra.

Moreover, the international community has identified the Certified Indicator as

the standard indicator microorganism for validating dry-heat processes. For example, the

British Pharmacopoeia, the European Pharmacopoeia, the Japanese Pharmacopoeia, and

28

---

the International Organization for Standardization all list the Certified Indicator as the

biological indicator to validate dry-heat sterilization treatments. The world-wide

acceptance of the Certified Indicator as the industry standard further supports BMTS'

assertion that the Certified Indicator is the most appropriate organism for the validation

of dry heat sterilization technologies.

XII.

Consistency with Federal Law

35 IAC 104.406(i) requires that the Petition provide supporting reasons that the

Board may grant the proposed standard consistent with federal Law. Section XI-D of this

Petition provides such a statement. Infectious waste treatment standards are not governed

at the federal level. There are no procedural requirements applicable to the Board's

decision on the petition that are imposed by federal law.

XIII. Supporting Documents

35 IAC 104.406(k) requires that the Petition cite supporting documents and legal

authority. With respect to documents, Exhibits A through Exhibit I are attached to this

Petition and are specifically referenced herein. In addition, for the convenience of the

Board, true and correct copies of relevant portions of the scientific authorities cited in this

Petition are attached collectively hereto as Group Exhibit J. The scientific literature

discussed in this Petition establishes that the Chemical and Certified Indicators are very

similar'', if not equivalent, with the Certified Indicator recognized internationally as the

most appropriate

biological indicator for the validation of dry heat sterilization processes.

12

In the scientific community, both the Certified and Chemical Indicators have been used to demonstrate

efficacy of a particular sterilization technology. The two substrains are very similar with the exception of

pigmentation response to certain culture conditions and, prior to 2004, were classified in the same

Bacillus

species. Nakamura and others state that, "[e]xcept for colour of the soluble pigment, all of the strains were

indistinguishable by the standard characterization method; i.e., they exhibited the traits typical of

B.

subtilis."

Group Exhibit J, Nakamura,

supra.

Blackwood reported that the RNA sequences of various

substrains of

B. subtilis are

indistinguishable with a reported sequence mapping of over 99%.

See

Group

29

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Most importantly, Gurney and Quesnel established that the Certified Indicator is

the preferred biological microorganism for the validation of dry heat treatment processes

in a definitive comparative study. This work surveyed the compendium of published

literature on dry heat resistance of

Bacillus subtilis

spores. Further, the authors

completed extensive comparative resistance studies on the Certified Indicator and a

generic

Bacillus subtilis

organism, typical of the Chemical Indicator, over a temperature

Exhibit J, Blackwood,

supra.

Moreover, Blackwood also reported that the only way to differentiate

between the substrains would be to observe oxidative activity since they are identical with the exception of

pigmentation differences.

See id.

In 2000, the European Commission Health and Consumer Protection

Directorate-General stated that

"B. atrophaeus

is distinguishable from

B. subtilis

only by pigmentation."

Group Exhibit J, European Commission, Health and Consumer Protection Directorate-General,

Opinion of

the Scientific Committee on Animal Nutrition on the Safety of Use of Bacillus Species in Animal Nutrition

(Feb. 17, 2000).

Both strains have been used in the validation studies for various oxidative sterilization technologies. In all

cases, there was no reported difference in the performance of the two substrains. The Chemical Indicator is

broadly used for the validation of disinfectants and chemical disinfection processes. The Certified

Indicator is broadly used and recognized as the preferred indicator organism for dry heat sterilization

processes due to its demonstrated superior dry heat resistance. The Certified Indicator is also recognized

for the validation of certain gas sterilization technologies, including ethylene oxide disinfection.

See

generally,

Group Exhibit J;

see Gurney and Quesnel, see

U.S. Food and Drug Administration,

Guidance on

Premarket Notification [510(k)] Submissions for Sterilizers Intended for Use in Health Care Facilities

(March 1993) (listing both the ATCC 9372 and the ATCC 19659

B. subtilis

samples as equivalent indicator

organisms to validate dry-heat sterilizers);

see also

U.S. Food and Drug Administration,

Guidance on

Premarket Notification [510(k)] Submissions for Sterilizers Intended for Use in Health Care Facilities;

Draft Guidance for Industry and FDA Reviewers (May

2001) (updated publication listing only the Certified

Indicator (ATCC 9372) to validate dry-heat sterilization treatments).

In a 2004 Environmental Technology Verification Report conducted by Battelle, both the Chemical and

Certified Indicators were used to validate the effectiveness of a formaldehyde-based decontamination

technology, and there were no reported qualitative differences in the resistance of the two samples.

See

Group Exhibit

J,

Battelle,

Environmental Technology Verification Report prepared for CERTEK, Inc.

(Aug. 2004).

In a 2001 comparative study by Khadre and Yousef, the resistance of both the Certified and Chemical

Indicators were shown to be equivalent during an evaluation of ozone and hydrogen peroxide sterilization

technologies.

See

Group Exhibit J, M.A. Khadre, A.E. Yousef,

Sporicidal Action of Ozone and Hydrogen

Peroxide: A Comparative Study, INT'L. J.

OF FOOD MICROBIOLOGY,

71, 131-138 (2001). In fact, Khadre

and Yousef concluded that "differences among these strains were not significant (p<0.05)."

Id.

Similarly, in a study by Sagripanti,

et aL,

the Chemical and Certified Indicators were evaluated along with

other various strains for sporicidal activity against a broad range of oxidative treatment technologies and

found to have resistances "within I Log

i°

of each other." Group Exhibit J, J-L. Sagripanti,

et al., Virulent

Spores of Bacillus Anthracis and other Bacillus Species Deposited on Solid Surfaces Have Similar

Sensitivity to Chemical Decontaminants, J.

APPLIED MICROBIOLOGY,

102, 11-21 (2007).

30

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range of 140 to 170°C. At all temperatures, the Certified Indicator demonstrated superior

heat resistance properties. Group Exhibit J, Gurney, T.R. & Quesnel, L.B.,

Thermal

Activation and Dry-heat Inactivation of Spores of Bacilus subtilis MD2 and Bacillus

subtilis var. niger, J.

APPLIED BACTERIOLOGY,

48, 231-247 (1980).

The following domestic and international standards list the Certified Indicator for

the validation of dry-heat processes. Each standard is developed by an expert panel of

microbiologists and sterility assurance specialists who review the body of scientific and

published literature to make recommendations based on overall resistance of organisms

to a specific sterilization technology. Manufacturers of certified carriers, such as those

used in the KSU Efficacy Study, must test each production lot of carriers to meet specific

heat resistance targets, as measured in D-value and z-values under specific conditions, to

ensure the proper standardization.

1.

US

Pharmacopoeia.

USP28-NF23 USP. Monographs: Biological Indicator for

Dry-Heat Sterilization, Paper Carrier; Rockville, MD; 2005.

2. FDA.

Guidance on Premarket Notification [510(k)] Submissions for Sterilizers

Intended for Use in Health Care Facilities. Infection Control Devices Branch,

Division of General and Restorative Devices (March 1993).

3. FDA.

Premarket Notifications [510(k)] for Biological Indicators Intended to

Monitor Sterilizers Used in Health Care Facilities; Draft Guidance for Industry

and FDA Reviewers; U.S. Department of Health and Human Services, Food and

Drug Administration, Center for Devices and Radiological Health, Infection

Control Devices Branch (March 2001).

4. British Pharmacopoeia Commission.

Methods of sterilization. London, UK:

British Pharmacopoeia Commission; British Pharmacopoeia Appendix XVIII

(2003).

5.

European Pharmacopoeia Commission.

Biological indicators of sterilization.

Strasbourg, France: European Pharmacopoeia Commission; European

Pharmacopoeia EP 5.1.2 (1997).

6.

Japanese Pharmacopoeia.

JP14e.partll.15 JP. Terminal

Sterilization and

Sterilization Indicators.

31

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7.

ISO and ANSI.

Sterilization of health care products – Biological indicators; Part

4: Biological indicators for dry heat processes. Geneva (Switzerland):

International Organization for Standardization/ANSI; ISO 11138-4:2006.

See

Group Exhibit J.

XIV. Waiver of Hearing

Pursuant to 35 IAC 104.406(j), BMTS hereby waives a hearing on the Petition.

CONCLUSION

BMTS

therefore asks that this Board, pursuant to its authority under Section 35 of

the Act and the Board's regulations under 35 IAC 104, grant BMTS an Adjusted

Standard from the provisions of 35 IAC 1422.Table B recognizing the Certified Indicator

as the most appropriate biological indicator organism for the validation of dry heat

sterilization technologies.

Specifically, BMTS requests that an Adjusted Standard be granted for the use of

Bacillus atrophaeus

(formerly

Bacillus subtilis var. niger,

also scientifically recognized

as ATCC 9372 or NRRL B4418) for the IET of dry heat treatment technologies.

Respectfully Submitted,

BIOMEDICAL TECHNOLOGY

SOLUTIONS, INC.

Dated: November 28, 2007

Neal H. Weinfield

Jason B. Elster

GREENBERG TRAURIG, LLP

(Firm No. 36511)

77 West Wacker Drive, Suite 2500

Chicago, Illinois 60601

312-456-8400 (Telephone)

312-456-8435 (Facsimile)

weinfieldn@gtlaw.com

elsterj@gtlaw.com

By:

One

?led

of Its Atto

ill/

32

---

d

Neal

xedet/KA/

H. Weinfi

Dated: November 28, 2007

BEFORE THE ILLINOIS POLLUTION CONTROL BOARD

BIOMEDICAL TECHNOLOGY SOLUTIONS,

)

INC., a Colorado Corporation,

Petitioner,

v.

PCB 07-

(Adjusted Standard Petition)

ILLINOIS ENVIRONMENTAL PROTECTION

AGENCY,

HEARING WAIVED

Respondent.

CERTIFICATE OF SERVICE

I, Neil H. Weinfield, an attorney, certify that I have caused a true and correct copy

of the foregoing ADJUSTED STANDARD PETITION and NOTICE OF FILING to be

served before 5:00 p.m. via First Class Mail, postage pre-paid, on the following:

Division of Legal Counsel

Illinois Environmental Protection Agency

1021 North Grand Avenue East

P.O. Box 19276

Springfield, Illinois 62794-9276

and

Kyle Davis

Illinois Environmental Protection Agency

1021 North Grand Avenue East

P.O. Box 19276

Springfield, Illinois 62794-9276

---

Exhibit A

---

aNtell-Smr

EliaMedical Technology Solutions, Inc.

September 28, 2007

Mr. Neal H. Weinfield, J.D.

Greenberg Traurig

77 West Wacker Drive, Suite 2500

Chicago, IL 60601

Re: Regulatory Approvals

Dear Neal:

Based on your recent conversations with Kyle Davis of the Illinois Pollution Control

Board, we are enclosing copies of the regulatory approvals in the State of Delaware and

the State of Arizona. These are the two states directly referred to in the draft Adjusted

Standard Petition that reference the ATCC 19659 strain in their infectious waste

regulations. Both states reviewed the Kansas State University efficacy data in 2006 and

reauthorized the approval of the upgraded technology.

In addition to these two states, the Demolizer® technology has been formally approved

in 20 states and 16 counties. Further, the technology meets or exceeds regulatory

requirements for treatment in 23 states that do not formally review technologies for the

onsite treatment of low volumes of medical waste. Instead most of these states either

formally recognize dry heat treatment consistent with the Demolizer® operating

conditions or require generators to have appropriate documentation onsite that

demonstrates compliance with state requirements.

We have also included listings from the few states that post approval status on their

agency websites. These states include California, Oregon, West Virginia, North

Carolina, and Michigan.

Diane R. Gorder

Director of Regulatory Compliance

Cc: Mr. Don Cox, BMTS

9800 Mt. Pyramid Court

Suite 350

Englewood, CO 80112

1-866.525.8MTS

P: 303.653.0100

F: 303.653.0120

bmtscorp.com

---

STATE OF DELAWARE

DEPARTMENT OF NATURAL RESOURCES

& ENVIRONMENTAL CONTROL

DIVISION OF AIR & WASTE MANAGEMENT

89 KINGS HIGHWAY

DOVER, DELAWARE 1990

SOLID & HAZARDOUS WASTE

MANAGEMENT BRANCH

TELEPHONE: (302) 739-9403

FAX No.:?

(302) 739-5060

S

eptember 12, 2006

Ms. Diane Gorder

BioMedical Technology Solutions, Inc.

9800 Mt. Pyramid Court, Suite 350

Englewood, CO 30112

Subject: Approval

of Request for Re-Issue and Transfer of Approval Letter for the

Demolizer Technology

Reference: File

Code: 09.A

Dear Ms. Gorder:

The Delaware Department of Natural Resources and Environmental Control (DNREC), Solid

and Hazardous Waste Management Branch (SHWMB) is in receipt of your letter dated

September 5, 2006, providing additional information regarding the efficacy data for your

company's alternative method to treat infectious waste. Based on the information provided in

the September 5, 2006 letter, your company's alternative method to treat infectious waste does

meet the regulations set forth in the

Delaware Regulations Governing Solid Waste

(DRGSW).

Please note, any facility installing the proposed system in the State of Delaware must fulfill all

the requirements of the DRGSW and will be evaluated on an individual basis. Also, there are

additional regulations that will apply if the treated waste is to be disposed of in a solid waste

landfill or compacted. Chapter 11, Sections G (1) and (2) state:

"1. Infectious waste may not be disposed at a sanitary landfill unless the waste has been

rendered noninfectious and non-recognizable.

2. Compactors, grinders or similar devices may not be used by a generator to reduce the

volume of infectious waste until after the waste has been rendered noninfectious, or unless

the device is part of an approved treatment process which renders the waste noninfectious."

While the DRGSW do afford disposal of infectious waste that is rendered noninfectious and non-

recognizable into a solid waste landfill, the decision to accept such waste lies with the operator

of the solid waste landfill. In Delaware, this entity is the Delaware Solid Waste Authority

(DSWA). Therefore, it is necessary to contact the DSWA at (302) 739-5361 to obtain written

disposal approval.

Deecteaatte" a

Toad

natant deoeinda on pa/

---

Approval of Request for Re-Issue and Transfer of Approval Letter for the Demolizer Technology

Page Two of Two

Should you have additional questions please feel free to contact me at (302) 739-9403.

Sincerely,

Nicole E. Hill

Environmental Scientist

Solid and Hazardous Waste Management Branch

NEN: jmr

NE110669.doc

cc: Karen

G. J'Anthony, Environmental Program Manager I, SHWMB

---

kAiztvrt&

Stephen

A. Owens

Director

Janet Napolltano

Governor

December 22, 2006

PRU 06-362

ARIZONA DEPARTMENT

OF

ENVIRONMENTAL QUALITY

1110 West Washington Street • Phoenix, Arizona 85007

(602) 771-2300

• www.azdeg.gov

CERTIFIED MAIL

Return Receipt Requested

Ms. Diane Gorder

Sr. VP of Operations

BioMedical Technology Solutions, Inc.

9800 Mt. Pyramid Court

Suite 350

Englewood, CO 80112

Re:?

Biohazardous Medical Waste Alternative Treatment

Method Registration No. MWAT221206.00

BioMedical Technology Solutions, Inc.

Dear Ms. Gorder:

The Arizona Department of Environmental Quality (ADEQ), Solid Waste Plan Review Unit (PRU),

received your application dated September 5, 2006, which included product information, efficacy test

results, and other supporting documents for the State of Arizona Biohazardous Medical Waste Alternative

Treatment Method Registration for the Demolizer II System manufactured by your company. PRU

reviewed your submittal and based on the information submitted, it appears that the biohazardous medical

waste treatment technology for Demolizer II System is capable of achieving high level disinfection of

biohazardous medical wastes without adversely impacting public health or the environment.

In accordance with Arizona Administrative Code (A.A.C.) R18-13-1414.A, ADEQ approves the

Demolizer U System unit outlined in the operator's manual as a biohazardous medical waste alternative

treatment technology system. This Biohazardous Medical Waste Alternative Treatment Method

Registration shall be deemed effective as of the date of

this

letter and the accompanying certificate.

Administrative, Operational, and Other Conditions

1.

This approval is granted only for the Demolizer Id System technology used in the efficacy studies

and should not be construed

as a

general endorsement of any other model(s) or system(s). Any

modification of the approved system will require separate approval by ADEQ and may involve

further efficacy testing.

2.

This approval does not relieve BioMedical Technology Solutions, Inc. or any person,

organization, or facility using or intending to use the Demolizer II System, from obtaining any

other approvals which may be required by federal, state, county or local agencies, or Indian

Nations which may have additional regulations within their respective jurisdiction.

Northern Regional Office

?

Southern Regional Office?

•

1801 W. Route 66 • Suite 117 • Flagstaff, AZ 86001

?400

West Congress Street • Suite

433 • Tucson, AZ 85701

(928) 779-0313

?

(520) 628-6733

Printed on recycled paper

---

BioMedical Technology Solutions, Inc.

December 22, 2006

Page 2 of 3

3.

This approval does not relieve BioMedical Technology Solutions, Inc. or any person,

organization, or facility using or intending to use the Demolizer II System of its responsibility to

comply with federal, state, county, or local requirements and shall not be construed as permission

to create a public health hazard, environmental nuisance, or cause contamination to the

environment as prohibited by Arizona Revised Statutes (A.R.S.) 49-141.A.8.

4.

This system is not to be used to treat chemotherapy waste, radioactive waste, and anatomical

wastes, or any identifiable human body parts.

5.

Notification of liquid discharge management practices shall be submitted to the local water/waste

water authority serving the facility where the Demolizer II System is installed prior to discharge.

All discharge must comply with pretreatment requirements.

6.

Arizona municipal solid waste landfills are prohibited from accepting liquids or waste that failed

the EPA paint filter test, in accordance with 40 § CFR 258.28. Therefore, processed waste must

be dewatered to the extent that it will pass the EPA paint filter liquid test prior to placement into

the landfill.

7.

Prior to operating a biohazardous medical waste treatment facility, the owner/operator shall

obtain a facility plan approval in accordance with A.A.C. R18-13-1410, and shall meet the

treatment standards as described in A.A.C. R13-1415, if off-site waste is to be received.

8.

This Certificate is not transferable and is valid for 5 years from the issuance date. Please notify

ADEQ within 14 days after any changes to the information in your application occur.

9.

This Arizona Biohazardous Medical Waste Alternative Treatment Method Registration

constitutes an appealable agency action pursuant to A.R.S. § 41-1092 et seq. To obtain an

administrative

hearing on ADEQ's decision, a Notice of Appeal ("Notice") must be filed with

ADEQ within thirty (30) days of receipt of this letter. The notice

must

contain the following:

The name of the person or party filing the appeal.

ii.

The address of the person or party filing the appeal. (The person or

party

must notify

ADEQ of any change of address within five (5) days of the change).

iii.

The name of the Agency whose decision is being appealed. (In this matter, the agency is

the Arizona Department of Environmental Quality).

iv.

The action being appealed.

v.

A concise statement of the reasons for the appeal.

The notice must be filed with ADEQ. Send the notice to:

Hearing Administrator

Office of Administrative Counsel

Arizona Department of Environmental Quality

1110

West Washington Street

Phoenix, Arizona 85007.

---

Sincerely

BioMedical Technology Solutions, Inc.

December 22, 2006

Page 3 of 3

Notice should be filed by either mailing the notice by certified mail, return receipt requested, or

by hand delivery to ADEQ. The hearing will be conducted by an administrative law judge from

the Office of Administrative Hearings. ADEQ will notify the parties of the hearing date at least

thirty (30) days prior to that date. If the appealing party wishes to try to settle this matter before

the administrative hearing occurs, that party must file a request for an informal settlement

conference with the Hearing Administrator. This request must be filed no later than twenty (20)

days before the hearing date. ADEQ will hold the settlement conference within fifteen (15) days

of receiving the request. Filing an informal settlement request does not change the date of the

hearing.

If you have any questions about this letter, please call Maria Sachs, Solid Waste Permits Plan Review

Unit, at (602) 771-4670 or toll free at (800) 234-5677, Ext. 771- 4670.

Atpif J S on

?

rector

este Programs Division

CC:

?

Mindi Cross, Manager, Inspection and Compliance Section

Enclosure

---

11?

i .

z--tstiffo.t

4 -,..._yym

w,.

•44a,....--

BIOHAZARDOUS MEDICAL WASTE

ALTERNATIVE TREATMENT METHOD

Registration No. MWAT221206.00

In accordance with Arizona Administrative Code Tale 18, Chapter 13, Article 14

Registration issued to

RioMedical Technology Volutions Ine.

This Registrationfor Arizona Biohazardous Medical-Waste

Alternative Treatment

is issued to the above named company or entity and is

to be usedfor treatment ofbiohazardous medical waste only in aceordance with instructions supplied by the company. This registration is

deemed effective on the Issue Date below and expires on the Expiration Date below (5 years after the Issue Date).

This registration is granted based upon the information provided in the Application for Arizona Bio ardous

Medical

Waste Alternative

Treatment Registration. This

registration is not

transferable

from

one company or entity to cmot

Amón a E. Stone,

-Waste Programs Division

ISSUE DATE: December 22, 2006

•

EXPIRATION DATE: December 22 2011

---

Alternative Medical Waste Treatment Technologies

Approved by the California Department of Public Health

Effective Date: August 22, 2007

The technologies listed below have been approved by the Department of Public Health to treat medical waste in California. The approval may

be limited to certain

types of wastes. Please review

the information provided to verify the approved uses of each technology. Individuals

interested in the products described in this document are encouraged to contact the company directly.

Alternative Technology Approval is based solely on a product's demonstration of pathogen destruction. Putting the technologies to use may

require permitting as on-site or off-site medical waste treatment. Sections

118130 and 118135 of

the Medical Waste Management Act require

that any offsite medical waste treatment facility obtain a permit from the Department before treating medical waste. Permitting of onsite

facilities is addressed in 117925 (a) and 117950 (a) of the Medical Waste Management Act.

Company

Device

Address

Telephone

Web Site

Type of

Treatment

Approved for

treatment

of:

BioMedical

Technology Solutions, Demolizer System

9800 Mt.

Pyramid Court, Suite

350

?

Englewood.

CO

303-653-0100

www.bmtscorp.com

Heat

red bag /sharps

Inc.

80112

866-525-BMTS

Earth-Shield Company

Sharp-Shield

304 Yampa Street

Bakersfield, CA 93307

661-322-0300

www.earth-shield.com

encase in

cement

sharps

GMS Marketing

Services

Sterimed

191Hempstead Turnpike

West Hempstead, NY

516 (800)483-

1403

www.globalmarketingservices.

OM

chemical

red bag/sharps

InEnTec Medical

Services, LLC

Plasma Enhanced

Melter

1935 Butler Loop Richland,

WA 99352

509-946-5700

949-472-3713

www.inentec.com

heat

red bag /sharps

/path /trace

chemo /pharms

International

Marketing

Needlyzer

2119 North Kenmore Ave.

Chicago, IL 60614

773-528-2652

www.needlyzer.com

heat

shams

Isolyser

Shams

Management

System (SMS)

6054 Corte Del Cedro

Carlsbad, CA 92009

866-436-9264

www.wcminc.net

encapsulir

e within

container

sharps

512 Lehmberg Road

662-327-1863

Isolyser

ORex Processor

Columbus, MS 39702

800-824-3207

www.orex.com

chemical

red bag

Encore 2000 RWP

116 Roddy Avenue South

Attleboro, MA 027037974

508-399-6400

chemical

red bag/sharps

Medical Innovations

P.O. Box 148 Wayland, MA

508-358-8099

heat

sharps

Inc

01778

508-358-2131

Medical Safe TEC

LFB 12-5,?

SF 15C

330 West Center Street

North Salt Lake, Utah

801-209-6582

801-936-0112 Fax

www.medwastetec.com

chemical

red bag/sharps

Metrex Research

Corp

PermiCide-CA

1717 W. Collins Ave.

Orange, CA92867

800-841-1428

www.metrex.com

chemical

Suction canister

only

---

Oregon

Theodore R. Kulongoski, Governor

Department of Human Services

Public Health Division

800 NE Oregon Street

Portland, OR 97232-2162

(971) 673-1111 Phone

(971) 673-1100 Fax

(971) 673-0372 TTY-Nonvoice

August 01, 2007

ALTERNATIVE INFECTIOUS WASTE TREATMENT PROCESSES APPROVED

BY THE PUBLIC HEALTH DIVISION FOR USE IN OREGON

PROCESS

DATE APPROVED

MEDWASTE TEC

11/18/1991

MODEL Z-5000 HC

MODEL Z-12,500

LFB 12-5/MST Z-12,500

11/19/2004

ECOMED I

12/24/1992

WINFIELD CONDOR

02/02/1993

MEDICLEAN IWP-1000

02/08/1993

STERICYCLE ETD

03/15/1993

DEMOLIZER

TWT

03/06/1995

BMTS

10/17/2006

ABB SANITEC MICROWAVE

03/21/1995

STERIS/ECOMED ECOCYCLE 10

07/24/1995

MEDICLAVE

11/13/1995

STI CHEM-CLAV/MODEL STI-2000CV

07/07/1997

STERIMED

02/17/1999

RED BAG SOLUTIONS SSM-150

03/27/1999

PREMICIDE

06/17/1999

STERIMED JUNIOR

07/02/2002

IET

PLASMA ENHANCED

MELTER

04/26/2004

Assisting People to Become Independent, Healthy and Safe

An Equal Opportunity Employer

---

West Virginia Infectious Medical Waste Program

Page 1 of 2

WV - DHHR - BPH - OEHS - PHS - Infectious Medical

Waste

DHHR Site Search - DHHR Site Map

Alternative Treatment Technologies

Information

Applications & Forms

Click here for information on how to submit a new alternative treatment

technology for approval.

Approved Solidifiers

State Zip

MS 39702

Autoclave Information

Definition of Infectious

Medical Waste

Documents

How Do I...

IMW Question & Answer

Forum

Incineration Information

Links

Medical Waste

Presentations

New Information

Obtain A Copy of the

Rule

Permitted Hauling

Companies

Program Contact

Information

Program Goals

Program Organization

Search our Site

Site Map

Spill Kit Requirements

http://www.wvdhhr.org/wvimw/alternative.asp

?

9/19/2007

Alternative Treatment

Products

Product

Isolyser LTS

Plus

Premicide

Notes Company

?

Address

?

City

24 hour Microtek Medical, 512 Lehmberg Columbus

hold timeInc.

? Road

12 hour OBF Industries, 2719 Curtiss Downers

hold timeInc.?

St.?

Grove

IL 60515

*All solidification products are subject to a hold time prior to

disposal. Hold times allow the product to effectively disinfect the

contents of a suction canister, as well as to set up. Healthcare

facilities are required to ensure the proper disposal of solidified

liquids.

At this time, only these two products can be used to solidify and

disinfect liquid infectious medical waste intended to be land

filled.

Any other solidification products may be used as long as suction

canisters are disposed in the infectious medical waste.

It is a violation of the West Virginia Infectious Medical Waste

Rule if you do not follow the manufacturer's instructions for use

for any of these products.

Alternative Treatment Technologies

Product

HGA-1005,

M, & -250M

Model HGA-

250-S

Demolizer II

System

DSI System

2000

ZMD-M3

Isolyser LTS

Plus

Notes

Microwave

treatment

Microwave

treatment

Heat treatment BioMedical

Technology

Solutions, Inc.

Heat treatment Disposal Sciences,

w/ reusable?

Inc.

sharps box

Stream?

GTH Roland North

treatment with America, Inc.

shredding

Liquid?

Isolyser Company

solidification

and

Company?

Address

ABB Sanitec, Inc. 155 Route 46,

West Plaza II

ABB Sanitec, Inc.

155 Route 46,

West Plaza II

9800 Mt.

Pyramid Court,

Suite 350

6352 320 Street Cannon

Way?

Falls

P.O. Box 487

7887 Katy

?

Houston

Freeway, Suite

200

4320

? Norcross

International

Blvd.

City

Wayne

Wayne

Englewood

State Zip

NI 07470

CO 80112

MN 55009

TX 77024

GA 30093

NJ 07470

---

North Carolina Depa

nt of

Environment and

N.C. Division of Waste Management - MEDICAL WASTE

?

Page 1 of 3

Home

?

About DWM? Contact Us

?

Site Map?

Search

Technical Assistance, Education

& Guidance

>>

Innovative Technology

>>

Medical Waste

Alternative Medical Waste Treatment Technologies

Many hospitals and medical waste treatment facilities have discontinued use of incinerators because of increased ci

costs of operating medical waste incinerators have risen mainly because of the recently enacted EPA regulations fo

Hospital/Medical/Infectious Waste Incinerators (HMIWI).

A number of new technologies have been developed which minimize or nearly eliminate environmental discharges.

summary of some representative technologies is presented in USEPA Alternate Treatment Technologies Fact Shee

Applying for State Approval in North Carolina

It is necessary to obtain state approval to operate a new treatment technology in North Carolina. Vendors who woull

seek approval of a medical waste technology may submit information to obtain approval. Vendors should submit:

•

General description of the treatment system

•

Efficacy data on representative microorganisms (a full report is required which includes complete information

experimental design, unsummarized and summarized data, and qualifications of the testing laboratory)

•

Description of any environmental discharges

•

Information on worker safety aspects

• Additional information may be required.

For further information please contact the Solid Waste Section at (919-508-8512).

Guidance and Assistance in Evaluating New Treatment Technologies

Guidance for Evaluating Medical Waste Treatment Technologies - EPA publication which provides guidance on hcm

evaluate new technologies

Some types of technology require registration under the Federal Insecticide, Fungicide, and Rodenticide Act

Technologies Approved for Use in North Carolina

The following is a list of alternative medical waste treatment technologies approved for use in North Carolina. Comp

products previously listed are either out of business or no longer market formerly approved products in North Carolir

Company

Product

Name

Technology Type

Contact Information

WPS Company

SSM 150/75

Superheated Water

Sanford A. Glazer Directo

Waste Processing

Solutions

Approved: Feb. 5, 2003

Technology

ph: 443-524-4245 ext.#14

3431 Benson Ave.

Fax: 443-524-4250

Suite 100

Ce11:301-254-2234

Baltimore, MD 21227

Email:sglazer@redbag.cc

redbagwps@aol.com

Website: www.redbag.cor

http://www.wastenotnc.org/swhome/medIst.htm

?

9/19/2007

---

N.C. Division of Waste Management - MEDICAL WASTE

?

Page

2 of 3

M.C.M Environmental

SteriMed

Chemical Disinfection

Karen Aibretsen

Technologies, Inc.

Approved: July 28,1999

Project Manager

One Parker Plaza

Ph: 201- 242-1222

Fort Lee, NJ 07024

1-800-783-7463

Fax: 201-592-0393

www.mcm-sterimed.com

Med WasteTec, Inc.

LFB-12-5

Chemical Disinfection

Dennis Cox

2200 South 400 West

(Medical SafeTEC "MST"

Ph:801-845-6550

Salt Lake City, Utah

series)

877-485-6550

84115

Approved: November 15, 1991

Cell:801-718-4904

Fax: 801-484-6417

Dennis.c©medwastetecx

www.medwastetec.com

Sterile Technology

STI ChemClave

Shred/Heat/ Chemical

Randall McKee, Pres., CE

Industries, Inc.

Approved: May 24,1996

(sodium hydroxide)

Ph:317-484-4200

5725 West Minnesota St.

Indianapolis, IN 46241

Fax: 317-484-4201

http://www.wr2.neti

Steris Corporation

Ecocycle 10 – no longer

Shred/chemical sterilant

Richard Snead

5960 Heisley Rd.

Marketed, but company will

(peracetic acid)

Ph:1-800-989-7575 Ext.2'

Mentor, Ohio 44060

continue to support models

still in use.

Fax:440-639-4450

www.steris.com

Approved: Oct. 20, 2001

Biomedical Technology

Demolizer

Thermal Treatment

Don Cox, Pres.

Solutions, Inc.

Approved: Feb. 2, 1994

Diane Gorden, Sr. VP of

9800 Mt. Pyramid Court,

Suite 350

Operations

Ph: 1 866-525-2687

Englewood, CO 80112

Fax: 303-653-0120

BMTSCORP.com

Waste & Compliance

Isolyser

Sharps Disposal System

Ken Tucker

Management, Inc.

Approved: Oct.13,1989

Ph: 866-436-9264

6054 Corte Del Cedro

Carlsbad, CA 92009

Fax: 760-930-9225

www.wcminc.net

email: service@wcminc.ni

Waste Reduction By

WR2 Animal Tissue Digester

Alkaline Hydrolysis,

Gordon I Kaye, Ph.D.

Waste Reduction, Inc.

Approved: Sept.18, 2001

Superheated water

Chairman

2910-D Fortune Circle

Ph:317- 484- 4200

West Minnesota St.

Fax: 317- 484- 4201

Indianapolis, IN 46241

Website: http://www.wr2.r

Ozonator Industries

Ozonator

Shred / Ozone

Randy Johnson

1850 Industrial Drive

Approved: July 17, 2006

Ph: 306-791-0900

PO Box 26030

Regina, Saskatchawan

Fax: 306-791-0905

www.ozonatorindustries.c

Canada S4R 897

Revised November 2006, send questions to: William.Patrakis©ncmail.net

Regulated Medical Waste Treatment Providers

http://www.wastenotnc.org/swhome/medlst.htm

?

9/19/2007

---

MICHIGAN DEPARTMENT OF ENVIRONMENTAL QUALITY

CURRENTLY APPROVED ALTERNATE TREATMENT TECHNOLOGIES

Baker, R.E., Inc.

NOLOGY

Autoclave/Shredder

EcTREATEnt '--

1,2,4

Biomedical Technology Solutions,

Inc.

Dry Heat Oven

2,3,4

BioSteril Technology, Inc.

(Biosiris)

Radiation Sterilization

(Electron Beam)

1-5

Disposal Services, Inc.

Dry_Heat

2,4

ECODAS

Autoclave

1,2,4

Healthcare Products Plus, Inc.

(Needlyzer)

Electric Arc/

Disintegration

4

IET Plasma Enhanced Melter

TM

(PEW")

Plasma Arc

2,3,4

Med Mark International, Inc

(Medaway 1

11")

Dry Heat (Infrared)

Chamber

2,3,4

Medical Safe Tec

Shredder/Chemical

2,3,4

NIC Americas, Inc. (Nic Safe

1800)

Electric Oxidation

4

OBF Industries Inc. (Premisorb,

Premicide, Vitalcide)

Solidifier/Sanitation

2

PEAT International, Inc. (Plasma

Thermal Destruction and

Recovery--PTDRTm)

Plasma Arc

2,3,4

Peerless Waste Reduction, Inc.

(formerly WR2)

Chemical

1,2,3,5

Red Bag Solutions (Formerly

Antaeus Group, Inc.) (SSM)

Autoclave/Maceration

1,2,4,5

San I Pack

Autoclave/Shredder

1-5

Sanitec

Microwave/Shredder

2,3,4

Spintech, Inc.

Dry Heat Oven

4

Stericycle, Inc.

Heat/Shredder

2,3,4

Sterimed

Chemical/Grinder/

Shredder

2,3,4

Steris Corp (Eco-cycle 10)

Chemical/Shredder

2,3,4

STI Chem Clave

Chemical/Autoclave

1,2,3,4

Tempico (Rotoclave ®)

Autoclave/Shredder

1-5

Thermokill, Inc.

Dry Heat Oven

2,3,4

*Medical Waste Categories

1.

Cultures & Stocks

2.

Liquid Human and Animal Waste

3.

Pathological Waste

4. Sharps

5. Contaminated Animal Waste

09/26/2007

---

Exhibit B

---

ILLINOIS ENVIRONMENTAL PROTECTION AGENCY

1021 NORTH GRAND AVENUE EAST,

P.O. Box 19276, SPRINGFIELD, ILLINOIS

62794-9276 -1217) 782-3397

JAMES

R.

THOMPSON CENTER, 100 WEST RANDOLPH, SUITE 11-300,

CHICAGO, IL

60601 - (312) 814-6026

Roo R. BIAGOJEvIcH, GOVERNOR?

DOUGLAS P.

SCOTT, DIREnoR

217/524-3300

January

5, 2007

Diane Corder

BioMedical Technology Solutions, Inc.

9800

Mt.

Pyramid Court

Suite 350

Englewood, Colorado 80112

Re: 9080000000 – Colorado

BioMedical Technology Solutions, Inc.

Log No. PS06-165

Permit File

Dear

Ms. Corder:

Thank you for your letter of October 19, 2006, in which you request to use an alternative method

to comply with the periodic verification test requirements in 35 Illinois Administrative Code

1422. You propose the use of continuous monitoring of critical control operating parameters as

an alternative periodic test for your Demolizer unit designed to treat potentially infectious

medical waste (PIMW).

The initial efficacy testing data included in your submittal indicated that testing was perfonned

on the Demolizer using several microorganisms. However, none of the microorganisms used

were the ATCC number required by 35 Illinois Administrative Code 1422.AppendixA.TableA.

For example, Demolizer testing used

Staphylococcus aureus

ATCC 33591, while the Illinois

regulations require

S. aureus

ATCC 6538. 35 III. Adm. Code 1422 requires the use of these

specific microorganisms, including ATCC number.

The composition and placement of challenge loads used in the efficacy testing appear to comply

with the PIMW regulations. The submitted data indicates that the Demolizer is capable of

achieving a 6 log ic, reduction for all of the microorganisms used.

An alternative periodic verification test (PVT) may be approved and used only when the initial

efficacy test (IET) has been performed completely in accordance with 35 Ill. Adm. Code 1422,

and the alternative PVT has been directly correlated to the results obtained in the 1ET.

Units designed to treat potentially infectious medical waste may be used in Illinois without a

permit from

the

Illinois

Environmental Protection

Agency (Illinois EPA),

provided the treatment

ROC/FORD -

4302 North Main Street, Rockford, IL 61103 -(815) 987-7760 • Da

PLAINES -

9511 W. Harrison St., Des Plaines, IL 60016 - (847) 294-4000

ELGIN - 595 South

State, Elgin, IL 60123 - (847) 608-3131 •

PEORIA -

5415 N. University St., Peoria. IL 61614 - (309) 693-5463

BUREAU Of

LAM) • PEORIA - 7620

N.

University St., Peoria, IL 61614 - (309) 693-5462 •

Ctinnewci4 -

2125 South First Street. Champaign, IL 61820 - (217)

270

-

5000

SlICINGORD -

4500 5. Sixth Street Rd., Springfield, 11 62706 -1217)

706-6092 • COLLP6viLLE - 2009

Mall Street, Collinsville, IL 62234 - (618) 346-5120

MAW.'-2309 W. Main St, Suite 116, Marlon, IL 62959 -(618) 993-7200

PRINTED ON REGYMED PAPER

---

Page 2

unit is accepting and treating only PIMW generated on-site. The efficacy testing results for the

treatment unit must be kept on-site and made available to the Illinois EPA upon request.

In order for the Demolizer treatment unit to be used in Illinois, testing must be performed on the

unit to demonstrate compliance with 35 Illinois Administrative Code: Subtitle M, or you could

seek an Adjusted Standard from the Illinois Pollution Control Board. They may be reached at

312/814-3620.

I hope this satisfies your inquiry. If you have further questions, please feel free to contact

Beverly Albarracin of my staff at 217/524-3289.

Sincerely,Stephe

F. Nightingale, P.E..E.

1

-0

Manager, Permit Section

Bureau of Land

SFNIRmls/073626.doc

---

Exhibit C

---

Page 1 of 1

Diane Gorder

From:

?

Diane Gorder (dgorder©bmtscorp.com]

Sent:?

Wednesday, January 10, 2007 12:04 PM

To:?

beverly.albarracin@epa.statellus

Subject:?

Demolizer technology and the Initial Efficacy Testing

Follow Up Flag: Follow up

Flag Status:?

Red

Beverly,

Attached is a response to your concerns expressed in the January 5, 2007 letter. We are sending this by email and hope

it provides the information you need to complete the evaluation of our alternative quality monitoring approach. While our

customers will likely not require a permit for use of the system within the state, we are diligently working to make certain

we have the appropriate approvals in place to provide the best guidance possible on state regulatory compliance issues.

If you need any more information or have any questions about the Information, please call me on my mobile at (719) 661-

2296. I currently work between two offices so my mobile is the easiest way to reach me.

Thank you again for your time and consideration. If you could please send me a quick reply that you have received the

information, I would greatly appreciate it.

Diane Gorder

BioMedical Technology Solutions, Inc.

9800 Mt. Pyramid Ct., Suite 350

Englewood, CO 80112

Direct: (719) 260-2331

Main Office: (303) 653-0100

Fax: (303) 653-0120

6/11/2007

---

SENT

VIA EMAIL

January 10, 2007

Ms. Beverly Albarracin

Illinois Environmental Protection Agency

PO Box 19276, BOL Permit #33

Springfield, IL 62794

Su: 5 January 2007 Letter from the Illinois EPA, Log No. PS06-165

Dear Beverly:

Thank you for returning my call so promptly this morning. As we discussed, I am responding in writing

to your letter dated 5 January 2007 related to your concerns about the Initial Efficacy Testing results

provided in our October submittal.

As we discussed, we recently completed supplemental efficacy testing under the leadership and guidance

of Dr. James Marsden, Regent's Distinguished Professor — Kansas State University. The purpose of this

effort was to substantiate the claim that the changes in the electronics, process control capabilities, and the

shape of the Collector do not affect the efficacy of the DemolizerTm technology that has been approved

and in use throughout the U.S. since the mid-1990s.

In developing a protocol in collaboration with the research team at Kansas State University, we diligently

reviewed the numerous protocols defined by various state agencies and developed a scientifically-sound

approach for this supplemental efficacy data based on the follow major criteria:

1. The waste load should be reflective of representative sharps or red bag waste loads and should

pose the most difficult challenge for a dry heat treatment process. The waste loads must also be

at full capacity.

2. The inoculation approach should represent a worst-case loading scenario

recovery rate.

3. Bacterial species should broadly meet state efficacy testing requirements

be selected based on their appropriateness for a dry heat treatment proces

4. Microbiological techniques, experimental design, and analytical analysis

generally accepted scientifically sound approaches.

5. The microbiological challenge must be conducted under normal

operating conditions for the device. This approach was

necessary since performing tests exactly conforming to general

requirements for alternative technologies (whether chemical,

microwave, etc.) and meeting the rigorous protocol guidelines

discussed above, would have required months of testing and

hundreds of thousands of dollars.

with a high bacterial

and, more importantly,

s.

should conform to

---

The recent efficacy testing of the Demolizerm

technology at Kansas State University was consistent with

Options 3 of Appendix A and Section 1422.122 (a)(1)(A) of the Illinois regulations. The regulations, as

we have interpret them, allow for the demonstration of a 6 log

i c, kill of indicator microorganism spores as

an alternative test for thermal treatment systems that maintain the integrity of the spore carrier. For the

efficacy trials, the tamper-resistant lid was altered to allow for retrieval of the carriers at the end of a

treatment cycle.

Bacillus atrophaeus

(formerly known as

Bacillus subtilis

var.

niger,

ATCC 19659) was

utilized as the USP recognized indicator spore organism for dry heat and ethylene oxide treatment

processes. Note: Spore strips of ATCC 19659 are no longer commercially available and have been

substituted with the species used in the trial.

Further the protocol largely conforms to the other requirements listed in the regulations. Specifically, five

carriers were used for each replication with the carriers placed near the geometric center of the load away

from the hot, radiating sides of the metal collector. The composition of the loads was selected to be both

representative

of the types of waste to be treated in the Demolizerm

system and to pose the most rigorous

challenge to a dry heat process. Specifically, we used both a sharps and red bag waste load. The Sharps

load was comprised of 370 g of syringes with a small amount of added residual liquid (—I 1% by weight).

This low moisture environment demonstrated a 6 log

i

c, reduction of resistant

B. subtilis

spores, the USP

indicator organism for dry heat processes. Reducing the moisture by a tablespoon to hit the target of 5%

moisture is not believed to be a meaningful difference and would therefore not impact the results.

The red bag waste load represented the greatest challenge and was thus evaluated using a broad array of

microbial species. The load was comprised of over 80% by volume of highly insulating adsorbent

material (3-ply gauze and cotton), about 8% by volume of non-adsorbent material (syringes and gloves),

and 12% by volume of organic material. By weight, the breakdown was approximately 42% moisture and

30% organic (equine serum and TSB broth). Importantly, even at this moisture level content (w/w%), the

waste load was very dry with only a small portion of the gauze moistened. The carriers were essentially

embedded within the dry, insulating gauze material near the geometric center of the load, representing a

worst-case loading challenge for the dry heat process. Note, the moisture content was very near the mid-

point guideline specified in the Illinois regulations. Reducing the content to 5% moisture would have

been an unrealistic loading condition for a typical bloody waste load (only 1 T of total liquid in the 1

gallon collector).

Finally, the requirement for a 70% organic load is not directly relevant for the dry heat process. For those

processes relying on an oxidizing chemistry, a high organic load could neutralize the reactive sites

thereby impacting the efficacy of the sterilization process. Dry heat does not rely on oxidative chemistry,

instead the kill is based on heat and dehydration of organisms. As such, increasing the organic load from

30% to 70% would have no impact on the results.

While not required under Option 3, we used a broad range of other representative microorganisms to

evaluate the effectiveness of the DemolizerTM treatment process. These included gram positive and

negative bacteria (Methicillin resistant

Staphylococcus aureus

and

Escherchla Candida

albicans,

Mycobacterium phlei

and

Bacillus subtilis.

While many of the specimens have ATCC numbers different

from those indicated in Table A, they are scientifically considered alternatives and represent the

commercially available equivalent. These organisms have been accepted by numerous state Departments

of Health and the Environment including New York, Delaware, West Virginia, Florida, Michigan,

Pennsylvania, South Carolina, North Carolina, Louisiana, etc.

The mycobacterium and bacillus species represent the toughest challenge for the Demolizeirm

technology

because these are the most heat resistant organisms. In fact, most states are modifying their regulations to

be consistent with the STAATT II and III guidelines that call for using the appropriate indicator spore

organism and one of three

Mycobacterium

species for the demonstration of efficacy of alternative

treatment technologies.

Bacillus subtilis

was selected because it is the recognized indicator organism for

dry heat processes and

Mycobacterium phlei

was selected due to its susceptibility to heat and its

BioSafety Level 11 classification.

Page 2 of 3

10 January 2007

---

In previous trials, the DemoEzell.'"

dry heat process has demonstrated a minimum 6 log

ic

reduction of the

following additional organisms:

Pseudomonas aeruginosa, Giardia

spp. (oocysts), Duck Hepatitis B,

Mycobacterium boviss

and

Mycobacterium fortuitum.

Please refer to the background information

provided in the October submittal.

We

did not specifically evaluate

Trichophyton metagrophytes

arthrospores because our research shows

that Illinois and Delaware are the only states that include this in the list of indicator organisms for a dry

heat process. This organism has been shown to be "extremely susceptible to moderate heat (above

50°C)" as reported by Hashimoto and Blumenthal (1978 Feb; 35(2):274-7; Apol Environ Microbiol).

Due to their low heat resistance, they are not considered appropriate indicator organisms for a dry heat

process.

We hope this provides additional information to substantiate our claim that the recent efficacy trials

conform to the Illinois requirements. While our customers will not fall under the permit requirements of

the state, we are seeking official acceptance of our quality control monitoring programs as a scientifically

sound alternative to periodic verification testing using spore strips. We strongly believe that the

continuous monitoring of critical control points provides a significantly higher level of assurance and is

consistent with other recognized quality programs such as 6-Sigma and HACCP initiatives.

Please keep me apprised of your review of this material. If there is any other information you wish us to

provide to support our submittal, please call me on my mobile at (719) 661-2296 or send me an email at

dgorder@bmtscorp.com.

Sincerely,

Diane Gorder

Director of Regulatory Compliance

Cc: Don Cox, President/CEO

Dr. James Marsden, KSU

Page 3 of 3

10 January 2007

---

Exhibit D

---

ILLINOIS ENVIRONMENTAL PROTECTION AGENCY

1021 NORTH GRAND AVENUE EAST, P.O. Box 19276,

SPRINGFIELD, ILLINOIS

62794-9276 - ( 217) 782-3997

lAmES

R.

THOMPSON CENTER,

100

WEST RANDOLPH, SUITE

11-300,

0-fic4co, IL

60601 - (312) 514-6026

ROD R. BLAGOIEVICH, GOVERNOR?

DOUGLAS P. SCOTT, DIRECTOR

217/524-3300

April 4, 2007

Diane Gorder

BioMedical Technology Solutions, Inc.

9800 Mt. Pyramid Court

Suite 350

Englewood, Colorado 80112

Re: 9080000000 – Colorado

BioMedical Technology Solutions, Inc.

Log No. PS07-022

Permit File

Dear Ms. Gorder:

Thank you for your e-mail with attached letter of January 10, 2007, in which you provide a

response to our letter of January 5, 2007.

Your letter stated that the testing performed was consistent with Option 3 of Appendix A and

Section 1422.122(0(1)(A) of 35 Illinois Administrative Code: Subtitle M. Your letter also

stated that

Bacillus atrophaeus

was used as a substitute for

Bacillus subtilis

var.

niger,

ATCC

19659 in the testing. You noted that spore strips of ATCC 19659 are no longer commercially

available and have been substituted with

B. atrophaeus.

Your letter also indicated that many of

the specimens used in your testing have ATCC numbers different from those indicated in 35 III.

Adm. Code 1422.Appendix A(Table A), but they are scientifically considered alternatives and

represent the commercially-available equivalent.

The regulations for efficacy testing found in 35 III. Adm. Code: Subtitle M require the use of at

least one of the Indicator Microorganisms found in Section 1422.Appendix A(Table B). It

appears as though

B. subtilis,

ATCC 19659, is still

available

through ATCC, although not in

spore strip form. In addition, if

B. subtilis

is not available, there are two other microorganisms

that can be used for the efficacy testing,

B. stearothermophilits

(ATCC 7953) or

B. pumilus

(ATCC 27142).

The initial efficacy testing data included in your submittal indicated that testing was performed

on the Demolizer using several microorganisms. However, none of the microorganisms used

were the ATCC number required by 35 Illinois Administrative Code 1422.AppendixA.TableA.

For example, Demolizer testing used

Staphylococcus aureus

ATCC 33591, while the Illinois

Rociactin - 4302 North Main Street, Rockford, IL 61103 - (815) 9877760 •

Do

PLAINES - 9511

W. Harrison

Si., Des Plaines, IL 60016 - (8471 294-4000

ELGIN

595 South Stale, Elgin, IL 60123 -

(847) 608

-

3131 •

PEORIA - 5415 N.

University SI., Peoria, t161614-(309)69)-5463

ammo Or LAND - PEORIA -

7620 N.

University SI., Peoria, IL 61614 -

(309) 693-5462 •

01MIPIUGN-

2125 South First Street,

Champaign, IL 61020 - (2171 278-5800

SPRINGFIND

4500 S. Sixth Street Rd..

Springfield, R 62706-1217) 786 .

6892 •

CouihsuLLE -

2009 Mall Street, Collinsville, IL 62234 - (618) 346-5120

MARION -

2309

W.

Main St., Suite 116, Marion, IL 62959 -1618) 993-7200

PRINTED ON RECYCLED PAPER

---

Page 2

regulations require

S. auretts

ATCC 6538. 35 III. Adm. Code 1422 requires the use of these

specific microorganisms, including ATCC number.

The composition and placement of challenge loads used in the efficacy testing appear to comply

with the PIMW regulations. The submitted data indicates that the Demolizer is capable of

achieving a 6 log i

c, reduction for all of the microorganisms used.

An alternative periodic verification test (PVT) may be approved and used only when the initial

efficacy test (IET) has been performed completely in accordance with 35 III. Adm. Code 1422,

and the alternative PVT has been directly correlated to the results obtained in the IET. In order

for an alternative PVT to be approved, the Initial Efficacy Test must be performed in accordance

with the regulations found in 35 Ill. Adm. Code: Subtitle M. Mother option is to seek an

Adjusted Standard from the Illinois Pollution Control Board. They may be reached at 312/814-

3620.

Units designed to treat potentially infectious medical waste may be used in Illinois without a

permit from the Illinois Environmental Protection Agency (Illinois EPA), provided the treatment

unit is accepting and treating only PIMW generated on-site. The efficacy testing results for the

treatment unit must be kept on-site and made available to the Illinois EPA upon request.

I hope this satisfies your inquiry. If you have further questions, please feel free to contact

Beverly Albarracin of my staff at 217/524-3289.

Sincerely,

Stephen F. Nightingale, P.E.

Manager, Permit Section

Bureau of Land

SEN:gekbjh

\072792s.doc

---

Exhibit E

---

Diane Gorder

From:?

William Ingersoll [WilliamIngersoll©Illinois.goy]

Sent:?

Monday, June 04, 2007 1:16 PM

To:

?

Diane Corder

Subject:?

RE: Inquiry re PIMW and Bacillus Subtilus

Diane,

I sat down with Bureau of Land Permit Section staff and management earlier today to

discuss this matter in more detail. To recap your issue, I believe that your company

proposed to use bacillus subtilus (ATCC 9362) in its performance testing, while the

Illinois regulations require the use of bacillus subtilsu (ATCC 19659).

One of the issues you raised was that the bacillus subtilsu (ATCC

19659) is no longer commercially available. I am told that while this strain may not be

"off-the-shelf" at this time, it can still be purchased. In addition, I am told that all

of the relevant facilities currently permitted in Illinois used the regulatorily required

strain.

Therefore, it seems that we are unable to help you in seeking an interpretive resolution

to your regulatory problem.

Bill Ingersoll

Manager of Enforcement Programs

Illinois EPA

217-782-9827

fax: 217-782-9807

Please note change of e-mail address to:

william.ingersoll@illinois.gov

>>> "Diane Corder" <dgorder@bmtscorp.com> 6/1/2007 10:09 AM >>>

Bill,

I just left a voicemail for you inquiring about whether we have made any progress on this

matter. I would very much appreciate if we could touch basis at the first of next week to

see if we can work on a resolution to move this forward.

Thank you and have a great weekend.

My direct number is 303-653-0111. I will be in the office Monday and Tuesday of next

week, but will be out of the office on Wednesday.

Sincerely,

Diane Corder

BMTS, Inc.

?From: Original

Diane Corder

Messagermailto:dgorder@bmtscorp.com)?

Sent: Tuesday, May 08, 2007 10:46 AM

To: 'William Ingersoll'

Subject: RE: Inquiry re PIMW and Bacillus Subtilus

Thank you for sending your information and working to see if we can come to a quick

resolution. My contact information is

Diane Corder

Director of Regulatory Compliance

BioMedical Technology Solutions, Inc. (BMTS) 9800 Mt Pyramid Court, Suite 350 Englewood,

1

---

CO 80112

(303) 653-0111 (direct)

(719) 661-2296 (mobile)

As a summary of our conversation, we used Bacillus subtilis var niger in our efficacy

studies. We used the commercially available isolate (ATCC 9372, NRRL 4E4418) since the

Bacillus subtilis var niger (ATCC 19659) is not commercially available in a certified

form.

In 1993, the FDA listed either ATCC 9372 or 19659 as appropriate organisms for testing dry

heat sterilization technologies. Multiple international organizations and state

departments of health and environment recognize the isolate of Bacillus subtilis var niger

used in our studies as the ideal species for demonstrating efficacy of dry heat treatment

processes, even though Many have outdated regulations that specify the ATCC 19659 isolate.

The following organizations recognize ATCC 9372 as the appropriate biological indicator

for dry heat processes: US Pharmacopia (USP), ISO 11138-4:2006, FDA pre-market clearance

requirements, and EN 866 guidelines.

All of the major U.S. and international biological indicator manufacturers market the

Bacillus subtilis (ATCC 9372) as THE indicator organism for dry heat. This includes STERIS

Corporation, Raven Laboratories, Charles River Laboratories, NAMSA, etc. We obtained our

spores strips from STERIS Corporation, the largest and most well-known company in this

industry.

We believe an adjusted standard should not be necessary since we used the organism listed

in Item 1 of Appendix A. It has been suggested that we repeat the extensive trials using

one of the other two organisms; however, that is not a scientifically sound recommendation

since B.

stearothermophilus is the USP indicator for steam sterilization processes and B. pumilus

is recognized internationally for radiation processes.

Neither is appropriate for dry heat processes.

If you need additional information, please give me a call or send me an email. Again,

thank you for your time and hopefully we can find a way to work through this over the near

term.

Sincerely,

Diane Gorder

?From: Original

William Ingersoll

Message ?

lmailto:William.Ingersoll@illinois.gov]

Sent: Tuesday, May 08, 2007 8:47 AM

To: dgorder@bmtscorp.com

Subject: Inquiry re PIMW and Bacillus Subtilus

Diane,

Here is my contact info:

Bill Ingersoll

Manager of Enforcement Programs

Illinois EPA

217-782-9827

fax: 217-782-9807

Please note change of e-mail address to:

william.ingersoll@illinois.gov

2

---

Exhibit F

---

trKSTATE

Kansas State University„

Food Science Institute

148 Waters Hail

Manhattan, KS 66506-4010

785-532-2202

Fax: 785-532-5861

E-mail: foochci0katate.edu

http://foodsci.k-slattedu

August 27. 2007

Diane Corder

Director of Regulatory Compliance

Biomedical Technology Solutions, Inc.

9800 Mt. Pyramid Court – Suite 350

Englewood, CO 80132

Dear Ms Corder:

I have evaluated the materials presented to the Illinois Environmental Protection Agency to substantiate

the claim that

Bacillus subtilis var. niger

(ATCC 9372), alas known as

Bacillus atrophaeus)

is the

most

appropriate

biological indicator organism for the validation of dry heat sterilization technologies.

Upon review of the large body of scientific citations and international standards, the following major

findings are provided for your consideration.

The use of dry heat for the sterilization of surfaces, medical devices, medical waste, etc., has been studied

extensively since as early as the late 1960s. The original research was performed primarily in the space

industry to address sterilization requirements in a space environment. Subsequently, dry heat sterilization

was broadly adopted for the sterilization of equipment and surfaces in the medical and dental applications.

In the mid-1990s, dry heat technology was adapted and demonstrated to effectively sterilize infectious

waste including infectious sharps waste consistent with state and local standards for disinfection.

Various scientific studies have focused on the identification of the most appropriate biological indicator

organism for the validation of dry heat technologies.[1-12] These studies evaluated the inactivation of

Bacillus subtlis

var.

niger

under various treatment conditions using various substrates including paper

strips, stainless steel, glass. epoxy resin, etc. By the late 1970s, the scientific community converged on

the selection of Bacillus

subtilis var. niger,

and more specifically ATCC 9372, as a spore-forming

organism exhibiting superior dry heat resistance. A wide variety of D-values and z-values had been

reported for this species based on the specific variables of the experimental design.

In 1980, Gurney and Quesnel [6] initiated a comprehensive comparison of a generic

Bacillus subtilis

organism and

Bacillus subtilis

var.

niger.

During this thneframe. both generic

B. subtilis

and the

subspecies

niger

had demonstrated excellence heat resistance in dry heat sterilization applications.

Gurney and Quesnel's work extensively studied the growth properties and the thermal inactivation

performance of both indicator organisms in a dry heat sterilization process and definitively concluded that

Bacillus subtilis var. niger

delivers superior growth and heat resistance properties. The purpose of this

extensive study

was

to

determine the optimal biological indicator organism for a dry heat treatment

extensive

process. The

D-value

authors

and

devised

z-value

a

determinations

very simple and

of

efficient

both organisms.

method for

Statistical

dry heat

methods

treatment

were

to support

employedthe

I

si

---

using appropriate experimental replicates (3-6 replications per condition) to derive regression lines

of

best

fit from these D-values at different temperatures.

Gurney and Quesnel definitely concluded that

'Bacillus subtilis

var.

niger

germinated more readily and

delivered higher percentage germination than

B. subtilis

MD2 on all media evaluated. - This is an

important finding for the selection of an optimal biological indicator since the objective of a validation

study is to assure that the most resistant organisms are inactivated by the anti-microbial technology under

evaluation.

The authors conducted extensive studies on decimal reduction times (D-values), comparing results with

those published by other investigators. Specifically, the decimal reduction times for each of the spores

were evaluated at various temperatures (between 140 and 160°C) using different media (TGE, CAA. and

MGR). At all temperatures var.

niger

showed greater dry-heat resistance than

B. subtilis.

The

difference in the shapes of the thermal death curves for

B. subtilis

var.

niger

and

B. subtilis

were found to

be significant, with a sharp drop-off in the death curve noted for the

B. subtilis

spore. The authors

reported that "The difference is due to the facts that MD2 spore germination is considerably enhanced by

heat activation, while

niger

spores germinate readily and rapidly with little or no heat activation...

In summary, this work definitely found that

Bacillus subtilis

var.

niger

is

the preferred and most

appropriate biological indicator organism for the validation of dry heat sterilization due to its enhanced

dry heat resistance at all temperatures evaluated and, importantly exhibited enhanced growth properties in

each of the growth media evaluated. Specifically.

"From this study the var.

niger

strain is clearly the organism of choice as an indicator of

dry-heat sterilization in the temperature range of 140 to 170°C."

Based on this research and the compendium of research by others in the field of sterilization and

microbiological control, multiple standards organizations have formally designated

Bacillus subtilis

var.

niger

(ATCC 9372) as the definitive preferred biological indicator organism for the validation of dry heat

sterilization processes.[I3-191 These standards are developed through review of the scientific literature

and a consensus of international experts in the field of sterility control. The committees assess the

morphological, growth, stability, and resistance characteristics of biological indicator organisms to select

the most appropriate organism for a given application. While numerous standards identify the ATCC

9372 organism for the validation

of

dry heat processes, the U.S. Pharmacopeia's Official Monograph and

the International Standard Organization (ISO) determinations are among the most notable.

U.S. Pharmacopeia (USP) is the official public standards-setting authority for all prescription and over-

the counter medicines, dietary supplements. and other healthcare products manufactured and sold in the

United States. These standards are recognized in over 130 countries. The USP Convention membership

has approximately 450 members who represent U.S. colleges and state associations

of

medicine and

pharmacy: governments

of

the U.S. and other countries: national and international health professional.

scientific. and trade organizations: the pharmaceutical industry; and consumer organizations. The

Council of Experts and Expert Committees are the bodies that make the USP's scientific and standards-

setting decisions. The Microbiology and Sterility Assurance Expert Committee is currently chaired by

Dr. James E. Akers. an internationally recognized expert in sterility assurance. Other distinguished

members of the committee include Mr. James P. Agalloco, Mr. Ivan W. Chin, Dr. Anthony M. Cundell,

Dr. Joseph K. Farrington, Dr. Dennis E. Guilfoyle, Dr. David Hussong, Dr. Leonard W. Mcstrandrea, Dr.

David A. Porter, Dr. Donald C. Singer, and Dr. Scott V.W. Sutton.

The official USP Monograph — Biological Indicator for Dry-heat Sterilization, Paper Carrier, USP28-

NF23, 2005, provides species identification, packaging and storage requirements, and resistance

performance testing standards for the production of biological indicators to be used to validate dry heat

sterilization processes. Specifically, 'Biological Indicator for Dry-Heat Sterilization. Paper Carrier, is a

defined preparation of viable spores from a culture derived from a specified strain of

Bacillus

subtilis

---

subspecies

niger,

on a suitable grade of paper carrier, individually packaged in a container readily

penetrable by dry heat, and characterized for predictable resistance to dry heat sterilization... The species

is further defined as "The biological indicator organism complies substantially with the morphological,

cultural and biochemical characteristics of the strain of

Bacillus subtilis,

ATCC No. 9372, designated

subspecies

niger,..."

This USP Monograph also provides D-value performance requirements for the

production of certified carriers to be used to validate city heat sterilization processes.

The International Standards Organization (ISO) has also formally recognized

Bacillus subtilis

var.

niger

organism as

the preferred and most appropriate

indicator organism for the validation of dry heat

sterilization technologies. ISO is a network of national standards institutes of 155 countries and is the

world's largest developer of standards publishing more than 16,000 diverse fields. ISO 11138-4:2006

provides specific requirements for test organisms, suspensions, inoculated carriers, biological indicators.

and test methods intended for use in assessing the performance of sterilization processes employing dry

heat as the sterilizing agent within the range of I20°C and I 80°C. This standard has specifically found

that

Bacillus atrophaeus

(known as CIP 77.18, NCIMB 8058, DSM 675, NRRL B-4418. and ATCC

9372) or

Bacillis subtilis

(DSM 13019 isolated from Hay in Denmark) are preferred biological indicator

organisms for the validation of dry heat processes. The standard further defines manufacturer quality and

test requirements to certify the D-value and z-value performance of given spore populations. Note the

American National Standards Institute (ANSI) has also formally adopted the ISO 11138-4:2006 standard

for the selection of

the

preferred and most appropriate biological indicator organism for the validation of

dry heat sterilization technologies.

Based on the overwhelming evidence, it is my expert opinion that

Bacillus subtilis var. niger

(ATCC

9372, also known as

Bacillus atrophaeus)

is the

most appropriate

biological indicator organism for the

validation of dry heat sterilization technologies. This specific subspecies of

Bacillus subtilis

demonstrates excellent growth and dry heat resistance characteristics. Standards for performance have

been established by USP. ISO. and others to ensure that certified biological indicators for dry heat

sterilization deliver predictable and standardized resistance.

The Demolizer® technology is an alternative infectious waste treatment system that employs dry heat as

the sterilization agent. As such. the most appropriate biological indicator organism for the validation of

the efficacy of the Demolizer® technology is the ISO and USP recognized standard.

Bacillus subtilis

var.

niger

(also known as

Bacillus atrophaeus).

Further, certified carriers manufactured under rigorous

quality standards should be used, wherever possible, since such carriers are tested for purity and

performance meeting defined D-value and z-value performance criteria.

Respectfully submitted,

C

Daniel Y. C. Fung, M.S.P.H., Ph.D.

Professor of Food Science, Professor of Animal Science and Industry, Kansas State University

Distinguished Professor, Universitat Autonoma de Barcelona, Spain

Food Microbiologist, Environmental and Public Health Microbiologist

---

References

Angelotti, R., Maryanski,111. Butler. TF and Peeler, iT. 1968. Influence of spore moisture on the dry-

heat resistance of

Bacillus subtilis

var.

niger. Appl. Microbiology,

16, 735-745.

?Brannen. J.P. and Garst, D.M. 1972. Dry-heat activation of Bacillus

subtilis

var.

niger

spores as a

function of relative humidity.

Appl. Microbiology,

23, 1125-1130.

?Bruch,

MK and Smith, FW. 1968. Dry heat resistance of spores of

Bacillus subtilis

var.

niger

on Kapton

and Teflon Film at high temperatures.

Appl. Microbial.

16, 1841-1846.

4Bruch,

C.W.. Koesterer. M.G. and Bruch, M.K. 1963. Dry heat sterilization: its development and

application to components of exobiological space probes.

Devel. and Indus. Micro.,

4.334-342.

'Drummond DW and Pflug. U. 1970. Dry-heat destruction of

Bacillus subtilis

var.

niger

spores on

surfaces: effect of humidity in an open system. 1970.

App! Microbial.

20, 805-809.

6Gumey. TG, Quesnel. LB. Thermal Activation and Dry-heat Inactivation of Spores of

Bacillus subtilis

IvID2 and

Bacillus subtilis

var.

niger, J. Appl. Batter.,

1980, 48. 231-247.

'Molin, G. 1977. Dry-heat resistance of

Bacillus subtilis

spores in contact with serum albumin.

carbohydrates or lipids. .1

Appl. Racier..

42, 111-116.

°Mohr:. G. 1977. Inactivation of

Bacillus

spores in dry systems at low and high temperatures. ..I.

General

Micro,

101, 227-231.

'Molin. G and Ostlund. K. 1975. Formation of dry-heat resistant

Bacillus subtilis

var.

niger

spores as

influenced by the composition of the sporulation medium.

Anionic van Leeivenclhoek,

42, 388-395.

"Molin, G. and Ostlund, K. 1975. Dry-heat inactivation of

Bacillus subtilis

spores by means of infra-red

heating..

Anionic van Leelvendhoek,

41, 329-335.

I I Paik WW. Sherry EJ, and Stern, JA. 1969. Thermal death of

Bacillus subtilis

var.

niger

spores on

selected lander capsule surfaces.

Appl. Microbial..

18.901-905.

"Simko, G.J., Devlin, J.D.. and Wardle, M.D. 1971. Dry-heat resistance of

Bacillus subtilis

var.

niger

spores on mated surfaces.

Appl. Microbial.

22, 491-495.

"US Pharmacopoeia. USP28-NF23 USP. Monographs: Biological Indicator for Dry-Heat Sterilization.

Paper Carrier; Rockville. MD: 2005.

"ISO: Sterilization of health care products - Biological indicators; Geneva (Switzerland): International

Organization for Standardization/ANSI: ISO ISO 11138-4:2006.

"Guidance on Premarket Notification [51 0(k)] Submissions for Sterilizers Intended for Use in Health

Care Facilities. Infection Control Devices Branch. Division of General and Restorative Devices. March

1993.

"FDA. Premarket Notifications [510(k)] for Biological Indicators Intended to Monitor Sterilizers Used in

Health Care Facilities: Draft Guidance for Industry and FDA Reviewers; U.S. Department of Health and

Human Services, Food and Drug Administration, Center for Devices and Radiological Health, Infection

Control Devices Branch, March 2001.

"British Pharmacopoeia Commission. Methods of sterilization. London, UK: British Pharmacopoeia

Commission; British Pharmacopoeia Appendix XVIII. 2003.

"European Pharmacopoeia Commission. Biological indicators of sterilization. Strasbourg, France:

European Pharmacopoeia Commission; European Pharmacopoeia EP 5.1.2, 1997.

"Japanese

Pharmacopoeia. JP14e.part11.15 JP. Terminal Sterilization and Sterilization Indicators.

---

Dr. Daniel Y. C. Fung

Biographical Background Information

Dr. Daniel Y. C. Fung, internationally recognized Food, Environmental and

Public Health Microbiologist, has published extensively in Food Microbiology, Applied

Microbiology and Rapid Methods with more than 700 Journal articles, meeting abstracts,

proceeding papers, book chapters and books in his career. He has served as the major

professor for more than 90 M.S. and Ph.D. graduate students. The Kansas State

University Rapid Methods and Automation in Microbiology Workshop, directed by Dr.

Fung, has attracted more than 3,500 participants from 60 countries and 46 states to the

program in the past 27 years.

Dr. Fung is a Fellow in the American Academy of Microbiology, Institute of

Food Technologists (IFT), International Academy of Food Science and Technology and

Institute for Food Science and Technology (UK). He has won more than 30 professional

awards which included the International Award from IFT (1997), Waksman Outstanding

Educator Award from The Society of Industrial Microbiology (2001), KSU College of

Agriculture Excellence in Graduate Teaching Award (2005), and the Exceptional

Achievement and Founder of the KSU International Workshop on Rapid Methods and

Automation in Microbiology Award given by the Director of the Center for Food Safety

and Applied Nutrition, U.S. Food and Drug Administration, 2005.

Dr. Fung received the B.A. degree from International Christian University,

Tokyo, Japan in 1965, M.S.P.H. at University of North Carolina-Chapel Hill in 1967, and

the Ph.D. in Food Technology from Iowa State University in 1969. He is currently a

Professor of Food Science, Professor of Animal Sciences and Industry and Ancillary

Professor of Biology at Kansas State University and Distinguished Professo,r Universitat

Autonama de Barcelona, Spain.

---

Exhibit G

---

TECHNICAL ASSISTANCE MANUAL: STATE REGULATORY

OVERSIGHT OF MEDICAL WASTE TREATMENT TECHNOLOGIft

April 1994

A Report of' the State and Territorial Association

on Alternate Treatment Technologies

---

EXECUTIVE SUMMARY

I. Introduction

The -purpose

of this report is to establish a framework or guideline that defines medical waste

treatment technology efficacy criteria and delineates the components required to" establish an

effective state medical waste treatment technology approval process. The recommendations made

in this report are an attempt to find commonality on many of the issues and criteria required in

the medical waste treatment technology review process. Recognizing that all states may not

totally agree with these recommended criteria or protocols, the guidelines developed should serve

only to provide guidance to the states in the development of an approval process for medical

waste treatment technologies.

The establishment of qualitative and quantitative parameters that ensure effective and safe

medical waste treatment are required in defining treatment technology efficacy criteria and

delineating the components necessary to establish an effective state medical waste treatment

technology approval process. Recommendations are provided in this report for the following:

•

Medical Waste Treatment Technology Efficacy Assessment

•

Medical Waste Treatment Technology Approval Process

Permitting and Site Authorization Issues

Research and Development

H. Medical Waste Treatment Technology Efficacy Assessment Criteria

This report

recommends that all medical waste treatment technologies meet the following

microbial inactivation criteria:

Inactivation of vegetative bacteria, fungi, lipophilic/hydrophilic

viruses, parasites, and mycobacteria at a 6 Logio

reduction or

greater; and inactivation of

D

L

stearotherrnoohilus

spores or

Q.

pubtilis

spores at a 4 Loglo

reduction or greater.

In meeting these criteria, selected pathogen surrogates which represent vegetative bacteria, fungi,

parasites, lipophilic/hydrophilic viruses, mycobacteria, and bacterial spores are recommended.

Formulas and methods of calculations are recommended and are based on microbial inactivation

("kiln efficacy as equated to "Log ic,

Kill", which is defined as the difference between the

logarithms of the number of viable test microorganisms before and after treatment.

---

report was distributed for review and comment to all state and territorial regulatory agencies

involved in medical waste regulatory activities.

To gain additional input into the development of a uniform guideline for the assessment of

medical waste treatment technologies, a third meeting was conducted on June 1416, 1993, in

Washington, DC with invited participants from all state and territorial medical wane regulatory

agencies. The report prepared from the Atlanta meeting served as a basis of dis6ussion. With

invited input from all state and territorial representatives, the primary objective of the meeting

was to sect consensus on the key topic areas listed above.

This report details the discussions and recommendations of the participants from the three

meetings. It should be emphasized that the recommendations made in this report are an attempt

to find commonality on many of the issues and criteria required in the medical waste treatment

technology review process. As such, consensus agreement was sought on key issues to

demonstrate support for the recommendations made in this report. However, consensus support

for a recommendation does not necessarily imply unanimity for the position taken. Recognizing

that all states may not totally agree with these recommended criteria or protocols, the guidelines

developed through this series of meetings should serve only to provide guidance to states in the

development of a review and approval process for medical waste treatment technologies.

Logistical support for all three meetings was provided by the USEPA. Roger Greene, Rhode

Island Department of Environmental Management, Diann I. Miele, Rhode Island Department of

Health, and Dr. Nelson S. Slavik, President, Environmental Health Management Systems, Inc.,

cofacilitated each of the meetings. A listing of all participants attending the New Orleans,

Atlanta, and Washington, D.0 meetings is found in Appendix D.

---

The committee realized that there might be circumstances under which a state may allow

relaxation of the Level M requirement. These exceptions would by necessity need to be made

on a ease-by-case

basis, would require the equipment manufacturer to provide a rationale for

relaxation, and would require adequate supporting documentation to substantiate that rationale.

The

commits= also

debated if laboratory wastes (i.e. discarded cultures and stocks of pathogenic

agents) should require

sterilization (i.e. meet Level IV criteria) on the basis that these wastes may

contain high concentrations of known pathogens. The committee took the position that Level

III

criteria

remained the

standard for all medical waste categories. The committee emphasized,

however, dm laboratories should be aware that cultures and stocks of disease-causing agents may

require sterilization before disposal. In addition to guidelines set by the Centers for Disease

Control in Biosafety in Microbiological and Biomedical Laboratories (1993) and standards of

the College of

American Pathologists (CAP), some states require laboratory cultures to be

incinaated or autoclaved (i.e., steam sterilized) before leaving the laboratory or before being

disposed of. Although no specific recommendations for medical waste disposal are made under

the

Clinical Laboratory

Improvement Amendments (CLIA), medical waste disposal practices are

receiving rased scrutiny during routine inspections.

2.3

Representative Biological Indicators

In the absence

of an ultimate pathogen surrogate to represent all defined microbial groups, the

selection of

pathogen surrogates representing vegetative bacteria, fungi, parasites, viruses,

mycobaderia, and bacterial spores was considered necessary to define and facilitate any state

approval process. Criteria defining surrogate selection should include that any surrogate

recommended:

•

Not affect healthy individuals;

•

Be

easily obtainable;

•

Be

an ATCC registered strain, as available;

•

Be

easily cultured and maintained; and

•

Meet quality control requirements.

Microorganism strains obtained from the American Type Culture Collection (ATCC) and methods

prescribed by the Association of Official Analytical Chemists (AOAC) assist in fulfilling these

recommendations by (1) providing traceable and pure cultures of known characteristics and

concentration and (2) providing recognized culturing protocols and detailed sampling and testing

protocols.

Provided

in

Table B are the biological indicators recommended by the committee for testing

microbial inactivation efficacy in medical waste treatment processes. The selection of these

representatives was based oo each microorganism:

12

---

•

Meeting, where possible, the criteria established above;

•

Representing, where possible, those organisms associated with medical

waste; and

•

Providing a biological challenge equivalent to or greater than that

associated with microorganisms found in medical waste.

Biological indicators selected to provide documentation of relative resistance to an inactivating

agent should be chosen after evaluation of the treatment process as it relates to the conditions

used during comparative resistance research studies described in the literature. Literature studies

support the assertion that the degree of relative resistance of a micrcorganism to an inactivating

agent can be dependent on various factors (i.e., pH, temperature). Conditions used in literature

studies that demonstrate a relatively high degree of resistance of a particular microorganism may

be significantly different to the conditions found within the treatment process. A comparison of

the conditions used in the literature to those used in the treatment process should be made to

determine if relative microbial resistance can be altered (i.e., lowered) as a result of treatment

process conditions.

The committee emphasized that although the microorganisms selected represent pathogen

surrogates, these selected surrogates may have the potential to be pathogenic under certain

conditions. As such, the committee recommended that all testing be conducted using recognized

microbial techniques. For those pathogen surrogates that still retain some higher degree of

pathogenicity (e.g.,

Crvntosvoridium, Giardia, and Mvcobacteria),

efficacy testing should be

conducted only by qualified laboratory personnel.

TABLE II - RECOMMENDED BIOLOGICAL INDICATORS

Vegetative Bacteria

Fungi

Staphylococcus pureus

(ATCC

6538)

Pseudomonas aeruginosa

(ATCC 15442)

Candida

albicans (ATCC 18804)

Penicillium chr

y

sonenum

(ATCC 24791)

Aspernillus

Riga

Viruses

Polio Z Polio 3

MS-2 Bacteriophage (ATCC 15597-B1)

Parasites

CrYptosporidium

222 oocysts

Giardia

am

cysts

Mycobacteria

Mycobacterium terra e

My

cobacterium

phisi

My

cobacterium.

bovis

(BCG)

(ATCC 35743)

13

---

Bacterial Spores

B. stearothermophilus

(ATCC 7953)

B. subtilis (ATCC 19659)

The committee recommended that one or more of the representative microorganisms from each

miaobial group

be used

in efficacy evaluation. Specific criteria for the selection of these

microorganisms are

provided below

in Table III:

TABLE III . BIOLOGICAL INDICATOR SELECTION CRITERIA

Vegetative Bacteria

Fungi

Viruses

Staphylococcus aureus and Pseudomonas aeru_ainosa

were selected to represent both gram-positive and

gram-negative bacteria, respectively. Both are

currently required by the Association of Official

Analytical Chemists (AOAC) use-dilution method

and both have been shown to be resistant to

chemical inactivation.

The selection of Candida albicans and Penicillium

chrvsoaenum was based on reported data indicating

these organisms representing yeast and molds,

respectively, are the most resistant to germicides.

Although 7rlchophytoq mentagronhvtes is the

AOAC test organism for molds, ?MACHU=

chrvsoaenum is reported to be more resistant to

germicides. The inclusion of Asperaillus Riga as

an indicator organism was based on its familiarity as

a common mold.

Lipophilic (enveloped) viruses are less resistant to

both thermal and chemical inactivation than the

hydrophilic (nonenveloped) viruses. As such,

enveloped viruses such as HIV, Herpes simplex

virus and Hepatitis B virus are less resistant than

enveloped viruses such as Poliovirus, Adenovirus,

and Coxsackievirus. Polio 2 (attenuated vaccine

strain) and Polio 3 virus were selected based on

their relative higher chemical and thermal resistance.

Additionally, the use of an enterovirus (e.g., Polio 2

or Polio 3) can provide a stringent measure of

efficacy for irradiation treatment processes. MS-2

bacteriophage was selected as a Hepatitis virus

surrogate in that this bacteriophage offers a

comparable degree of chemical and thermal

resistance, is safe to handle and easy to culture.

14

---

Parasites

Mycobacteria

Bacterial Spores

Both Crvotosvoridiuqi

sgo.

oocysts and Glardia,1224

cysts are used as test organisms to demonstrate

germicidal effectiveness. Crvotosooridium has been

demonstrated to have a higher chemical resistance

and Crvotosooridium spy oocysts are more readily

available than Giardia

sm

cysts. Both are

significantly pathogenic (both have an infeeilous

dose of 10 cysts) and care is advised when 'using

these microorganisms as parasitic biological

indicators.

Mvcobacterium

lot

has

a demonstrated measure of

disinfectant resistance, is a rapid grower and is

pigmented for easy identification.

mh bovis (BCG)

is used in the AOAC Tuberculocidal Method and is

analogous to M. tuberculosis in that it is in the same

group or complex. Individuals exposed to a

(BCG, ATCC strain) may skin test convert although

no actual infectivity or disease occurs. Risk it

exposure would come from those mechanisms that

grind the waste. Mycobacterium(

tense is equivalent

to m,

tuberculosis in resistance to chemical

inactivation. In Europe it is recommended for

disinfectant testing. a terrae does not grow as

rapidly as m„

bovis or tuberculosis.

Both B. stearothermoohilus and a subtilis spores

are commonly used as biological indicators for both

thermal and chemical resistance. IL,

stearothermophilus spores exhibit more thermal and

chemical resistance than spores from B. subtilis.

After discussion on the rationale for selection of the representative biological indicators presented

above, consensus by the committee was attained on recommending the use of these biological

indicator strains for treatment technology efficacy testing.

2.4 Quantification of Microbial Inactivation

Establishing the mechanisms to quantify the level of microbial inactivation is essential in

developing the format and requirements of the guidance protocols. As presented and discussed,

microbial inactivation ("kill") is equated to "Log

loKill"

which is defined as the difference between

the logarithms of number of viable test microorganisms before and after treatment. This

definition is translated into the following formula:

15

---

Roger

Greene, Rhode Island Department of Environmental Management, Diann J. Miele, M.S.,

Rhode bland Department of Health, and Nelson S. Slavik, Ph.D., President, Environmental

Health Management Systems, Inc., were primarily responsible for facilitating consensus among

participants during each of the three meetings that were held to discuss state review of medical

waste treatment technologies.

Nelson S. Slavik, Ph.D., prepared this final document which reflects the discussions and

amen= reached at these meetings.

The following state officials served as a steering committee for these meetings:

Charles IL Anderson

Louisiana Department of Health and Hospitals

Lawrence Chadzynski, M.P.H.

Michigan Department of Public Health

Robert AL Confer

New Jersey Department of Environmental Protection & Energy

Carolyn Dinger

Louisiana Department of Environmental Quality

Roga Greene

Rhode Island Department of Environmental Management

Diann J. Miele, M.S.

Rhode Island Department of Health

Phillip R. Morris

South Carolina Department of Health and Environmental Control

Ira F. Salk* Ph.D.

New York Department of Health

Wayne Turnberg

Washington Department of Ecology

John Winn, R.F,.H.S.

California Department of Health Services

A complete listing of all participants attending the New Orleans, Atlanta, and Washington, D.C.

meetings may be found in Appendix D.

---

Exhibit H

---

EHMS

Ike

ENVIRONMENTAL HEALTH MANAGEMENT SYSTEMS, INC.

2617 Rom Street, Niles, Michigan 49120

269/6834444(0), 269/6834441(F)

June 11, 2007

Diane Gorder

BioMedical Technology Solutions, Inc.

9800 Mt. Pyramid Court, Suite 350

Englewood, CO 80112

Dear Ms. Gorder,

I am writing pursuant to your request for historical background concerning biological

indicator strains used during treatment efficacy studies on medical waste treatment

devices and equipment. I hold a doctorate in microbiology from the University of Illinois

at Urbana-Champaign and I served as co-facilitator and medical waste consultant to the

State and Territorial Association on Alternate Treatment Technologies (STAATT). This

was a select group of state regulatory representatives gathered to prepare and adopt a

cohesive approach to evaluate the microbiological inactivation effectiveness of medical

waste treatment equipment. This group was first convened in late 1992 and was

supported and funded by the U.S. Environmental Protection Agency with the primary

mission to establish qualitative protocols and quantitative measures by which to evaluate

the efficacy of microbial kill of these devices. This effort culminated in the document

entitled

Technical Assistance Manual: State Regulatory Oversight of Medical Waste

Treatment Technologies

published in April of 1994. I was the author of that document.

This document was the first attempt at creating a comprehensive protocol and evaluative

mechanism to determine the treatment efficacy of medical waste treatment equipment.

We relied on documents that provided a semblance of guidance as they related to clinic

evaluation of microbial kill. We realized that in creating this document that revisions

would be required as knowledge advanced or as necessary to enhance the use of the

protocols. We also realized that states might view this document as a path to regulatory

development and incorporate portions of the document into regulatory language. As

such, we stated clearly in the document's "Introduction" that "the guidelines developed

through this series of meetings should serve only to provide guidance to states in the

development of a review and approval process for medical waste treatment

technologies." The document was never intended to be the final word on treatment

efficacy of medical waste treatment equipment, but rather a first start of a work-in-

progress.

As part of our qualitative measure, it was required that we assign specific challenge

(surrogate) organisms to each microbiological category requiring testing (i.e., vegetative

bacteria, viruses, fungi, parasites, mycobacterium, and bacterial spores). The

aforementioned categories (with the exception of bacterial spores) represented the types

---

of microorganisms that could be found in medical waste that potentially could transmit

disease. Bacterial spores were to be tested to provide a "margin of safety from the

variables inherent in the treatment of medical waste (is., waste packaging, waste

composition, waste density, and factors influencing the homogeneity of the treatment

process)" since

"B. subtilis

and

B. stearothermophilus

spores both display significantly

more heat resistance than microorganisms in the aforementioned groups? There was no

effort to single out a specific strain of

B. subtilis

and

B. stearothermoplulus

as the most

resistant for chemical or thermal resistance or as having a specific desirable

characteristic. The recommended strains selected were those that met the following

criteria:

•

"Not affect healthy individuals;

• Be easily obtainable;

•

Be an ATCC registered strain, as available;

•

Be easily cultured and maintained; and

•

Meet quality control requirements?

It was recognized by the committee that other strains not provided in the "Technical

Assistance Manual" could also meet these criteria and be acceptable as microbial

challenge surrogates.

I have reviewed your efforts to use the appropriate spore surrogate for your device and

have found that your selection of

B. subtilis

ATCC 9372 spores is consistent with the

criteria provided

by

STAATT in their publication. This strain provides the dry-heat

resistance which is appropriate for your treatment process. It is readily available through

a certified manufacturer and each manufactured lot has a traceable background and

certification analysis that quantifies dry-heat resistance (e.g., D-value) to demonstrate

the quality assurances required of the STAATT criteria.

I hope that this brief summary into the development of the "Technical Assistance

Manual" and its recommendations will provide you with the information you need. I can

be reached at the numbers listed above or by e-mail at enmed0aol.com.

Sincerely,

Nelson S. Slavik, Ph.D.

President

---

Exhibit I

---

191\41-Syr

Bidlaical Technology Solutions. Inc.

September 25, 2007

Mr.

Neal H. Weinfield, J.D.

Greenberg Traurig

77 West Wacker Drive, Suite 2500

Chicago, IL 60601

Re: Estimate for Repeating Demolizere Validation Study using B. subtilis ATCC 19659

Dear Neal:

As you requested, we have obtained a firm estimate from Dr. James Marsden consistent

with the verbal estimate provided in June 2007 for repeating the Demolizer0 Validation

using

B.

subtilis

ATCC 19659 instead of

B.

subtilis (111, atrophaeus)

ATCC 9372, the

most appropriate and preferred

B. subtilis

organism for the validation of dry heat

sterilization processes. A copy of the formal estimate from Dr. Marsden is included with

this letter,

as

requested.

The first phase involves stabilizing a culture population of the

B. subtilis

spores and

"certifying" its resistance properties through exhaustive D-value studies. Dr. Marsden

would use standard protocols for validating the resistance of the culture similar to those

used by industry. It is very possible that this study would need to be repeated several

times until a population is grown to the standards comparable to those obtained from

certified manufactures. Manufacturers such as STERIS Corporation have a complete

research unit dedicated to such efforts. Dr. Marsden provided an estimate of a minimum

of $60,000 for a single D-value evaluation of a population. It is very possible that

repeated trials could result in a total cost approaching $250,000 to properly certify the

population with a total time frame of up to 2 years.

The second phase involves repeating the Demolizer® Efficacy study using appropriate

replicates, load conditions, etc. This requires a minimum of 2-4 months to coordinate

and report the study. Upon completion of both phases, validation results comparable to

those already reported could be obtained. The estimate provided by Dr. Marsden for the

validation study using ATCC 19659 is $40,000.

In addition to these costs, BMTS would incur direct costs totally

more than $30,000 which includes the cost of three dedicated

systems, supplies and other consumables, and the cost of BMTS

staff time to be onsite at Kansas State University to facilitate and

witness the trial. This practice is necessary to support

representations BMTS makes in relation to the efficacy of its

products.

9800 Mt. Pyramid Court

Suite 350

Englewood, CO 80112

1-866-525.8MTS

P: 303.653.0100

F: 303.653.0120

bmiscorp.com

---

Sincerely,

BioMedical Technology Solutions, Inc.

Page 2 of 2

September 25, 2007

Therefore the total cost for repeating the study using a different, non-certified,

B. subtilis

isolate is estimated between $130,000 and $320,000 dollars. The timeframe to complete

this work would range from 9-12 months to as much as 2.5 years.

Diane R. Gorder

Director of Regulatory Compliance

Cc: Mr. Don Cox, BMTS

---

Peo S

Kansas

TA

State University

TE

Food Science Institute

148 Waters Hall

Manhattan, KS 66506

-4 010

785.532-2202

Fax: 785-5325861

Email: Inadsci@kntate.edu

htip://foodsci.k.staie.edu

September 23, 2007

Diane Gorder

Director of Regulatory Compliance

BioMedical Technology Solutions, Inc.

9800 Mt. Pyramid Court, Suite 350

Englewood, CO 80112

Re: Proposal for additional spore validation testing on the Demolizer® technology

Dear Ms. Gorder,

As requested, this letter provides a formal proposal for the validation of the Demolizer

technology using

Bacillus subtilis

ATCC 19659 consistent with the verbal estimates

provided earlier this year.

The new validation study would be carried out using the same protocol utilized in the

original trial conducted in 2006. The objective would be to perform an equivalent

validation trial using the Illinois designated spore organism, an organism that is not

recognized for the validation of dry heat sterilization technologies. The certified USP

,intemationally-recognized and most appropriate

B. subtilis

substrain was used in the

original study and is well understood to deliver superior resistance properties in thy heat

applications.

The spores used in the July 2006 study were comprehensively tested and certified by

Steris Corporation for purity and performance as represented by D-value resistance

consistent with FDA, USP and other international standards requirements. Since the

ATCC 19659 spore is not available in a certified form, we would need to complete an

exhaustive D-value trial to replicate the standards achieved in the original study. This

type of a study is extensive and is typical of the level of effort for

a

Ph.D research

initiative. As is the nature for microbiological studies, it is possible the growth of the

population and D-value certification may need to be repeated several times if satisfactory

results cannot be easily obtained.

Upon acceptance of a given population, the trial would then be conducted. All

experiments would be conducted in triplicate to support meaningful findings. This effort

would be led by either a Ph.D. candidate or a post-doctoral research associate. The work

would be

supervised

by me along with the

remainder of our scientific team.

Please find

below an estimate of this scope of work. There is substantial effort involved in

coordinating such an effort. It is reasonable to expect that the study could involve a

fit

---

minimum of 6 months up to 2 years should the culture prove difficult to grown to the

standards obtained in the original study. As with the previous study, BMTS is

responsible for delivery of three Demolizer 11 Systems and associated supplies for

representative waste loads. We would also recommend that you provide a member of the

BMTS engineering team be onsite in support of the effort particularly during the

completion of Phase IT of this effort.

Phase 1 – Growth and stabilization of

B.

$60,000 per repetition for up to $180,000

subtlis

ATCC 19659 to a minimum

population density of 2.0 x 106 to a

maximum population density of 5.0 x 106;

D-value study reporting the heat resistance

of the population and comparisons with

published standards (may need to be

repeated up to 3 times prior to accepting a

given population)

Phase 2 – Validation study of the

Demolizer technology against

B.

subtilis

ATCC 19659 using representative waste

loads

$40,000

Total Estimated Cost

$100,000 to $220,000

Lead time: minimum 6 months up to 2

years

As discussed with the Illinois representatives in our recent conference call, this effort is

not only expensive in both money and time, it is not scientifically warranted because the

Bacillus subtilis

certified spore used in the original study is the most appropriate spore

organism for the Demolizer® sterilization process. Please refer to Dr. Daniel Y.C.

Dung's letter of authority on this matter for additional details.

Please advise if additional information is required.

Si•ely,

James L. Marsden, PhD.

Regent's Distinguished Professor

Kansas State University

---

Exhibit J

---

ction of chlorhe

le mode of actin

28-32.

m the electroph

ails of

Escherich?

18,

569-578

.

?

le!

al activity

of

Oben°

tbstrate and medi

the wall of

Bacil

anent on resistan

.ondon: John Wil

'aeon of phosphitt

Uochemical Journal

ttion of polar

lipids

olar

FEMS

lipid

Letters

c

omposi.

27,

--

ons of polar

lipids

9-1003.

ttions at various

.3ôs octyl phenol.

c

ondition on the

e

r

elationship to

cal

Journal

106,

the

composition

se

ngineering 11,

I

I

Wild

of

Applied Bacteriology

1980,

48,

231-247

?

582/04/79

Thermal Activation and Dry-heat Inactivation

of Spores of

Bacillus subtilis

MD2 and

Bacillus subtilis

var.

niger

Spores of

Bacillus subtilis

MD2 and

Bacillus subtilis

var.

niger

were heat activated for different

times at 60° and 80°C. Strain MD2 required considerable heat activation while

B. subtilis

var.

niger

did not. Maximum germination rates increased with heat activation dose and declined subse-

quently without loss of germinability. Germination rates and percentages were considerably

greater in tryptone glucose extract (TGE) than in nutrient broth. The addition of 2% dimethyl

sulphoxide did not increase germination in nutrient broth. The spores of var.

niger

are more

resistant to dry-heat than MD2 although they are less resistant to moist heat. Survivor curves in the

dry-heat range I40°-170°C gave D

.

-values from 4123 to 0

.

106 min for MD2 and 5

.

679. to 0233 min

for var.

niger

recovered on TGE agar. D-values were lower on poorer media. The z-values for MD2

and var.

niger

on TOE were 18

.

7°C and 21-25°C respectively.

THE

LITERATURE

on moist heat sterilization has been vast; that on dry-heat sterilization

less so but is increasing, partly because of interest in its use for sterilization of

space-craft components. There has also been considerable interest in recent years in the

biochemical nature of the lesions caused by heating bacteria under sublethal and lethal

time/temperature conditions. The nature of heat damage to bacterial spores (Keynan

1969; Gould 1970; Brown

&

Melling 1971; Russell 1971) and vegetative cells (Allwood

& Russell 1970; Ingram 1971;

Corry

1973;

Mossel & Corry 1977) has been reviewed at

various times and damage to membrane, protein, DNA and RNA are recorded. Most

of the records relate to moist heat damage.

The indicator organism for moist heat sterilization is

Bacillus stearotherrnophilus;

the organism commonly used for the evaluation of dry-heat killing procedures is

B.

subtilis

var.

niger.

For the spores of both organisms a wide variety of D-values has been

reported, varying with the experimental procedures used. Results for dry-heat condi-

tions have been notoriously variable, largely because of the lack of attention to the

thermal lags encountered in heating-up times..

On the basis of results obtained by H.M. Darlow and W.R. Bale (unpublished

report: 'Observations on a high speed hot air instrument sterilizer') who used an equal

mixture of

B. subtilis

var.

niger

and

B. subtilis

MD2 and found the var.

niger

strain to be

`somewhat less resistant', Quesnel

et al.

(1967) used the same organisms for the

evaluation of dry-heat sterilization at 200°C. The study reported in this paper was •

designed

to

compare the dry-heat resistance of the

B. subtilis

MD2 and var.

niger

strains using a simple but efficient method of dry-heat treatment. A subsequent paper

will report on the nature of dry-heat damage to these spores.

'

Present address: Department of Science, Salford College of Technology, Salford 6, U.K.

0021-8847/80/020231 + 17$01.00/0

1231] ©

1980 The Society for Applied Bacteriology

TESSA R. GURNEY'

AND

L. B. QU ESN EL

Department of Bacteriology and Virology, University of Manchester, Manchester

M13 9PT, U.K.

Received 12 April 1979 and accepted 10 October 1979

---

232

T. R. GURNEY AND L. B. QUESNEL

Materials and Methods

Organisms

Two strains of

Bacillus subtilis

selected for their high resistance to dry-heat killing were

originally supplied by Dr H.M. Darlow, Microbiological Research Establishment,

Porton. These strains,

B. subtilis

MD2 and

B. subtilis

var.

niger

were biochemically

similar in activity to

B. subtilis

as characterized by Cowan & Steel (1974) except that

neither strain fermented xylose, and the

niger

strain did not reduce nitrate. Strain MD2

gave a circular, buff-white, matt textured colony with irregular margin on nutrient agar

at 36°C, while the

niger

strain gave a smaller circular, orange-chestnut, shiny, raised

colony. Morphology and colour varied quite considerably on different media,

especially after heat treatment.

Preparation

of

spore suspensions

The sporulation medium of Ohye & Murrell (1962) was used, solidified with 1.2%

Oxoid Agar No. 3. Each of 30 plates were spread with 0-2 ml overnight Nutrient Broth

(Oxoid CM 67) culture of test organism grown at 36°C, in each case, and the plates

were incubated at 36°C for

4

d. Samples from plates were removed daily for visual

evaluation of sporulation by phase-contrast microscopy. After 2 d strain MD2 showed

98% sporulation, while strain

niger,

even after 4 d, was only 80% sporulated.

Spores were washed froth the plates with three rinses of distilled water, centrifuged

and washed once in distilled water then stored at 4°C for 2d, when the suspensions were

treated with 50 pg lysozyme/mI to remove remaining cells. Microscopic examination

showed that after 1 h all cells had lysed in the case of strain MD2; 2 h treatment was

required for var.

niger.

Spores were then centrifuged and washed six times in distilled

water before storing at 4°C. From these stock suspensions working suspensions were

made by diluting in sterile distilled water to give suspensions of absorbance 1

.

0 at 660

nm (Cambridge Unicam SP 600 spectrophotometer). Spore viable counts were per-

formed on both Nutrient Agar (NA, Oxoid) and Tryptone Glucose Extract Agar

(TGE, Difco) using a surface spread method with unactivated and heat-activated

(60°C for 10 min) samples.

Germination studies

Germination and outgrowth was tested on four different media: Nutrient Broth (NB,

Oxoid CM 67), Nutrient Broth + I% glucose; Nutrient Broth +2% dimethylsulphox-

ide (NB +2% DMSO) and Tryptone Glucose Extract Broth (TGE, Difco).

Germination was followed by measuring loss of retractility as

a

decrease in optical

density (OD) at 660 nm (Unicam SP 600 spectrophotometer). For the experiments 1.4

ml of working spore suspension was added to 10 ml of the appropriate medium in a

side-arm flask equilibrated at 37°C. OD readings were taken at zero time and at 5 min

intervals using sterile medium as the blank. Between readings flasks were returned to a

water bath at 37°C and shaken at 50 strokes/min.

For activation, 1

.

4 ml of spore suspension was added to 2 ml of medium in a sterile

test tube held at 60 or 80°C for the required activation time, after which the spores were

---

NEL

THERMAL RESPONSE OF

BACILLUS

SPORES 233

; to dry-heat killing w

=arch Establisltha

.8er

were

biocherntea

Steel (1974) except

ice nitrate. Strain

nargm on

n

utrient a

chestnut, shiny

,

rais

• on different m

night

solidified

Nutrient

with

Broth'

1.27

,

I case,

and the

plates :rte

wed daily for visual '

strain MD2 showed

sporulated.

d water, centrifuged

he

suspensions

were

scopic examination

; 2 h treatment was

ax

times in distilled

8

suspensions

were

;orbance 1

.

0 at 660

It

counts

were

per-

zse Extract Agar

nd heat-activated

Tient Broth (NB,

lifco).

crease in optical

i

ite medium in a

experiments

1.4

me

and at 5 min

re

returned

to a )

hum in a sterile

the

spores were

sled in iced water for 2 min before adding to 8 ml of medium in a flask pre-warmed to

°C.

errnination

is recorded in the graphs as per cent fall in OD against time. Rates of

ermination were derived as per cent fall in OD/min from the straight line portions of

the curves.

Before and after heat activation, the percentage germination of spores was derived

by

direct observation under phase contrast, scoring 100-200 spores on each occasion.

The final per cent germination was always taken when OD ceased to fall.

Dry-heat inactivation

Preparation

of

spore samples

Squares of side 6 mm were cut from aluminium foil and sterilized by dry-heat at

160°C for I h. Standard spore suspensions were distributed by calibrated Microtiter

pipette (Dynatech Laboratories, Billingshurst, Sussex), allowing one drop (0

.

0254 ml)

to dry on the dull side of each square at 37°C for 1 h. The inoculated foil squares were

equilibrated to an RH value of 32% in a sealed cabinet containing saturated calcium

chloride solution at 20°C for 14 d before use.

For use the foil squares were enclosed in a foil envelope formed from a 30 mm square

of

sterile aluminium foil, by folding as illustrated in Fig. 1 with the dull surface inside.

1. 6 mm foil square

with sample, on

30 mm foil square

4.

Third and fourth

folds — short edges

inwards

2. First fold

5. Each edge folded

inward again to

trap wire along

long edge

11

11 1

111

1111

111

111

1

11 1

1

a

Second fold long

edges inwards

Fig.

I.

Method

of

enclosing spore sample

in

aluminium

foil

envelope.

---

234

T. R. GURNEY AND L. B. QUESNEL

The foil enclosure was flattened with the side of a pair of forceps to minimize the

amount of air trapped inside the foil envelope. A 120 mm length of nichrome wire was

inserted under the fold along one edge of the foil envelope to enable transfer to and

from the hot oil bath. The entire wrapping procedure was carried out in the humidity

cabinet with the gloves provided and the wrapped spore samples were removed from

cabinet to oil-bath in the minimum amount of time.

Heating procedure

The wrapped spore samples were treated for the required times in hot oil carefully

maintained at the required temperature. The oil (Edwards High Vacuum oil) was filled

to a depth of

ca.

45 mm in 16 x 95 mm Pyrex test tubes held in the aluminium heating

block of a Techne Dri-Block DB3-H heater (Techne Ltd., Cambridge, England). The

block was loaded with 12 such tubes in holes of 16

.75 mm diameter, and was placed

between two other aluminium heating blocks in the same apparatus.

The apparatus was calibrated (by adjustment of two controls in the electronic

control unit) so that the temperature on a thermometer placed in the oil was also

recorded on the instrument meter. Slight adjustment to the level of oil in some tubes

was needed to achieve the same temperature an each tube (indicating that heating was

not even throughout the block). The minimum temperature available was 90°C and the

maximum 170°C, and thebeating-up time from ambient to 170°C was 40 min.

Recovery media

The three media used were: (1) TrYptone Glucose Extract Agar (TGE, Difco); (2)

casamino acids medium (CAA) (8/1)

;

NH4C1,4;

Na2HPO4, 12; KH

2PO4

, 6; NaC1, 6;

MgSO4. 7H20,

0. 1; Difco vitamin-free casamino acids, 5; dextrose, 10; Oxoid agar No.

1, 10; pH 7-7

.

2; dextrose was added as a sterile solution to the other sterilized

ingredients cooled to 50°C; (3) minimal growth requirement agar (MGR) as for CAA

medium except that casamino acids are replaced by alanine, 100 pg/m1; aspartic acid,

50 pg/m1; glycine, 100 pg/m1; methionine

100

pg/ml. L-Isomer amino acids were used

unless otherwise stated.

Recovery

of

spores

Spore .'envelopes' removed from hot oil were plunged immediately into a mixture of

ice and water for

ca. 5

s and dried on a sterile paper towel. Two sides of the envelope

were then cut with sterile scissors and the spore sample removed with sterile forceps to

10 ml of sterile distilled water in

a

screw-capped bottle.

Spores were removed from their foil support by placing the sealed screw-capped

bottle in a Megason ultrasonic cleaning bath (Shuco Scientific, London) for 5 min

which was shown by experiment to be an adequate procedure for removal of spores.

The 10 ml suspension of spores this produced was called 'neat' and suitable dilution

for plating were made in distilled water. Duplicate samples of 0 .2 ml were spread on

one or More of three recovery media: TGE, CAA or MGR. All plates were incubated at

30°C for 3 d before counting. (A

4%

increase in count was found between days 2 and 3,

but no increase after 3 d.)

The thermal death curves and values derived from them are based on the mean

values obtained by performing each determination between 3 and 6 times.

•

---

to a mixture of

f

the envelope

:rile forceps to

screw-capped

m) for 5 mini

/al of

dilution

spores.

able

re spread on

lays 2 and 3,

I -

incubated

at

n the mean

MIIIRTI1 MEM ■110I■

EL

THERMAL RESPONSE OF

BACILLUS

SPORES 235

reeps to min'

of nichrome wire

ena

ble

tr

ansfer

t

NJ out in the h

:s were removed

s in hot oil carefu

acuum oil)

was fill

idge,

aluminium

England).hea

t',

ter, and

was pla

s in the electronic'

in the oil was also:

f

oil in some

tubes

g

that heating was

a was 90°C and the

vas 40 min.

TGE,

Dike); (2)

PO4

, 6; NaC1, 6;

Oxoid

agar

No.

other sterilized

3R) as for CAA

:I; aspartic acid,

acids were used

Measurement of thermal lag

To measure the time taken for heat penetration through the aluminium envelope

grounding the spore samples a fast-response fine-wire (0

.

139 mm diam.) thermocou-

ple, type K76 P1 (Comark, Rustington, Sussex) was used with a Comark Electronic

'thermometer

(160 series). The thermocouple was enclosed in the foil envelope which

was then placed in the oil bath under the experimental conditions used and readings of

temperature and time taken for Dri-Block instrument settings of 90°C rising in 10°C

steps to 170°C.

The response time lag of the electronic thermometer was 02 s and any remaining lag

was assumed to be due to time required for heat transfer from oil and through the

aluminium

envelope.

The temperature rise with time followed a logarithmic curve

asymtoting to the set maximum. The heat transfer time was found to be

ca.

15 sat 90°C

and ca. 20 s at 170°C. At each set temperature 90% of the maximum was reached in

ca.

2 s. For the values plotted and used to calculate killing rates, no allowance was made,

therefore, for heating-up time.

Derivation of data

The data from 'the thermal inactivation experiments were subjected to a computer

analysis to derive regression lines of best fit and from these the D-values at different

temperatures on different media were derived. Similarly z-values and Y intercepts were

also calculated from the computer determinations.

Results

Typical germination curves for

B. subtilis

MD2 and var.

niger

strains are shown in Figs

2 and 3. Table 1 gives the rates of germination (calculated as per cent loss of OD/min)

and final germination (as per cent phase dark when 010 ceased to fall) for the two

strains after various activation treatments and germination in several media.

The data show that the

B. subtilis

var.

niger

strain germinates more readily and gives

a higher percentage germination than

B. subtilis

MD2 on all media and under all the

,conditions

tried. In both cases TGE was a far superior medium to the others and was

necessary for germination approaching 100%. Increasing the time of activation at 60°C

increased the rate of germination to a maximum at 10-15 min, with concomitant

increases in percentage germination on TGE. On the other hand increasing the

activation time beyond 5 min at 80°C reduced the germination rate'of MD2 in all three

media with a decrease in percentage germination except in TGE, which was clearly the

superior medium. It is interesting that the germination of 99% obtained after 80°C for

15 min was obtained at a slower rate which presumably reflects the need to repair a

damaged germination system. The thermal death curves resulting from dry-heat

treatment of MD2 and

niter

spores at 160°C are given in Figs 4 and 5. The curves are of

greatly different form, MD2 curves having pronounced shoulders which were absent in

the case of

niger

spores. Table 2 gives the D-values obtained for the strains at

temperatures of 140, 150,160 and 170°C on several media. Clearly the highest D-values

were obtained for recovery on TGE medium indicating the ability of TGE to allow

repair of heat-damaged spores. At all temperatures var.

niger

showed greater dry-heat

resistance than strain MD2. Table 3 shows that the Y axis intercepts, obtained by

---

236

T. R. GURNEY AND L. B. QUESNEL

r

..s

—• — • .--

?

• --

•

46

40

E

•

0

ID

a

• 32

a

-0 24

a

..E

•?

16

•

/•—•°-._•—

• — • —

r

♦—♦—

-•

/3

20 30 90

1

50

Minutes

Fig. 2. Germination of

Bacillus subrilis

MD2 spores activated in various media at 60°C for

5

min

and germinated to completion in the media at 37°C.., nutrient broth; s

i

nutrient broth +2%

DMSO; tryptone glucose extract broth. Extrapolations (dashed lines) intersect at points

arrowed to give 'completion times' (see Table 1).

10 20 30 40

Minutes

Fig. 3. Germination of

Bacillus subtilis var. niger

spores activated in various media at 60°C for

5

min and germinated to completion in the media at 37°C: a, nutrient broth; 4, nutrient

broth +20/0

DMSO; tryptone glucose extract broth (see Table I).

---

a 9

TABLE

1

The effect of heat activation and medium composition on the germination of spores of Bacillus

subtilis MD2

and

Bacillus subtilis

var.

niger

TGE?

NB+ I% glucose?

NB

?

NB +2% DMSO

Max.?

%?

Completion?

Max.?

% Completion?

Max.?

%, Completion?

Max.?

% Completion

germ. rate•

Germ.t

?

time:?

germ. rate Germ.

?

time?

germ. rate Germ.

?

time?

germ. rate Germ.?

time

Bacillus subtilis

MD2

unactivated

1-54

80

38

1.0

54

40

0.69

49

399

0.48

28

43

6015 min

2.94

89

21.4

.

1 -

43

52

18.2

0.66

24

232

60°/10 min

4.0

93

18

0.91

34

19.5

0.87

26

22

60°/15 min

4.0

96

18.6

N.D.

N.D.

N.D.

N.D.

N.D.

ND.

8075 min

3.57

94

21

1.66

60

19

0.87

35

27

80°/10 min

3.13

97

19-5

0.83

30

24

0-83

25

23

80°/15 min

2.94

99

24

'?

0

.34

24

42.5

0.42

17

25-5

B. subtilis

var.

niger

unactivated

2.38

95

18

2-34

85

20.5

1.64

76

22

1.43

69

26

60'15 min

4.0

93

12

227

70

15.6

227

66

152

* Maximum germination rate measured as per cent OD fall/min, calculated from the straight line region of the curve.

t Per cent germination recorded as per cent phase-dark spores present when OD ceased to fall.

t Completion time obtained by finding the intersect of extrapolations of straight line 'fail' and terminal OD values (see Fig. I). Times in minutes from end of

activation time.

N.D.=- not determined.

-a

Fr

---

238

T. R. GURNEY AND L. B. QUESNEL

107

I 0 6

105

104

103

o

2

—

t

,

0. 25 0

.

5 0.75 1 .

00 1-25

Minutes

•

Fig. 4. Thermal death curves of

Bacillus subtilis

MD2 spores dry-heated at 160°C and recovered on

various media:., tryptone glucose extract agar; u, casamino acids agar; minimal growth

requirement agar. 1)-values are given in Table 2.

10' -1

0-25

I

0-50

I

0.75

I

1 . 00

1

1

.

25

1

1.50

I

.

Minutes

Fig. 5. Thermal death curves of

Bacillus subrilis var. niger

spores dry-heated at I60°C and recovered

on:., tryptone glucose extract agar; im, casamino acids agar (see Table 2).

1

1 .50 I . 75 2.00

---

1

THERMAL RESPONSE OF

BACILLUS

SPORES 239

UESNEL

TABLE

2

Decimal reduction times for spores of

Bacillus subtilis

MD2

and

Bacillus

subtilis

var.

niger

heated at several temperatures and recovered on different

media

B. subtilis

var.

niger

Bacillus subtilis

MD2, D-value (min)D-value min)

Temperature

(°C)

?

TGE? CAA?

MGR?TGE?

CAA

140

4 123

3-493

2-190

5-679

4791

150

'

?

1307

0889

0.511

1600

1259

160

0412

0.284

0-201

0654

0-501

170

0106

0081

N.D.

0 233

0166

TABLE

3

Y-axis intercepts* for

Bacillus subtilis

MD2

dry-heat death

F

5

2-0

0

Iprves, and

Y

0

/N

o

t

ratios, at various temperatures for different

media

160°C and recovered on

agar;

A

,

minimal growth

TGE?

CAA?

MGR

Temperature

(°C)?

?

Y intercept

YolNo

Y intercept

Yo/ No

Y intercept

Yo/ No

140ISO

?

?

9.45

1.43

x

x

101076?143

?2

.

16 39

.

.

89

06

x

x

101067?4?149

.

62

1-22

923

x

x

10107

7?1.841394

160?

3.94 x 107?3

.35 9 .58 x 107?1447

1 .57

x 108 23-72

170?

228 x 108 34-44 7

.

82 x 108 11813?

N.D.

" Recorded as the hypothetical original population of spores, Yo.

t

No

is the actual number of spores in the sample (value derived from viable

counts arid direct microscopic determination of germination percentages).

TABLE

4

z-values for

Bacillus subtilis

MD2

and

Bacillus subtilis

var.

niger

spores reco-

vered on different media

z-value (°C)

Medium

B. subtilis

MD2

B. subtilis

var.

niger

0°Candrecovered

TGE?

18-75?

21-25

CAA?

1825?

21.0

•?

MGR?

18-25?

N.D.

---

240

T. R. GURNEY AND L. B. QUESNEL

extrapolation of the logarithmic regions of the death curves, increased significantly as

the temperature of inactivation was increased.

From Table 4 it can be seen that the z-values for var.

niger

are some 2

.

5°C higher

than the MD2 strain, while the z-values on different media differ but the curves are

essentially parallel in both strains (Figs 6 and 7).

To our knowledge this method of dry-heat treatment has not been used before and it

was important to assess the accuracy and precision of the techniques. From the

prepared stock suspensions of spores, one drop (as distributed to foil squares) was

suspended in 10 ml distilled water and a 5 x 10-

0

dilution in water made in ten fold and

two fold steps. This was done three times and duplicate 0

.

2

ml

samples

were

spread on

TGE

agar

plates on

each

occasion for each strain; colonies were counted after

incubation at 36°C for 24 h. Table 5 gives the estimated number of spores per drop.

Table 6 shows the number of spores recovered after sonication of sample-loaded

aluminium foil squares as described above, using the same methods for diluting and

plating. Sonication times of 2, 4, 6, 8 and 10 min were used and yielded essentially the

same number of viable spores as obtained from an equivalent drop of suspension not

sonicated.

2

loTI

11

?

i

?

I

?

I

140?

. 150?160

Temperature (°C)

Figs 6

(above) and

7

(below). Derivation of z-valucs

for

Bacillus subtilis

MD2

spores

(above) and

Bacillus subtilis

var.

niger

spores (below) from survivor curves

of

cells

recovered on: •,

tryptone glucose extract agar; ., casamino acids agar; a, minimal growth requirement agar.

Dashed lines denote intercepts at each log unit of ordinate (see Table 4).

170

---

re some 2

.

5°C hi

er but the cure

iL

THERMAL RESPONSE OF

BACILLUS

SPORES 241

[-eased

si

gnifican

TABLE 5

Calibration of spore count per drop of stock

suspension

B. subtilis

MD2?

B. subtilis

var.

niger

Sample?

count?

mean?

count?

mean

a

?

5.35 x 106

770x106

b?

5.40 x 106?535 x 106?790 x 106?

7

.

77

x 106

c

530 x 106?7.70 x 106

ds for diluting

an

?

j

TABLE 6

•

lded

of

sue

ssentially

spension

nt;

th

?

Effect

spores

of sonication

dried on to aluminium

on the recovery

foil

of

ehmq ues. From

en

usedbefore

to foil

s

quares)

Wade in ten fold

f spores per drop.

l of sample load

au

pies were spread:

ere

c

ounted

a

B. subtilis

MD2

B. subtilis

var.

niger

Exposure recovered count recovered count

time (min) (x

106)?

( x 106)

2

545

795

4

520

815

6

560

78

8

540

7.8

10

5.25

7,95

Discussion

above) and

:red on: •,

ment agar.

While both of these strains have been recommended for use in dry-heat sterilization

i‘

tests they are clearly different in their characteristics. These differences can be seenboth

in their germination and thermal inactivation behaviour. The var.

niger

spores ger-

minated in the three germination media without appreciable lag while there was a

1 significant

lag for MD2 spores (Table 1). The difference is typified by the data for

! germination in TGE broth. Activation of 60°C

for 5 min gave a germination rate of

194, a germination percentage of 89 and a completion time of 21

.

4 min for strain

MD2, while the corresponding values for

niger

spores were 4

.

0, 93 and 12. The

difference in germination mechanism between these strains is further shown by the fact

! that

var.

niger,

but not MD2, may be germinated by subtilisin alone (Quesnel

et al.

1977).

The increase in germination rate with increased activation dose (e.g.

at

60°C, Table 1)

followed by a decrease in germination rate as the thermal dose is increased (e.g.

at

80°C) has been found for other species as well (Curran

&

Evans 1945; Powell 1955;

Keynan 1969;

Levinson &. Hyatt 1970; Hashimoto

a al.

1972). It is significant,

however, that the increased dose at the higher temperature gives decreased rates and

lower percentages of germination on the poorer media, but an increased percentage

germination on the richer medium.

---

242

T. R. GURNEY AND L. B. QUESNEL

Hashimoto

et al.

(1972) have shown that the kinetics of germination of individual

B.

cereus

spores are biphasic and that after about a 42% loss of refractility, individual

heat-damaged spores exhibited a secondary microlag period related to the heat dose,

before the second phase of microgermination produced the phase dark spore. Such

heat-damaged spores showed no loss of viability until a lethal dose of heat was applied.

Lethal heating at 90°C for 30 min did not, however, prevent germination as measured

by loss in OD of a spore suspension, although outgrowth and colony formation was

impossible on trypticase soy agar.

In this study phase-grey spores were classed as ungerminated (as indeed they are) but

to judge from their number many of the phase-grey (damaged) spores found in nutrient

broth after treatment at 80°C were arrested in the secondary microlag and would have

proceeded to the second phase of microgermination in TGE medium. Clearly some

heat-damage is reversible, or such damage

can

be ignored in the presence of additional

germinant compounds present in the richer medium, and germination then proceed by

an alternative mechanism as suggested by Sogin

et al.

(1972).

While the specific nature of the biochemical lesion which inhibits progress to the

second phase of germination is not known, Schacter & Hashimoto (1975) have

suggested that extended heat treatment may cause the alteration of the structural

components of spores in such a manner that the rapid degradation of these structures

, may become difficult. These structures would not involve those responsible for heat

resistance or dipicolinic acid retention as both these properties have been lost by the

time of secondary microlag (Schacter & Hashimoto 1975). Dring & Gould (1975) have

provided convincing evidence for the initiation of endogenous metabolism during

germination and shown the importance of the membrane-linked electron-transport

chain in germination. They suggest this as the motive force for bound ion translocation

necessary for germination, but leave open the question whether germinative amino

acids are themselves metabolized, or function allosterically to trigger metabolism of

endogenous reserves.

Although Quesnel

et a!.

(1971) found that low concentrations of DMSO enhanced

recovery in a simple glucose-peptone medium this result was not confirmed here,

although it can be seen that the adverse effect of DMSO at non-damaging temperatures

was eliminated when damaging conditions were used (Table 1).

The method of dry-heating devised for this study allowed fairly accurate measure-

ment and great ease of handling. The numbers of spores in each sample showed slight

but not significant differences and recovery by sonication in distilled water proved to be

adequately reproducible (Table 6).

While every effort was made to control accurately the temperature of the oil baths

used to immerse foil-wrapped spore samples it was found that increasing the level of oil

(e.g. by immersion of a thermometer) caused a slight lowering of the temperature of

2-3°C. However, the foil samples raised the level of oil to a much lower extent than did

a

thermometer and thermocouple measurements indicated that the heating tempera-

tures were accurate to within < I°C, when the oil level was maintained below the top of

the Dri-Block.

At temperatures below 140°C, where holding times in excess of 15 min were used, it

was found that oil sometimes entered the foil envelope.

While

experiments indicated

that the oil was non-toxic even to heated spores, it did appear to interfere with the

precision of the sonication technique used to release the spores from thin foil supports.

---

thibits progress to

the

ishimoto (1975) hav -

lion of the structura1

ion of these structures;

e

responsible

for heat

have been lost by the

;

&

Gould (1975) have

s metabolism during

xi electron-transport

and ion translocation

r

germinative

amino

-igger metabolism of

I

of DMSO enhanced

notconfirmed

here,

caging temperatures

r accurate measure-

tmple showed slight

!water

proved to be

ure of the oil baths

ming the level of oil

the temperature of

Her extent than did

heating tempera-

xi below the top of

Ii

t

;NEL

THERMAL RESPONSE OF

BACILLUS

SPORES 243

urination of individ

of refractility, indiv

related to the heat d

phase dark Sporn

s'

dose of heat was ap

termination

as mea

d colony formation

1(as indeed they are

spores found in nuttie

icrolag and would ha

medium. Clearly so

c presence of addition'

nation then proceed b

treatments

at or above 140°C, where no oil seepage was experienced, were

rded here. Sonication has been used successfully to remove spores from films dried

glass coverslips (Molin & Ostlund 1975, 1976; Molin & Svensson 1976; Molin

197

7a,b). The use of an ultrasonic probe inserted into the liquid was found to be totally

tisfactory presumably because of aerosolization on to the cotton plug surround-

g the probe at the neck of the container.

4-

A preliminary inactivation experiment gave the same thermal death times (TDT) of

t34 mM at 150°C, 3

.0 mM at 160°C and 1

.

5 mM at 170°C for spore populations of

9 x 106

MD2 spores and 7

.

77 x 106 var.

niger

spores, which might indicate a greater

`dry-heat

resistance for MD2 spores which was previously reported for MD2 (Quesnel

et at

1967). In fact the thermal death curves showed the opposite to be true although

mpris more resistant to moist heat (Fig. 8). The fact that var.

niger

spores are less

resistant to moist heat but more resistant to dry-heat might indicate different killing

mechanisms under the two types of condition. More experiments would be needed to

confirm this.

The recovery medium greatly influenced the survival of the organisms. Decimal

reduction times for MD2 spores recovered on TGE were almost twice as great as for

spores recovered on MGR (Table 2). Independent experiments (not reported) showed

that 100 tg L-almine/ml caused complete germination of both strains of spores. While

germination of > 97% was obtained in liquid-MGR medium, the count on solidified

MGR medium was only 85% of that on TGE medium for undamaged spores, and the

greater the amount of heat-damage the smaller was the fraction of spores recovered on

MGR relative to TGE.

107

106

lo°

to

o4

min were used, it

riments

indicated

interfere with the

hin foil supports.

•

los ?

0.25 0.50

0-75 1-00

Minutes

Fig. 8.

Thermal death curves for

Bacillus subtilis

MD2 (•) and var.

niger

(e)

spores in moist heat at

110°C. Survivors were recovered on tryptone glucose extract agar.

---

244

T. R. GURNEY AND L. B. QUESNEL

The death curves derived by using CAA medium lay between those for MGR and

TGE (Figs 4, 5 and Table 2). As CAA contains amino acids not present in MGR, and

TGE contains amino acids and vitamins not present in CAA it is possible to identify

requirements for specific growth factors consequent upon dry-heat damage to these

spores. (The nature of these growth factors has been investigated and will be reported

separately.)

Obviously, the magnitude of D-values at any temperature, and of z-values, depends

on the medium used for recovery (Table 3) and comparison with published data should

compare like with like. Data for

B. sublilis

MD2 spores are not available, but Table

7

lists data for var.

niger

spores for

D160

values obtained under several different condi-

tions. The values range from 0

.

33 to 347 min, which compares with the value of 0.654

min obtained in this study. The value for MD2 recovered on TGE is D

160

=0.

412 min.

Similarly the z-value

(var.niger)

obtained by various workers using TGE range from

12.

9°C to 32°C compared with 21.25°C obtained by our methods (Table 8). The z-value

graphs are linear (Figs 6 and 7) and in good agreement with those published by Molin

(1977b) using the same temperature range. Molin & Ostlund (1976) have shown that

spore density affects the rate of kill of spores dried on to glass surfaces heated by

infra-red radiation and this is an additional cause of variation between dry-heat killing

data. The relative humidity of the spores at time of treatment also markedly affects

inactivation rates and the driest conditions do not give the lowest D-values. Brannen &

Garst (1972) found that as the r.h. was raised from 0 .

003 to 1 . 67% the D-values rose

from 1 . 4 to 2. 5 min at 105°C for

B. subtilis

var

niger

spores. According to Murrell &

Scott (1966) spores are most heat resistant in the range

of

020-0

.

40 water activity and

0.32 was chosen for equilibration of the spores in this study. During the heating process

changes in water activity relationships would occur, but no attempt was made to

control this during heat treatment.

TABLE

7

Published

D160

values for dry-heated

Bacillus

subtilis

var.

niger

spores recovered on TGE

medium

Supporting

?

Dm value

material?(min)? Reference

Glass coverslip?

037? Molin

(1977a)

Glass coverslip+

soybean oil?

045

? Molin (1977a)

serum albumin?

107? Molin

(1977a)

sucrose?

I.00

?

Molin (1977a)

starch?

04I 7?Molin

(1977a)

Glass coverslip-

(spores produced

on different

media)?

033-7I7 Molin & Svensson (1976)

Aluminium foil

?

0.654?

Gurney & Quesnel

(this paper)

---

M. MI

JESNEL

THERMAL RESPONSE OF

BACILLUS

SPORES

245

p

etween

those for MG

ds not present in MG

AA it is possible to i

a

dry-heat

damage to

tigated and will be repo

re, and of z-values, d

with published data sh

not available, but To

der several different

tres with the

value

of 0•

n TGE is Dm-4.412

trs using TGE range

f

tr

Cods (Table 8). The not

those published by M

d (1976) have shown that

glass surfaces heated -I:84H

between dry-heat killint

ent also markedly affece

vest D-values. Brannen & ' 1

1 .

67% the D-values rosé

According to Murrell &

0-0

.

40 water activity and'

urmg the heating process

o attempt was made to

TABLE

8

blished z-values for dry-heated

Bacillus subtilis

var.

niger

spores recovered

on TGE medium

Supporting

z-value

material

(°C)

Paper strips

12.9

Stainless steel

strips

?

208

In lucite rods

20.7

In epoxy rods

214

On steel washers

32.0

(under 150 in -lb torque)

Glass coverslips

23B

Glass coverslips

25.0

Glass coverslips

22B

Filter paper strips

2722

Aluminium

foil

?

21.25

The difference in shape of the thermal death curves is also significant. Inactivation

curves for strain MD2 are all shouldered while those for var.

niger are

not, in spite of

the fact that unsonicated samples of the stock suspension showed significant clumping

while MD2 spores did not. The difference is-due to the facts that MD2 spore germina-

tion is considerably enhanced by heat activation, while

niger

spores germinate readily

and rapidly with little or no heat activation (Table 1). Table 3 gives the increase in

Y-intercept values with temperature rise shown by MD2 in all three recovery media.

Similarly, for any given temperature Y-intercepts increase as the medium gets poorer,

indicating that the number of 'targets' inactivated in rich media are lower than for

poorer media. This difference is a measure of the ability to repair damage, or, more

probably, to ignore damaged molecules because of the availability of growth factors in

the recovery medium. Interestingly enough both strains yielded shouldered curves for

moist

heat inactivation at 110°C (Fig. 8), but only one such experiment was performed.

Adams & Busta (1972) provide strong evidence that the germination inactivation of

a strain of

B.

subtilis

spores stimulated by L-alanine was due to protein denaturation,

and drew attention to the difference between germination inactivation and thermal

injury inhibiting outgrowth. Similar differences apply here and the nature of the injury

in these two strains under similar conditions may, moreover, be different. From this

study the var.

niger

strain is clearly the organism of choice as an indicator of dry-heat

sterilization in the temperature range 140-170°C.

Reference

Angelotti

et

at

(1968)

Angelotti

et

at

(1968)

Angelotti

et

al.

(1968)

Angelotti et

at

(1968)

Angelotti

et

al.

(1968)

Man

(1977b)

Molin & Svensson (1976)

Molin & Ostlund (1976)

Bruch

et

al.

(1963)

Gurney & Quesnel (this paper)

References

ADAMS,

D.

M.

&

BusrA,

F. F. 1972 Heat injury as the selective inactivation of a

Bacillus subtilis

spore germination system. In

Spores V

ed. Halvorson, HD., Hanson, R. & Campbell, L. L.

pp. 368-372. Washington: American Society for Microbiology.

ALIN/00D,

M.

C. & RUSSELL,

A. D.

1970 Mechanisms of thermal injury in non-sporing bacteria.

Advances

in

Applied Microbiology

12,89-119.

ANGELorri,

it,

MARYANSKI,

J. H.,

Bunn,

T. F. &

PEE1.ER,

J. T.

1968 Influence of spore

moisture on

the dry-heat resistance Of

Bacillus subtilis

var.

niger. Applied Microbiology

16,

735-745.

cillus

TGE

76)

---

246

T. R. GURNEY AND L. B. QUESNEL

BRANNEN,

J.

P. & GARST,

D. M. 1972 Dry-heat activation of

Bacillus subtilis

var.

niger

spores as a

function of relative humidity.

Applied Microbiology

23,1125-1130.

BROWN, M.

R. W. &

MELLING, J. 1971 The destruction of bacterial spores. In

Inhibition and

Destruction of the Microbial Cell

ed. Hugo, W. B. pp. 451-460. London: Academic Press.

Baum C. W.,

KOESTERER, M. G. & BRUCH, M. K. 1963 Dry-heat sterilization: its development

and application to components of exobiological space probes.

Developmental and Industrial

Microbiology

4,334-342.

CORRY,

J. E. L. 1973 The water relations and heat resistance of micro-organisms.

Progress in

Industrial Microbiology

12,73-108.

COWAN, S.

T. &

STEEL,

K.

J.

1974

Manual for the Identification of Medical Bacteria.

Cambridge:

Cambridge University Press.

CURRAN,

H. R. & EvANS,

F. B. 1945 Heat activation inducing germination in the spores of

thermo-tolerant and therrnophilic aerobic bacteria.

Journal of Bacteriology

49,335-346.

DRING,

& GOULD, G.W. 1975 Electron transport-linked metabolism during germination of

Bacillus cereus

spores. In

Spores VI

ed. Gerhardt. P., Costilow, R. N. & Sadoff, H. L. pp.

488-494. Washington: American Society for Microbiology.

GOULD,

G. W. 1970 Germination and the problem of dormancy.

Journal of Applied Bacteriology

33,34-49

HAsmMoro, T., FRLEBEN, W. R. & CONTI, S.

F. 1972 Kinetics of germination of heat-injured

Bacillus cereus

spores. In

Spores V ed.

Halvorson, H. 0., Hanson, R. & Campbell, L.

L.

pp.

409-415. Washington: American Society for Microbiology.

INGRAM, M.

1971 The microbiology of•food pasteurisation.

Pastorisering avkivsmedel. SJX.

Rapport

292, A l-A43.

KEYNAN,

A. 1969 Activation. In

The Bacterial Spore

ed. Gould, G. W. Hurst, A. New York:

Academic Press. •

LEVINSON,

H. S. & Thom, M. T. 1970 Effects of temperature on activation, germination and

outgrowth of

Bacillus megaterium

spores.

Journal of Bacteriology 101,

58-64.

MoLIN,C. 1977a Dry-heat resistance of

Bacillus subtilis

spores in contact with serum albumin,

carbohydrates or lipids.

Journal of Applied Bacteriology

42,111-116.

MOLIN,

G.

1977b

Inactivation of

Bacillus

spores in dry systems at low and high temperatures. '

Journal of General Microbiology

101,227-231.

Mot"

G. &

OSTLUND,

K. 1975 Dry-heat inactivation of

Bacillus subtilia

spores by means of •

infra-red heating.

Antonie van Leeuwenhoek

41,329-335.

MOLIN,

G. &

OSTLUND,

K. 1975 Dry-heat inactivation of

Bacillus subtilis

spores by means of

special reference to spore density.

Canadian Journal of Microbiology

22,359-363.

MOLIN,

G. &

SVENSSON, M.

1976 Formation of dry-heat resistant

Bacillus subtilis

var.

niger

spores as influenced by the composition of the sporulation medium.

Antonie van Leeuwen-

hoek

42,388-395.

Massa, D. A. A.

&

CORRY,

J.

E. L. 1977 Detection and generation of sublethally injured

pathogenic and index bacteria in foods and water processed for safety.

Alimenta

16,

Sondemummer Mikrobiologie, 19-34.

MURRELL

W.

G.

& Scott, W. J. 1966 Heat resistance of bacterial spores at various water

activities.

Journal of General Microbiology

43,411-425.

OHYE, D.

F. & MURRELL, W.

G.

1962 Formation and structure of the spore of

Bacillus coagulans.

Journal of Cell Biology

14,111-123.

POWELL,

J.

F. 1955 Spore germination in the

Bacillus, the

modification of germination require-

ments as a result of pre-heating.

Journal of General Microbiology

13,59-67.

QUESNEL, L. B., HAYWARD,

J.

M. & Bxmarr,

J.

W. 1967 Hot air sterilization at 200°C.

Journal

of Applied Bacteriology

30,518-528.

QUESNEI,

L. B., Scum B. L. &

TAYLOR,

J.

L. 1971 The effect of dimethyl sulphoxide on the

survival and germination of

Bacillus subtilis

spores. In

Spore Research 1971

ed. Barker,

A. N., Gould, G. W. & Wolf, J. pp. 303-314. London: Academic Press.

QUESNEL, L.

B.,

OwERS,

J.

A.,

FARMER,

V. E. & Cow's, D. 1977 Subtilisin induced germination

of

Bacillus cereus

Px spores and the

effects of

dimethylsulphoxide.

In Spore Research 1976

Vol.

IL

ed. Barker, A. N., Wolf, L. J., Ellar, D. J., Dring, G.

J.&

Gould, G. W. pp. 753-770

London: Academic Press.

---

THERMAL RESPONSE OF

BACILLUS

SPORES 247

;lige

I

In

cad

is d

1a

ss.

p

Q C.

the

19, 33

oft: H.

Bacterk

ell, L. L.

ledel 8:1„

New York:

nation aid

n albumin,

iperatures.

I

• means

of

means of

var.

nigir "

I

Leeuwen-

1

y injured

lento

16,

us water

agulans.

require-

Journal

on the

,

3arker,

nation

h1976

1

3-770

A.

a

1971 The destruction of bacterial spores. In

Inhibition and Destruction of the

obial Cell

ed. Hugo, W. B. pp. 451-612. London: Academic Press.

crot,

S. M. & HAstitmoTO, T. 1975 Bimodal kinetics of germination of

Bacillus cereus

T

spores. In

Spores VI

ed. Gerhardt, P., Costilow,

R. N.

&

Sadoff, H. L. pp. 531-538.

Washington: American Society for Microbiology.

M. L., McCAUL, W. A. &

ORDAL,

Z. J. 1972 Effect of heat activation conditions on

the

germinal response of

Bacillus cereus

T spores. In

Spores Ved.

Halvorson, H. D., Hanson, R.

&

Campbell, L. L. pp. 363-367. Washington: American Society for Microbiology.

---

Guidance on

Premarket Notification (510(k)) Submissions

for

Sterilizers

Intended for Use in Health Care Facilities

Infection Control Devices Branch

Division of General and Restorative Devices

March, 1993

---

H.?

Biological Performance Tests

1.

General

The applicant must unequivocally demonstrate that the device

can sterilize, to an acceptable SAL, all the medical

products identified in the labeling, when used.in accordance

with the directions for use.

2.

Test Organisms

since a consistent type and concentration of bioburden

cannot be assured or realistically evaluated in a health

care facility, an overkill sterilization is necessary. The

sterilization cycle is based upon an initial concentration

of at least 106 CFU (or Plaque Forming Units - PFU)/unit of

a highly resistant organism to the process. Typically, the

most resistant organism to a sterilization process is used

based upon determination of D-values. Table 1 lists the

commonly recognized test organisms for the classified

sterilizers.

TABLE 1

TEST ORGANISMS FOR CLASSIFIED STERILIZERS

Sterilizer

?

organism

steam

dry heat

EtO

Bacillus stearothermonhilus (ATCC 7953)

Bacillus subtilis var. niger (ATCC 9372 or

19659)

bacillus

subtilig var. niger (ATCC 9372 or

19659)

The biological lethality profile of a nontraditional

' sterilization technology must be exhaustively evaluated since the

most resistant organism is initially

unknown. Table 2 identifies

recommended organisms to test for determination of the most

resistant organism.

17

---

TABLE 2

TEST ORGANISMS FOR NONTRADITIONAL STERILIZERS

A.

Bacterial Spores

Bacil l

u

s?

aget

(ATCC 9372 or 19659)

Bacillus stearothermoPhilus (ATCC 7953)

lgalisusmapigrast2 (ATCC 3584)

B. Mycobacteria

MVOobacterium tuberculosis var. 1?ovis

(or other representative mycobacterium)

C.

Nonlipid Viruses

poliovirus Type II

D. Fungi

Tricophvton

mentaorophytes (with conidia)

E.

Vegetative Bacteria

Staphyl ococcus animus

Salmonel la choleraesius

.ES-142E2nsl

F.

Lipid Viruses

herpes simplex

G.

THE LITERATURE OR OTHER

INPORKATION

MAX SUGGEST ADDITIONAL

TEST

ORGANISMS DEPENDING UPON THE TECHNOLOGY OR THE TYPICAL

BIODURDEN ENCOUNTERED

BY TEE ARTICLES INTENDED FOR

REPROCESSING IS

TEE

STERILIZER.

1 tt

---

Premarket Notifications [510(k)] for Biological Indicators Intended to Monitor Sterilizers Used in Health... Page 1 of 33

Food and Drug Administration

n■J

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's,

:t:

CENTER Ptia DOMES AND,

litAmowatett Mani

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Premarket Notifications [510(k)] for

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Draft Guidance for Industry and FDA

Reviewers

Sae

tutongationRelated

-1

Draft Guidance — Not for Implementation

This guidance document is being distributed for comment purposes only.

Draft released for comment on May 21, 2001

This document will supersede the document "FDA Guide for Validation

of Biological Indicator Incubation Time" dated January 1, 1986 once

this draft guidance is finalized.

U.S. Department of Health and Human Services

Food and Drug Administration

Center for Devices and Radiological Health

Infection Control Devices Branch

Division of Dental, Infection Control and General Hospital Devices

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Preface

Public Comment

Imp://wvvw.fda.gov/cdrh/ode/guidance/1320.html

6/22/2007

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Premarket Notifications [510(k)] for Biological Indicators Intended to Monitor Sterilizers Used in Flea... Page 15 of 33

16. Emergency and additional information: Provide a telephone number for

emergencies or for additional information.

H. Efficacy Data

Health care facilities use biological indicators to monitor sterilization processes. As defined in 21 CFR §880.2800, a

biological indicator accompanies devices through sterilization processes and monitors sterilization adequacy by its

growth or failure to grow. Biological indicators do not indicate that any given sterilizer load or device is rendered

sterile. Instead, biological indicators indicate that conditions to inactivate the biological indicator organisms were

achieved at the biological indicator location in a particular cycle. When the user places biological indicators in the most

difficult to sterilize location in a device load, the biological indicator result provides some assurance that organisms in

devices were inactivated. Health care facilities use biological indicators as part of an infection control quality assurance

program along with physical and chemical monitoring.

Biological indicators are of two types: paper strip, which require a separate culture medium; and self-contained, which

include the culture medium. Some self-contained biological indicators include growth indicators such as pH sensitive

dyes. Some biological indicators include two spore species to allow the same product to monitor either steam or

ethylene oxide processes. Additionally, biological indicators are marketed in test packs (see Section

with

separate chemical indicators (see Section f..1.3), or with indicators that allow for rapid interpretation (prior to the

visible growth of spores) on the basis of an enzyme or chemical reaction (see Section

1. Indicator (Test) Organisms

Bacterial spores are used as indicator organisms because they have high resistance to

the various sterilization processes. Spore resistance is complex and many aspects are

not well understood. Factors involved include: intrinsic (innate) resistance of the

spore species and strain, environmental conditions during sporulation, biological

indicator preparation, storage, exposure, incubation, and recovery, and biological

indicator carrier and packaging materials. The following Bacillus species and strains

are accepted for the uses listed in Table 2 (USP, 2000).

Table 2

Sterilizer type:

Indicator Organism/Spore:

Steam

Bacilluslius (ATCC 7953 or 12980)

Dry Heat

Bacillus

var. niger

(ATCC 9372)

Ethylene Oxide

Bacillusau_bliiiS var. niger (ATCC 9372)

For biological indicators intended to monitor sterilization processes other than those

listed in Table 2, you should justify the indicator organism using valid science. To

do so, you may conduct testing and submit data, or rely on published literature, if an

adequate body of knowledge exists.

Because resistance involves many factors other than spore species and strain, you

should characterize and validate biological indicators in the final finished form for

your specific indications for use (see Section 111.14.3 below).

2. Efficacy Study Reports

Efficacy study reports should provide complete details and include data to support

product effectiveness claims. Study reports should meet standards for publication in

peer-reviewed scientific journals. Reports should include the following information:

http://www.fda.gov/cdrh/ode/guidance/1320.html

6/22/2007

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JOURNAL OF CLINICAL MICROBIOLOGY,

Apr. 2004, p. 1626-1630

Vol. 42, No. 4

0095-I 137/04/508.00+0 DO/: 10. 1128/1CM.42.4.1626-1630.2004

Copyright 2004, American Society for Microbiology. All Rights Reserved.

Reassessment of Sequence-Based Targets for Identification of

Bacillus

Species

K. S. Blackwood, 1 * C. Y. Turenne, 1 D. Harmsen,2 and A. M. Kabani•

National Reference Centre for Mycobacteriology, National Microbiology Laboratory, Population and Public Health

Branch, Health Canada,' and Department

of

Medical Microbiology, University

of

Manitoba, 3 Winnipeg Manitoba,

Canada, and Institut far Hygiene and Mikrobiologie, Universinit Munster, Munster, Germany'

Received 13 August 2003/Returned for modification 7 October 2003/Accepted 14 December 2003

The

Bacillus

genus is a large heterogeneous group in need of an efficient method for species differentiation.

To determine the current validity of a sequence-based method for identification and provide contemporary

data, PCR

and sequencing of a 500-bp product encompassing the VI to V3 regions of the 16S

rRNA gene were

undertaken using 65 of the 83 type strains of this genus. This

region proved discriminatory between most

species (70.0 to 100% similarity), the exceptions being clinically relevant

B.

crass

and

B.

anthraces

as well as

nonpathogenic

B.

psychrotolerans

and

B. psychrodurans.

Consequently, 27 type and clinical strains from the

B.

cereus

group were used to test alternate targets

(spal, rrrA,

and the 16S-23S spacer region) for identification.

The

spoil

gene proved the best alternate target, with a conserved 4-nucleotide difference between

B. cants

and

B.

anthraces.

The high 16S rRNA gene sequence similarities between some strains demonstrated the need for

a polyphasic approach to the systematics of this genus. This approach is one focus of the Ribosomal Differ-

entiation of Medical Microorganisms mandate. Accordingly, the 16S

rRNA

gene sequences generated in this

study have been submitted for inclusion into Its publicly accessible, quality-controlled database at

http://www.ridom_rdna.dei.

The

Bacillus

genus is an extensive heterogeneous group en-

compassing 83 validly described species to date (http://www

.bacterio.cict.fr/b/bacillus.html). Many species in this taxon are

of major clinical importance, such as the

B. cereus

group (com-

prised of

B. cereus, B. anthraces,

B.

thuringiensis, B. mycoides,

and

B.

weihenslephanensis),

but unfortunately, members of this

group share a great deal of morphological and biochemical

similarities (3, 8, 16). In contrast, the environmental and non-

pathogenic species of this genus exhibit a wide range of phys-

iology, DNA base content, and nutritional requirements (2, 4,

15). Since the biochemical approach for species identification

can be tedious, expensive, and inaccurate, a rapid, definitive

method is greatly needed. Molecular procedures are increas-

ingly being used for rapid species identification. However,

some methods used for this genus such as restriction digests of

a target gene (i.e., 16S rRNA gene) (11) or randomly amplified

polymorphic DNA analysis (22) are limiting in discriminating

between a large group of species (6). Sequencing of the 16S

rRNA gene and select housekeeping genes has shown to be

particularly useful, generating large public sequence databases

due to the tangible, exact nature of sequence data. With the

increasing use of these methods and decreased expense of

running sequencing reactions after the initial equipment in-

vestment, more laboratories are relying on sequence data for

species identification (21).

A previous study using the 16S rRNA gene for rapid iden-

tification of the

Bacillus

genus was undertaken by Goto et al.

(6). At this time, the validity of using a hypetvariable region

• Corresponding author. Mailing address: National Reference Cen-

tre

for Mycobsocriology,

Canadian Science Centre for Human and

Animal Health, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E

3R2. Phone: (204) 789-6039. Fax: (204) 789-2036. E-mail:

kym_blackwood@he-sc.gc.ca.

(nucleotides Intl 70 to 344) of the gene was proven adequate to

discriminate between all the species except between

B. cereus

and

B. anthraces

and between

B. mofavensis

and

B. atrophaeus.

However, new sequence data were only acquired for 19 of the

species, with the rest obtained from preexisting sequences

available from the National Center for Biotechnology Infor-

mation GenBank. The GenBank nucleotide database is well

known for the non-quality-controlled nature of its data, includ-

ing base errors, ambiguous base designation, and incomplete,

short sequences. Several recent studies have examined the

problems surrounding the use of non-quality-controlled data-

bases such as GenBank and the Ribosomal Database Project

for identification purposes and have shown the benefits of

standardized, maintained databanks

that

include subsidiary in-

formation, such as Ribosomal Differentiation of Medical Mi-

croorganisms (RIDOM) (7, 21).

With the available data on this genus incomplete and the

many problems associated with public database use for simi-

larity searches, a fragment of the 16S rRNA gene

(Escherichia

coil

nt 54 to 510) for species of the

Bacillus

genus was se-