XENOBIOTIC DETECTION SYSTEMS, INC.

Presents EPA Method 4435

The XDS-CALUX Assay

Rapid, Accurate, and Affordable Dioxin/Furan and PCB Analysis
Mission Statement:
XDS exists to develop and advance the commercial acceptance of biological methods that will facilitate assessment of environmental and human health risks. A Message from Our President...
On behalf of myself and the staff at Xenobiotic Detection Systems, Inc. I would like to take this opportunity to thank you for expressing interest in our company and its products. Our primary goal is to use the latest advances in biotechnology to develop accurate and cost-effective assays for environmental testing. This is becoming increasingly important as both government and industry realize that frequent testing is necessary to safeguard the environment and human health. XDS is proud to offer you the patented Chemical Activated Luciferase Gene Expression (CALUX) assay. The XDS-CALUX assay (EPA Method 4435) is the culmination of over 30 years of research in biotechnology and environmental testing. The result is a test for dioxins and dioxin-like compounds that offers fast results and superior accuracy at only a fraction of the cost of standard high resolution mass spectrometry analysis. XDS is committed to providing quality analysis and developing biological assays to their fullest potential. I am confident that our testing methods will meet your expectations. We look forward to fulfilling your testing needs. Sincerely, George Clark, Dr. P.H. President

George Clark brings over 30 years of experience to XDS. His education includes a Ph.D. in toxicology from the University of North Carolina at Chapel Hill and Masters degrees in immunology from the University of California at Berkeley and biochemistry from North Carolina State University. He has previously worked for GlaxoSmithKline, and the National Institutes of Health.

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I. What can the XDS-CALUX Assay Measure?

Persistent Bioaccumulating Toxins (PBT) PBTs are chemicals that remain in the environment for long periods of time and accumulate in living organisms. Repeated low-level exposure to these chemicals result in toxic effects. Toxic effects are the result of the group's ability to alter normal biological processes. Substances classified in this group include PCDH, mercury, and polycyclic aromatic compounds. The EPA has proposed new reporting requirements with lower thresholds for PBT. As a result of these changes, industries that produce PBT will be forced to increase their reporting. XDS has developed a low cost, accurate screening assay to meet these needs. The XDS-CALUX assay has the potential to detect any of the compounds within this group. Polychlorinated Diaromatic Hydrocarbons (PCDH) PCDH are a subset of PBT. This subset includes dioxins and dioxin-like substances such as PCB and furans. XDS has developed a dioxin specific XDS-CALUX assay that can measure dioxins and dioxin like compounds such as PCBs and furans. This dioxin specific assay can determine total concentration of all dioxins and dioxin-like

Our Separation Guarantees Accuracy In order to accurately measure low-level contaminants like dioxins, separation methods are critical. XDS has developed and patented (US 6,720,431 B2) methods that allow us to accurately separate PCDH, PCB, dioxins, furans, and other biological toxicants. The new separation methods combined with the XDS-CALUX assay enable us to measure the total biological response of either specific classes of PCDH or total PCDH.

II. Uses
The XDS-CALUX assay is ideal for use as a low cost research tool to determine biological activity of a specific substance. It can also be used as a screening assay to measure contamination levels at industrial sites. Once contamination has been determined using the XDSCALUX assay, the identity of contaminants can be determined using techniques like GC-MS. The XDSCALUX assay saves customers money by decreasing the number of GC-MS samples needed, which are higher in cost than XDS-CALUX.

PBT

Other Biological Toxicants

PCDH

PCBs Dioxins & Furans

Figure 2: Diagram of XDSs separation scheme and the activity

III. Summary of Research

Background information
Figure 1: Dose response curves of the XDS-CALUX assay to various PCDH

compounds, or it can determine the concentrations of a specific class of compounds in a sample. Figure 1 shows luciferase response to several dioxins, furans and PCB congeners at various concentration levels.

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The effects of polychlorinated diaromatic hydrocarbons (PCDH) have been widely studied. PCDH include dioxins and dioxin-like compounds such as 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), PCB and furans. PCDH have been known to accumulate in animals and cause species- and tissue-specific toxic effects. Birth defects, immune system disorders, tumor production, and death have all been observed as a result of high-level exposure to PCDH.

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Mechanism of Action
The mechanism of action for these compounds depends on their ability to bind to an intracellular receptor called the aromatic hydrocarbon receptor (AhR). The PCDHAh Receptor complex then travels to the nucleus of the cell and binds to specific sequences in DNA called dioxin

Sensitive and Accurate

In a study by Garrison et al., the firefly luciferase gene used by XDS-CALUX has been shown to activate dioxins in a time, dose and AhR dependent manner. XDS routinely measures dioxins below one part per trillion concentrations by XDS-CALUX.

Environmental Ligands: PCDHs, AhR Complex PCBs, Dioxins, and Furans

Recombinant Cell
DRE
ARNT

CYP1A1 or other genes

Translocation

DRE

Luciferase

Translation
New Polypeptides
Increased Cytochrome P-4501A1 and Other Proteins

mRNA

Luciferase Activity

DRE =Dioxin Responsive Element =Dioxin-like compounds: PCDHs, PCBs, Dioxins and Furans ARNT =AhR Nuclear Translocator protein

Induction of light is directly proportional to concentration of dioxin TEQ in the sample.

AhR Complex =Aryl hydrocarbon Receptor Complex

Figure 3: XDS-CALUX cell mechanism

responsive elements (DRE). Binding of the PCDH-Ah Receptor complex to a DRE causes expression of the associated gene to be altered. It is this alteration in gene expression that causes observed toxic effects. The XDS Chemical Activated Luciferase Expression Assay (XDS-CALUX) applies the knowledge of the toxic mechanisms of PCDH. Using recombinant technology, the gene for firefly luciferase has been placed under the control of the Ah Receptor and inserted into a mouse cell line. Using this cell line, the presence of PCDH in a sample can be identified because PCDH will bind to the Ah Receptor, which will then bind to DRE in the nucleus and cause expression of firefly luciferase. The amount of PCDH in the sample can be related to how much light is produced by the activated cells.

Flexible and Versatile

The XDS-CALUX bioassay was designed to detect biological and environmental toxicants in any medium. Studies have shown that XDS-CALUX has accurately detected the concentration of dioxins in a number of matrices. Figure 4 (following page) illustrates the versatility of XDS-CALUX and provides a sample of the various samples analyzed. The figure also shows the close correlation with HR GC/MS results.

Certification / International Reputation

U.S. - by the U.S. EPA (Method 443) and U.S. FDA European Union - by BELLTEST Japan - by the Japanese Ministry of the Environment

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XDS CALUX has been selected by the Japanese, Belgian and Polish Governments as a screening tool for protecting their food supply and environment. XDS has licensed and provided training to the US FDA, the Hiyoshi Corporation of Japan and many others to use the patented XDS-CALUX technology.

Xenobiotic Detection Systems, Inc.

IV. Pricing
The price of XDS-CALUX analyses are 40%-70% lower than HR GC/MS fees. These substantial savings make XDS-CALUX an excellent screening tool and allow researchers, laboratories, and government agencies the option to use expensive HR GC/MS analyses only when XDS-CALUX results indicate dioxin levels are above client-established threshold levels. Savings exist even above 50% HR GC/MS follow-ups. Prices for analyses are determined based on the quantity and type of samples ordered, and the time requested to deliver results. Please email (info@dioxins.com) or call us (1-888-DIOXINS) for a written estimate. We offer order and volume discounts.

1000000 100000 10000 1000 100

x=y

HRGCMS-TEQ (ppt)

x = 10y
10 1 0.1 0.01 0.001 0.01 Gas A sh Soil Water Fish Blood Fat tissues Human Milk Bio Solid (US EPA)

0.1

10

100

1000

10000

100000

1000000

Bioassay-TEQ (ppt)

Correlation of XDS CALUX Bioassay and HRGCMS TEQ (Data and graph provided by Hiyoshi Corporation, Shiga, Japan)

Figure 4: Comparison of XDS-CALUX to HR GC/MS

Costs and Savings

For XDS-CALUX v.s. GC/MS
Note: Based on 100 samples with a $1100 per sample rate for GC/MS and $425 per sample rate for XDS-CALUX. Neither XDS-CALUX nor GC/MS pricing reflects volume discounts.

V. Selected References
1. Garrison, P.M. et al., (1996). Species-Specific Recombinant Cell Lines as Bioassay Systems for the Detection of 2,3,7,8Tetrachlorodibenzo-p-dioxin-like Chemicals. Fundamental and Applied Toxicology. 30, 194-203. Denison, M. S., Clark, G., et al., (1996). Application of the CALUX Bioassay System for the Detection of Dioxin-Like Chemicals (Ah receptors) in Whole Serum Samples and in Extracts From Chemical and Consumer Products. Organohalogen Compounds. 27, 280-284. Van Overmeire., et al., (1999). A Comparative Study of GCHRMS and CALUX TEQ Determinations in Food Samples by the Belgium Federal Ministries of Public Health and Agriculture. Organohalogen Compounds. 57, 196-199. Clark, G., et al., (1999). A Novel Low-Cost Air Sampling Device (AmbStack Sampler) and Detection System (CALUX Bioassay) for Measuring Air Emissions of Dioxin, Furan, and PCB on a TEQ Basis Tested With a Model Industrial Boiler. Organohalogen Compounds. 42, 309-312. Clark, G., et al., (2001). Comparison of CALUX Bioassay TEQ Determinations with High Resolution GC/MS Analyzing Soil Samples from a Hazardous Waste Site. United States - Vietnam Scientific Conference on Human Health and Environmental Effects of Agent Orange/Dioxins, March 3-6, 2002, Hanoi. Kayama, F. et al., (2001). CALUXTM Assay Is Applicable to Human Epidemiological Studies. Organohalogen Compounds. 54, 48-50.

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$120,000.00

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$100,000.00 $80,000.00 $60,000.00 $40,000.00 $20,000.00 $XDS-CALUX XDS-CALUX plus 10% GC/MS Confirmation GC/MS Price Savings with Confirmation

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For additional information and research abstracts please contact Xenobiotic Detection Systems, Inc. at www.dioxins.com or (919) 688-4804.

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