[Federal Register Volume 82, Number 121 (Monday, June 26, 2017)]
[Notices]
[Pages 28858-28860]
[FR Doc No: 2017-13296]

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

[Docket No. FDA-2008-N-0312]

Agency Information Collection Activities; Proposed Collection;
Comment Request; Extralabel Drug Use in Animals

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

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SUMMARY: The Food and Drug Administration (FDA or Agency) is announcing
an opportunity for public comment on the proposed collection of certain
information by the Agency. Under the Paperwork Reduction Act of 1995
(PRA), Federal Agencies are required to publish notice in the Federal
Register concerning each proposed collection of information, including
each proposed extension of an existing collection of information, and
to allow 60 days for public comment in response to the notice. This
notice solicits comments on the reporting requirements associated with
extralabel drug use in animals.

DATES: Submit either electronic or written comments on the collection
of information by August 25, 2017.

ADDRESSES: You may submit comments as follows. Please note that late,
untimely filed comments will not be considered. Electronic comments
must be submitted on or before August 25, 2017. The https://www.regulations.gov electronic filing system will accept comments until
midnight Eastern Time at the end of August 25, 2017. Comments received
by mail/hand delivery/courier (for written/paper submissions) will be
considered timely if they are postmarked or the delivery service
acceptance receipt is on or before that date.

Electronic Submissions

    Submit electronic comments in the following way.
     Federal eRulemaking Portal: https://www.regulations.gov.
Follow the instructions for submitting comments. Comments submitted
electronically, including attachments, to https://www.regulations.gov
will be posted to the docket unchanged. Because your comment will be
made public, you are solely responsible for ensuring that your comment
does not include any confidential information that you or a third party
may not wish to be posted, such as medical information, your or anyone
else's Social Security number, or confidential business information,
such as a manufacturing process. Please note that if you include your
name, contact information, or other information that

identifies you in the body of your comments, that information will be
posted on https://www.regulations.gov.
     If you want to submit a comment with confidential
information that you do not wish to be made available to the public,
submit the comment as a written/paper submission and in the manner
detailed (see ``Written/Paper Submissions'' and ``Instructions'').

Written/Paper Submissions

    Submit written/paper submissions as follows:
     Mail/Hand delivery/Courier (for written/paper
submissions): Dockets Management Staff (HFA-305), Food and Drug
Administration, 5630 Fishers Lane, Rm. 1061, Rockville, MD 20852.
     For written/paper comments submitted to the Dockets
Management Staff, FDA will post your comment, as well as any
attachments, except for information submitted, marked and identified,
as confidential, if submitted as detailed in ``Instructions.''
    Instructions: All submissions received must include the Docket No.
FDA-2008-N-0312 for ``Agency Information Collection Activities;
Proposed Collection; Comment Request; Extralabel Drug Use in Animals.''
Received comments, those filed in a timely manner (see ADDRESSES), will
be placed in the docket and, except for those submitted as
``Confidential Submissions,'' publicly viewable at https://www.regulations.gov or at the Dockets Management Staff between 9 a.m.
and 4 p.m., Monday through Friday.
     Confidential Submissions--To submit a comment with
confidential information that you do not wish to be made publicly
available, submit your comments only as a written/paper submission. You
should submit two copies total. One copy will include the information
you claim to be confidential with a heading or cover note that states
``THIS DOCUMENT CONTAINS CONFIDENTIAL INFORMATION.'' The Agency will
review this copy, including the claimed confidential information, in
its consideration of comments. The second copy, which will have the
claimed confidential information redacted/blacked out, will be
available for public viewing and posted on https://www.regulations.gov.
Submit both copies to the Dockets Management Staff. If you do not wish
your name and contact information to be made publicly available, you
can provide this information on the cover sheet and not in the body of
your comments and you must identify this information as
``confidential.'' Any information marked as ``confidential'' will not
be disclosed except in accordance with 21 CFR 10.20 and other
applicable disclosure law. For more information about FDA's posting of
comments to public dockets, see 80 FR 56469, September 18, 2015, or
access the information at: https://www.gpo.gov/fdsys/pkg/FR-2015-09-18/pdf/2015-23389.pdf.
    Docket: For access to the docket to read background documents or
the electronic and written/paper comments received, go to https://www.regulations.gov and insert the docket number, found in brackets in
the heading of this document, into the ``Search'' box and follow the
prompts and/or go to the Dockets Management Staff, 5630 Fishers Lane,
Rm. 1061, Rockville, MD 20852.

FOR FURTHER INFORMATION CONTACT: Ila S. Mizrachi, Office of Operations,
Food and Drug Administration, Three White Flint North, 10A63, 11601
Landsdown St., North Bethesda, MD 20852,<PHONE_NUMBER>,
<EMAIL_ADDRESS>
SUPPLEMENTARY INFORMATION: Under the PRA (44 U.S.C. 3501-3520), Federal
Agencies must obtain approval from the Office of Management and Budget
(OMB) for each collection of information they conduct or sponsor.
``Collection of information'' is defined in 44 U.S.C. 3502(3) and 5 CFR
1320.3(c) and includes Agency requests or requirements that members of
the public submit reports, keep records, or provide information to a
third party. Section 3506(c)(2)(A) of the PRA (44 U.S.C. 3506(c)(2)(A))
requires Federal Agencies to provide a 60-day notice in the Federal
Register concerning each proposed collection of information, including
each proposed extension of an existing collection of information,
before submitting the collection to OMB for approval. To comply with
this requirement, FDA is publishing notice of the proposed collection
of information set forth in this document.
    With respect to the following collection of information, FDA
invites comments on these topics: (1) Whether the proposed collection
of information is necessary for the proper performance of FDA's
functions, including whether the information will have practical
utility; (2) the accuracy of FDA's estimate of the burden of the
proposed collection of information, including the validity of the
methodology and assumptions used; (3) ways to enhance the quality,
utility, and clarity of the information to be collected; and (4) ways
to minimize the burden of the collection of information on respondents,
including through the use of automated collection techniques, when
appropriate, and other forms of information technology.

Extralabel Drug Use in Animals--21 CFR Part 530 OMB Control Number
0910-0325--Extension

    The Animal Medicinal Drug Use Clarification Act of 1994 allows a
veterinarian to prescribe the extralabel use of approved new animal
drugs. Also, it permits FDA, if it finds that there is a reasonable
probability that the extralabel use of an animal drug may present a
risk to the public health, to establish a safe level for a residue from
the extralabel use of the drug, and to require the development of an
analytical method for the detection of residues above that established
safe level (21 CFR 530.22(b)). Although to date, we have not
established a safe level for a residue from the extralabel use of any
new animal drug and, therefore, have not required the development of
analytical methodology, we believe that there may be instances when
analytical methodology will be required. We are, therefore, estimating
the reporting burden based on two methods being required annually. The
requirement to establish an analytical method may be fulfilled by any
interested person. We believe that the sponsor of the drug will be
willing to develop the method in most cases. Alternatively, FDA, the
sponsor, and perhaps a third party may cooperatively arrange for method
development. The respondents may be sponsors of new animal drugs,
State, or Federal and/or State Agencies, academia, or individuals.
    FDA estimates the burden of this collection of information as
follows:

                                 Table 1--Estimated Annual Reporting Burden \1\
----------------------------------------------------------------------------------------------------------------
                                                     Number of
         21 CFR section              Number of     responses per   Total annual   Average burden    Total hours
                                    respondents     respondent       responses     per response
----------------------------------------------------------------------------------------------------------------
530.22(b), Submission(s) of                    2               1               2           4,160           8,320
 Analytical Method..............
----------------------------------------------------------------------------------------------------------------
\1\ There are no capital costs or operating and maintenance costs associated with this collection of
  information.

    The burden for this information collection has not changed since
the last OMB approval.

    Dated: June 20, 2017.
Anna K. Abram,
Deputy Commissioner for Policy, Planning, Legislation, and Analysis.
[FR Doc. 2017-13296 Filed 6-23-17; 8:45 am]
BILLING CODE 4164-01-P


LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 1 Appendix A Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone A water system may perform a site specific study to generate a set of chlorine dioxide or ozone CT values for that site if it believes those developed by EPA do not reflect the true inactivation achieved. Such a study would involve measuring actual Cryptosporidium inactivation under site conditions, with a full range of temperature and contact times. If accepted by the State, the CT values may be used instead of those developed by EPA.

The LT2ESWTR does not specify any requirements for the chlorine dioxide or ozone sitespecific study, only that it be approved by the State ( 40 CFR 141.729( b)( 3) and ( c)( 3)). This appendix describes the different elements of a study and discusses some of the issues involved in the statistical analysis of the results.

A. 1 Experimental Design Experiments should be conducted with water that is representative of the water to be treated with respect to all conditions that can affect Cryptosporidium inactivation. Inactivation experiments should be performed with water exerting the highest oxidant demand ( i. e. spring run off or summer conditions) at high temperature to obtain the worst case scenario in terms of chlorine dioxide or ozone demand/ decay rate. In addition, experiments should also be conducted with water obtained during the winter months at the lowest temperatures observed at the treatment plant. These experiments would allow for the determination of the highest CTs that would be necessary to achieve the required level of inactivation. Additional experiments may be necessary to characterize the effects of other water quality parameters.

In order to obtain the most challenging water to assess the chlorine dioxide or ozone process, a predetermined testing schedule should be established based on source water TOC and UV 254 levels. Testing can occur when source water values for these parameters fall within defined worst case ranges. Experiments should then be performed in the laboratory at worst case temperatures for a given month.

In order to obtain a complete data set, testing should occur at least every other month over the course of an entire year. Each sample date should be determined by the first time the TOC or UV 254 levels are within 75 percent of the maximum historical value for that month. At the time of sampling, sufficient water should be acquired to allow for three sets of experiments to be conducted, with each experiment having six data points ( CT values) and a control. Two independent sets of experiments should be conducted with the water. Should significant discrepancies develop between the data sets, a third set of experiments would need to be conducted. An example experimental matrix is provided in Table A. 1. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 2 Table A. 1 Example Experimental Test Matrix Date Temperature to be Tested ( Historical Record) Water Quality Criteria Schedule of Experiments Test 1 Test 2 Test 3 February Lowest Annual TOC or UV 254 > 75% of max historical value X X If Required April Highest in April Same X X If Required June Highest in June Same X X If Required August Highest Annual Same X X If Required October Highest in October Same X X If Required December Lowest in December Same X X If Required A. 2 Experimental Procedure A. 2.1 Preparation of oocysts High oocyst quality is imperative to the success of the study because sub standard oocysts could dramatically affect the data in a way that would underestimate the CT required to achieve a desired level of inactivation. Traditionally, Cryptosporidium parvum oocysts are derived from two host sources, bovine and rodent. The most common strain of Cryptosporidium parvum used to date is the Iowa strain, developed by Dr. Harley Moon. It is recommended that the utility perform all experiments using fresh (< 1 month old) Iowa strain oocysts obtained from a reputable supplier. The utility should ensure that after purification the supplier stores the oocysts at 4

C in a solution of dichromate or 0.01 M phosphate buffer saline solution ( pH 7.4) containing two antibiotics ( 1,000 U/ mL penicillin, and 1,000 mg/ mL streptomycin), and an antimycotic ( 2.5 mg/ mL amphotericin B). The oocysts should be shipped in a cooler on ice to the utility via nextday service. Upon arrival, the oocysts should be placed in a refrigerator and stored at 4

C until needed.

When ready for use, the oocysts should be suspended in 0.01 M pH 7 buffer and centrifuged at a relative centrifugal force of approximately 1,100 for at least 10 minutes. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 3 Following centrifugation, the oocysts should be aspirated and re suspended in the buffer, then centrifuged again at the same conditions. This step should be repeated once more to remove as much of the antibiotic or dichromate solution as possible. Following the last aspiration, the oocysts should be re suspended in approximately 10 mL of the pH 7 buffer. The oocysts should then be stored at 4

C until the experiment is initiated. The oocysts should be vortexed thoroughly prior to initiation of the experiment. Additional details regarding this procedure can be found in Rennecker et al. 1999.

A. 2.2 Source Water Preservation Testing should be conducted as close as possible to the date that the experimental water is collected. If testing is to be performed at a location other than the utility where the water was collected, the water should be sent to the laboratory via an overnight delivery service and stored at 4 degrees Celsius until the start of testing.

A. 2.3 Experimental Apparatus A. 2.3.1 Chlorine Dioxide It is recommended that chlorine dioxide be generated using the equipment and procedures outlined in Standard Methods for the Examination of Water and Wastewater, APHA 1998. With this as a basis, all inactivation experiments using chlorine dioxide should be performed using a batch reactor configuration. An example of such a system is provided by Ruffell et al. 2000. This system uses an enclosed recirculating water bath to maintain the desired temperature inside the reactor vessels, which consist of 2 liter amber glass bottles. During the experiment, care should be taken to minimize the exposure of the reactors to light. Mixing of the reactor contents is provided with a magnetic stir bar and stir plate.

A. 2.3.2 Ozone Inactivation experiments can be performed with either a semi batch or batch reactor configuration. When performing experiments with a semi batch system, it is recommended that analytical components similar to those described by Hunt and Mariñas ( 1997) be used. Using this system, the reactor vessel containing the experimental water is maintained at the experimental temperature by immersion in a water bath. Ozone can be generated from either compressed air or oxygen and passed through a continuously stirred glass bottle, which serves to dampen the effect of fluctuating ozone concentration. The ozonated gas leaving the dampening bottle is then introduced to the experimental water via a fine bubble diffuser. The ozonated water is stirred continuously using a magnetic stirring plate and a stir bar. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 4 It is recommended that inactivation experiments performed using a batch reactor configuration use analytical components similar to those described by Kim ( 2002). This reactor used a 100 mL gas tight syringe to prevent ozone in solution from volatilizing into the atmosphere. The temperature inside the reactor is held constant by immersion in a recirculating water bath, and mixing is provided by a stir bar in the syringe controlled by a magnetic stir plate. Ozone can be produced from either compressed air or oxygen. A concentrated ozone stock solution should be prepared using distilled de ionized or reverse osmosis filtered water.

Other, less complex, batch reactor systems are also available which simply use an open vessel such as an Erlenmeyer flask or beaker ( Finch et al. 1993a). With these systems, the reactor containing the experimental water is typically maintained at the desired temperature using a water bath. An ozonated solution, prepared with distilled de ionized or reverse osmosis water, is added to the experimental water, and the ozone dose is measured from the diluted experimental water. When using this type of batch reactor configuration that is open to the atmosphere, the user should take into account that ozone is lost to volatilization. This loss of ozone should be considered and minimized when performing any inactivation or demand/ decay experiment.

A. 2.4 Inactivation experiments The CT values obtained from each of the site specific inactivation experiments are expected to be similar to those provided in the standard LT2ESWTR tables. Therefore, utilities wishing to determine site specific inactivation data are advised to use the standard tables as a baseline. Each experiment should be designed such that six data points span the range of the " standard" inactivation curve for a given temperature. One " control" point with no disinfectant should also be taken.

A. 2.4.1 Chlorine Dioxide An experimental protocol developed from Ruffell et al. 2000 is provided here as an example. The reactor bottle should be filled with experimental water to a total volume corresponding to the desired sample volume times the number of samples expected per bottle ( 6 is recommended). The bottle is then placed in the water bath and allowed to equilibrate to the target experimental temperature. At this point, chlorine dioxide stock solution is added to the reactor bottle at the target dose. The reactor bottle is then capped to minimize chlorine dioxide volatilization. The chlorine dioxide concentration is measured approximately 10 min after dosing. An experiment was started by adding approximately a pre determined number of oocysts to the reactor that will be sufficient for at least six data points. Note the volume of the oocyst aliquot should be less than 1 mL. Samples are then taken periodically at the contact times that correspond to the desired CT. The samples are immediately filtered through a1 µ m filter. The filter is then placed in a clean 50 mL beaker and rinsed with approximately 15 mL of the dilute surfactant. The resulting oocyst suspension is transferred into a sterile 15 mL centrifuge tube. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 5 These steps are repeated at various contact times corresponding to target CT parameters. After the last sample is taken, the chlorine dioxide dose is measured again. " Control" samples are also taken for each experiment by placing a sample of oocysts inside a similar reactor containing the experimental water minus the disinfectant at the target temperature. The oocysts are typically exposed to this condition for the duration of the experiment and subsequently processed for viability assessment with methods similar to those for the disinfected samples.

A. 2.4.2 Ozone If a semi batch reactor configuration is used, the protocol described by Rennecker et al. ( 1999) is recommended. The protocol is described briefly as follows. Ozonated gas is applied to the temperature acclimated experimental water via a fine bubble diffuser. The ozone gas concentration is adjusted to achieve steady state at dissolved ozone concentrations representative of what would be observed at the facility. The actual dissolved ozone concentration achieved for each experiment is measured. Mixing of the ozonated water is performed with a magnetic stir bar and stirring plate. An inactivation experiment is initiated by injecting a suspension containing a sufficient number of oocysts into the reactor, and ends by simultaneously removing the bubble diffuser and injecting a quenching agent. It should be noted that the number of oocysts necessary for each data point is dependent on the viability assessment method selected. Oocysts are then removed from the quenched solution by filtration through a 1 µ m filter. The reactor is then rinsed with approximately 50 mL of a dilute surfactant, and then again with approximately 100 mL of the experimental water to remove any residual surfactant. Both eluents are passed through the filter that is then placed in a clean 50 mL beaker and rinsed with approximately 15 mL of the dilute surfactant. The resulting oocyst suspension is transferred into a sterile 15 mL centrifuge tube. These steps are repeated at various contact times corresponding to target CT parameters ( i. e., the product of dissolved ozone concentration and contact time).

Control samples are prepared with each daily experimental set by shutting off the ozone generator, but allowing the oxygen gas to flow through the system. Oxygen gas is allowed to bypass the semi batch reactor after shutting off the generator to purge residual ozone gas from the system. All other conditions used for the control are consistent with the experimental conditions previously described. The " contact" time for control samples is 1 minute. After completion of the experiment, the samples are generally centrifuged at 1,100g for 10 minutes and stored in a phosphate buffer solution for a period of time not to exceed 48 hours prior to viability assessment procedures.

Experiments performed with a head space free reactor can follow the following protocol ( described previously in Kim 2002). The experimental temperature is maintained by immersing the 100 mL syringe, which serves as the reactor in a water bath. Mixing inside the reactor is provided using a stir bar and magnetic stir plate. The syringe is filled with the experimental water containing enough oocysts for all six data points. At this point, an aliquot of temperature Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 6 adjusted ozone stock solution of known concentration is added. Samples are then taken at time intervals corresponding to the pre determined estimated CT using a syringe containing a quenching reagent. The samples are then processed using filtration and centrifugation, similar to those described above. A " control" should be performed for each experiment by placing the sample number of oocysts in the experimental water at the desired temperature. The oocysts should remain there for a period of time equal to the duration of the inactivation experiment. After this time, the oocysts should be processed in a manner consistent with the disinfected samples.

Experiments performed with batch reactor components that are not head space free typically follow a similar, although less complex protocol. An example of such a system and the associated experimental protocol can be obtained from Finch et al. 1993a.

It should be noted that for all batch reactor systems, a careful characterization of the ozone demand and decay kinetics of the experimental water should be performed prior to any disinfection testing. In addition, it is also recommended that ozone concentration samples be procured alternately between inactivation samples to verify ozone concentrations observed during the disinfection study.

A. 2.5 Sample Processing After procuring each sample point, the samples should be stored at 4

C until the end of the experiment. At the end of each experiment, the samples should be centrifuged at a relative centrifugal force of 1,100 for at least 10 minutes to remove quenching agents or surfactants. Following centrifugation, the samples should be carefully aspirated and re suspended in 0.01 M pH 7 buffer solution. The samples should be stored at 4 degrees until the time of viability assessment.

A. 2.6 Viability Assessment Determining the viability of oocysts for varying levels of disinfection is one of the most critical components of the inactivation experiments. At present, there are three methods available to assess Cryptosporidium parvum viability, each presenting unique advantages and disadvantages. These methods include the following techniques:

° Animal infectivity ° Cell culture ( in vitro infectivity)

° In vitro excystation Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 7 The most established of these methods is animal infectivity. This viability assessment method typically involves inoculating immuno suppressed neonatal mice with varying numbers of oocysts exposed to a particular CT. After a certain " incubation" period, the mice are then sacrificed and their intestinal tracts are examined for signs of Cryptosporidium induced infection ( cryptosporidiosis). The primary benefit of this method is that it demonstrates that the treated oocysts are capable of reproduction inside a mammalian host and therefore are able to induce an infection. One criticism of this method is that although an infection is capable of being observed, mouse infectivity has not been correlated to human infectivity. In addition, the protocol associated with this method is difficult and expensive. It requires specialized laboratory training, facilities, and equipment. An example of this protocol can be found in Finch et al. 1993b.

A second method used to assess the viability of Cryptosporidium parvum is known as in vitro infectivity or cell culture. At present, cell culture methodologies used for this purpose are based on either microscopic evaluation ( Slifko et al. 1997) or polymerase chain reaction ( PCR) ( Rochelle et al. 1997). The first step in using cell culture to assess oocyst viability involves applying the treated oocysts to a lawn of cells ( typically derived from human or canine cell lines). After an incubation period, using microscopic evaluation based culture methods, the cells are stained with fluorescent chemicals and then examined microscopically for various cryptosporidium life stages. The presence of these life stages suggests that the oocysts were capable of reproduction and thus were viable and likely able to cause an infection in humans.

When using a PCR based technique, after incubation the cells are processed and the Cryptosporidium parvum RNA is extracted. Infectivity is then determined by targeting specific genetic sequences in the RNA. The primary advantage of using cell culture to assess Cryptosporidium parvum infectivity is that it can measure very low concentrations of oocysts. Therefore, cell culture is capable of demonstrating high levels of inactivation. In contrast, the disadvantages associated with using cell culture include a lack of agreement over the preferred cell lines and viability assessment technique. In addition, there has been no extrapolation between cell culture techniques and human infectivity. Lastly, cell culture techniques are complex and typically require specialized equipment and rigorous training, which makes this procedure somewhat expensive.

A third method known as in vitro excystation has also been developed to assess the viability of Cryptosporidium parvum ( Rennecker et al. 1999). This method involves exposing oocysts to a simulation of a mammalian digestive tract. Following the simulation, the oocysts are then examined microscopically for oocyst life stages that are indicative of viability. The advantages of this method are that it is cost effective, offers the ability to rapidly develop data, and requires minimal training. The main disadvantage of the method is that of the three methods described, in vitro excystation has the least similarity to an actual infection. However, it should be noted that in spite of this fact, two published studies have shown that inactivation data Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 8 obtained with in vitro excystation closely matches animal infectivity and/ or cell culture data ( Rennecker et al. 2000, Owens et al. 1999).

A. 3 Statistical Analysis A general approach for calculating a set of CT values involves the following steps:

1) Fitting an inactivation model( s) to the experimental inactivation data ( for the entire year).

2) Calculating the predicted average CT requirements from the best fit model.

3) Calculating and applying a factor of safety for the average predicted CT requirement.

One approach by Clark et al. ( 2002) used a one parameter Chick Watson model to fit experimental data sets and develop standard CT curves, relative to inactivation level and temperature. As described in the LT2ESWTR Preamble, EPA used the Clark et al. approach for developing CT values but adjusted the analysis to account for different types of uncertainties and variability inherent in the data. EPA wanted to account for variability among different water matrices and oocyst strains, but not variability within the same group ( i. e., same oocyst lot and water), and uncertainty in the regression. While such a complex approach may not be necessary for a site specific study, the water system should be aware of the uncertainties and variability of the experimental data and use a statistical method that builds in a reasonable safety factor to ensure public health is protected.

Two types of confidence bounds that are commonly used when assessing relationships between variables, such as disinfectant dose ( CT) and log inactivation, are confidence in the regression and confidence in the prediction. Confidence in the regression accounts for uncertainty in the regression line ( e. g., a linear relationship between temperature and the log of the ratio of CT to log inactivation). Confidence in the prediction accounts for both uncertainty in the regression line and variability in experimental observations   it describes the likelihood of a single future data point falling within a range. Bounds for confidence in prediction are wider ( i. e., more conservative) than those for confidence in the regression. Depending on the degree of confidence applied, most points in a data set typically will fall within the bounds for confidence in the prediction, while a significant fraction will fall outside the bounds for confidence in the regression. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 9 References American Public Health Association, American Water Works Association, and Water Environment Federation. 1998. Standard Methods for the Examination of Water and Wastewater. Washington D. C.

Clark, R. M.; Sivagenesan, M., Rice; E. W.; and Chen, J. ( 2002). Development of a Ct equation for the inactivation of Cryptosporidium oocysts with ozone. Wat. Res. 36, 3141 3149.

Finch, G. R.; Black E. K.; Gyurek, L.; and Belosevic, M. ( 1993a). Ozone inactivation of C. parvum in demand free phosphate buffer determined by in vitro excystation and animal infectivity. J. Appl. Environ. Microbiol. 59( 12), 4203 4210.

Finch, G. R.; Daniels, C. W.; Black, E. K.; Schaefer III, F. W.; and Belosevic, M. .( 1993b). Dose response of C. parvum in outbred neonatal CD 1 mice. J. Appl. Environ. Microbiol. 59( 11), 3661 3665.

Hunt, N. K.; and Mariñas, B. J. ( 1997) Kinetics of Escherichia coli inactivation with ozone. Wat. Res. 31( 6), 1355 1362.

Kim, J. H.; Tomiak, R. B.; Rennecker, J. L.; Mariñas, B. J.; Miltner, R. J.; and Owens, J. H. ( 2002). " Inactivation of Cryptosporidium in a Pilot Scale Ozone Bubble Diffuser Contactor. Part I I: Model Verification and Application." ASCE Journal of Environmental Engineering, 128( 6), 522 532.

Li, H.; Finch, G. R.; Smith, D. W.; and Belosevic, M. ( 2000). Chemical inactivation of Cryptosporidium in water treatment. AWWA Research Foundation, Denver, CO.

Owens, J. H.; Miltner, R. J.; Slifko, T. R.; and Rose J. B. ( 1999). In vitro excystation and infectivity in mice and cell culture to assess chlorine dioxide inactivation of Cryptosporidium oocysts. Proceedings of the AWWA WQTC Conference, Tampa.

Rennecker, J. L.; Mariñas B. J.; Owens J. H.; and Rice E. W. ( 1999) Inactivation of C. parvum oocysts with Ozone. Water Res. 33 ( 11), 2481  2488.

Rochelle, P. A.; Ferguson, D. M.; Handojo, T. J.; De Leon, R.; Stewart, M. H.; and Wolfe, R. L. ( 1997). An assay combing cell culture with reverse transcriptase PCR to detect and determine the infectivity of waterborne C. parvum. J. Appl. Environ. Microbiol. 63( 5) 2029  2037.

Ruffell, K. M; Rennecker, J. L.; and Mariñas, B. J.( 2000). Inactivation of C. Parvum oocysts with chlorine dioxide. Wat. Res. 34 ( 3), 868  876. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 10 Slifko, T. R.; Friedman, D.; Rose, J. B.; and Jakubowski, W. ( 1997). An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture. J. Appl. Environ. Microbiol. 63( 9) 3669  3675. Appendix A  Site Specific Determination of Contact Time for Chlorine Dioxide and Ozone LT2ESWTR Toolbox Guidance Manual Proposal Draft June 2003 A 11 A. 1 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 1 A. 2 Experimental Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 2 A. 2.1 Preparation of oocysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 2 A. 2.2 Source Water Preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 3 A. 2.3 Experimental Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 3 A. 2.3.1 Chlorine Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 3 A. 2.3.2 Ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 3 A. 2.4 Inactivation experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 4 A. 2.4.1 Chlorine Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 4 A. 2.4.2 Ozone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 5 A. 2.5 Sample Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 6 A. 2.6 Viability Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 6 A. 3 Statistical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 8 Table A. 1 Example Experimental Test Matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A 2