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Product testing is an integral part of ensuring control of the manufacturing process and lot-to-lot
consistency.  Therefore, it is important to identify critical parameters in the manufacturing
process and critical product attributes to ensure the desired clinical effect of the final product.
Refer to the “FDA Guidance Concerning Demonstration of Comparability of Human Biological
Products, Including Therapeutic Biotechnology-Derived Products” (Ref. 10) for additional
information.
Note to FDA Reviewers:  Document the testing performed.  Assess the appropriateness of that
testing and the acceptance criteria, based on any results previously obtained by the sponsor.
A.
Microbiological Testing
We recommend that you perform microbiological testing on cell banks, in-process
intermediates, and the final product, as appropriate.
1.
Sterility Testing (Bacterial and Fungal Testing)
Current practices for sterility testing.
a. Test Method
Suitable sterility tests include the test described in 21 CFR 610.12 and the test
described in United States Pharmacopoeia (USP) <71> Sterility Tests (23
rd
edition, 1995), (Ref. 11).  Later versions of the USP<71> are also suitable.  If you
are using another test method, you should describe its suitability.  Note that under
21 CFR 610.9, prior to product licensing, the alternative method must be shown
to provide assurances of the safety, purity, potency and effectiveness of the
biological product equal to or greater that the assurances provided by the method
or process specified in the 21 CFR 610.12 method.
If you use antibiotics in manufacturing, you should provide documentation that
the antibiotics were removed prior to sterility testing.  If the antibiotics cannot be
removed from the final product, we recommend that you assess the validity of the
sterility assay using the bacteriostasis and fungistasis testing as described in USP
<71> Sterility Tests.  Use of this assay is designed to ensure that any residual
antibiotic present in the product does not interfere with the results of sterility
testing.
Note to FDA Reviewers:  If the Sponsor proposes to use an alternative method in
place of the traditional sterility methods described in 21 CFR 610.12, then you
will need to assess the adequacy of this alternative test method and the data
provided to provide assurances of the safety, purity, potency and effectiveness of
the biological product equal to or greater than the assurances provided by the
method or process specified in the general standard (21 CFR 610.9).  The
completeness of the validation study for the alternative method used during the
17

Contains Nonbinding Recommendations
product development should reflect an assessment of risk associated with the
application of the method (e.g., stage of the manufacturing process).
b. Test Timing
Sponsors frequently perform in-process sterility testing at critical points during
manufacturing, such as during purification, or after ex vivo gene modification or
extended culture periods.  You should identify when in-process sterility testing is
performed during manufacturing and the test method used.  The test method that
you choose for in-process sterility testing should be adequate to provide assurance
of product sterility.
If you freeze the final product before its use, we recommend that you perform
testing on the product prior to cryopreservation, so that results will be available
before the product is administered to a patient.  However, if the product
undergoes manipulation (e.g., washing, culturing) after thawing, particularly if
procedures are performed in an open system, you may need to repeat sterility
testing.  You should incorporate the results of in-process sterility testing into your
acceptance criteria for final product specifications.
If your product has a short dating period and must be administered to patients
before sterility test results of the final product are available, then you will need to
develop an alternate approach to provide sterility assurance.  As an alternative
approach, we recommend that you perform all of the following tests:
x
in-process sterility testing on a sample taken 48 to 72 hours prior to final
harvest or after the last re-feeding of the cultures
x
a rapid microbial detection test such as a Gram stain or other procedure on
the final formulated product
x
sterility testing compliant with 21 CFR 610.12 on the final formulated
product
Under this alternative approach the release criteria for sterility would be based on
a negative result of the Gram stain and a no-growth result from the 48 to 72 hour
in-process sterility test.  Although in this situation the results of the sterility
culture performed on the final product will not be available for product release,
this testing will provide useful data.  A no-growth result will provide assurance
that an aseptic technique was maintained.  A positive result will provide
information for the medical management of the subject, and trigger an
investigation of the cause of the sterility failure.  The sterility culture on the final
formulated product and when possible the in-process culture should be continued
to obtain the full 14 day sterility test result even after the product has been given
to the patient.
In all cases where product release is prior to obtaining results from a full 14 day
sterility test, the investigational plan should address the actions to be taken in the
18

Contains Nonbinding Recommendations
event that the 14 day sterility test is determined to be positive after the product is
administered to a subject.  You should report the sterility failure, results of
investigation of cause and any corrective actions, in an information amendment
submitted to your IND in a timely manner, within 30 calendar days after initial
receipt of the positive culture test result (21 CFR 312.31).
The investigator should evaluate the subject for any signs of infection that may be
attributable to the product sterility failure.  If the patient experiences any serious
and unexpected adverse drug experience that could be from administration of the
sterility failure of the gene therapy product, then you must report this information
to FDA in an IND safety report no more than 15 calendar days after your initial
receipt of the information (21 CFR 312.32).  If you determine that an
investigational drug presents an unreasonable and significant risk to subjects, you
must discontinue those investigations that present the risk, notify FDA, all
Institutional Review Boards, and all investigators (21 CFR 312.56(d)).
2. Mycoplasma
There are several potential sources of mycoplasma contamination.  Two major
sources include animal serum products used in culture and the culture facility
environment, particularly with open culture systems.  We recommend that you
perform mycoplasma testing on the product at the manufacturing stage when the test
is most likely to detect contamination, such as after pooling of cultures for harvest but
prior to cell washing.  Testing should be conducted on both cells and supernatant.
We recommend that you inform FDA whether there is in-process testing for
mycoplasma during extended culture procedures.  Due to the limited dating period of
many ex vivo genetically modified cellular products, it is frequently not feasible for a
sponsor to perform the recommended culture-based assay (see Ref. 4) for release
testing.  In those cases, we recommend the use of polymerase chain reaction (PCR)-
based mycoplasma assays or another rapid detection assay during product
development.  As part of your BLA, you should submit appropriate data to
demonstrate that the PCR or alternative test has adequate sensitivity and specificity.
3.
Adventitious Agent Testing
As appropriate, you should perform and describe in your IND adventitious agent
testing as set out below.  For more information on adventitious agent testing, refer to
“Points to Consider in the Characterization of Cell Lines Used to Produce
Biologicals” and ICH guidance Q5A:  “Guidance on Viral Safety Evaluation of
Biotechnology Products Derived From Cell Lines of Human or Animal Origin” (Refs.
4 and 12).
a.
In Vitro Viral Testing
When cell lines are used, you should describe the cell lines and perform in vitro
viral testing.  In vitro viral testing should be performed on the MCB, WCB, MVB,
19

Contains Nonbinding Recommendations
WVB, and final vector product, and as a one time test on the EOP.  Testing should
be conducted by inoculating the test sample (MCB, WCB, MVB, WVB, final vector
product) onto various susceptible indicator cell lines such as the human cell line
MRC-5 or Vero cells which are primate in origin.  The choice of cells used would
depend on the species of origin of the product to be tested.  An appropriate test
should include monolayer cultures of the same species and tissue as that used for
production of the product, as well as a human and a non-human primate cell line
susceptible to human viruses.  In addition, the test would include a measure of both
cytopathic and hemadsorbing viruses.
In the event that the product is a cytolytic virus, it will need to be neutralized using
specific antibodies prior to in vitro adventitious testing of the MVB, WVB and final
product.  If neutralizing antibodies are not available, then only in vivo adventitious
agent testing needs to be performed on the MVB, WVB and final product until
appropriate neutralizing antibodies are developed.
b.
In Vivo Viral Testing
When cell lines are used, we recommend that you perform and submit data on in
vivo viral assays carried out by inoculating the test sample (MCB, MVB) into
animals such as adult and suckling mice and embryonated hen eggs.  You should
consider whether to include additional testing of guinea pigs, rabbits, or monkeys.
Such studies would assess the test animals for any indication of illness.  If such
additional testing is appropriate, you should describe and explain the suitability of
the animals used.
c.
Selected Testing for Adventitious Viruses
We recommend that you test your MCB and MVB for appropriate, species-specific
viruses.  You should describe the testing that is performed, the different stages of
manufacturing where those tests are performed (e.g., cell banks, viral banks, final
product), and the test methods used.
1)
Species-Specific Viruses
You should describe all species-specific virus testing performed on the MCB
and MVB.  We believe that all rodent cell lines used during product
manufacturing should be tested for rodent specific viruses.  These viruses are
usually detected by antibody production tests, murine antibody production
(MAP), rat antibody production (RAP), or hamster antibody production (HAP).
If human cell lines are used in the therapeutic product, we recommend that you
perform testing for human pathogens (CMV, HIV-1 & 2, HTLV-1 & 2, EBV,
HBV, HCV, and B19) and other human viral agents, as appropriate. We
recommend such testing because these cells are manipulated (cultured for
extended time periods) and human pathogens can be introduced or propagated
20

Contains Nonbinding Recommendations
during the extended culture periods.  Human viral agents may be tested using a
PCR-based test system.
When the gene therapy product is produced in a human cell line, e.g., an
adenoviral vector produced in human 293 cells, you should test for the presence
of additional human viruses such as adenovirus and adeno-associated virus
(AAV), and describe those tests in your IND.
2) Testing for Retroviruses
When the MCB and MVB are used for production of vectors other than
retroviral vectors, you should test the MCB and MVB for retroviral
contamination using Reverse Transcriptase (RT) assays and electron
microscopic analysis, and include a description of those tests in your IND.
We recommend that you perform testing for replication competent retrovirus
(RCR) in the production of retroviral vectors at multiple points in production,
including MVB, WVB, vector supernatants, end of production cells, and ex vivo
modified cells.  For further information on retroviral testing refer to the
“Guidance for Industry:  Supplemental Guidance on Testing for Replication
Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and
During Follow-up of Patients in Clinical Trials Using Retroviral Vectors” (Ref.
13).
For cells that produce vectors containing amphotropic murine leukemia virus
envelope, we recommend that you test for RCR using a permissive cell line such
as Mus dunni and describe that testing in the IND.  If an ecotropic packaging
cell line is utilized during retroviral vector production, you should conduct and
describe an ecotropic retroviral assay for the detection of low-level viral
contamination in the MCB.  Murine ecotropic viral contamination can be
detected using either XC or D56 plaque assay methods.
You should describe how vector supernatant is tested.  An appropriate test of
vector supernatant lots would be by amplification on a permissive cell line such
as Mus dunni, followed by detection in an appropriate indicator cell assay such
as PG-4 S+L-.  An appropriate test of the pooled End of Production (EOP) cells
would be by co-culture with a permissive cell line such as Mus dunni for
amphotropic virus, and the amplified material assayed in an appropriate
indicator cell assay.  We have determined that detection of RCR in ex vivo
modified cells requires cells to be cultured for at least 4 days post transduction.
Therefore, ex vivo gene modified cells that are cultured for
t
4 days should use
RCR testing as a release assay.  If ex vivo gene modified cells are cultured for <
4 days, archiving cells would be appropriate in place of active RCR testing.  If it
is not possible to have results from the RCR assay prior to treatment, we
recommend that you initiate the culture assay and perform an alternative method
(such as PCR) for product release.
21

Contains Nonbinding Recommendations
3) Adenoviruses
For studies using adenoviral vectors, we recommend that you conduct tests for
replication competent adenovirus (RCA) on MVB as well as the final vector.
We believe that an appropriate maximum level of RCA contamination would be
< 1 in 3 x 10
10
viral particles, and that the adenoviral particle to infectious unit
(iu) ratio would be
d
30 to 1.
4)
Adeno-Associated Virus (AAV)
For studies using AAV, we recommend that you conduct tests to determine
the amount of replication competent AAV present in the final vector product
and report the results in your IND.
B.
Identity
We recommend that you verify the identity of the MCB/MVB, WCB/WVB, and the final
product by assays that will identify the product and distinguish it from other products
being processed in the same facility.  For the final product, identity testing is important to
ensure that the contents of the vial are labeled appropriately.  For additional information
on labeling, refer to Section VII.B below.
If the final product is an ex vivo genetically modified cell product, we recommend that you
test the final cell product for identity.  Testing should include an assay to measure the
presence of vector (i.e., expression assay, restriction digest) and an assay specific for the
cellular component of the final product (i.e., cell surface markers).
C.
Purity
Product purity is defined as relative freedom from extraneous material in the finished
product, whether or not harmful to the recipient or deleterious to the product (21 CFR
610.3(r)).  Purity testing includes assays for pyrogenicity/endotoxin (see below), residual
proteins or reagents/components used during vector manufacture, such as cytokines, growth
factors, antibodies, and serum, and in the case of ex vivo modified cells any unintended
cellular populations or cell debris.
1.
Residual Contaminants
The appropriate purity testing should include assays for residual proteins, DNA,
RNA, and solvents used during production and purification, and reagents used
during manufacture such as cytokines, growth factors, antibodies, and serum. If the
product is a genetically modified ex vivo cell therapy product, appropriate purity
testing should include a measurement of contaminating cell types or cell debris.  For
22

Contains Nonbinding Recommendations
further information, refer to ICH Q3 on “Impurities” (Ref. 14).  You should describe
in your IND the purity testing you conduct and your specifications for release.
2. Pyrogenicity/Endotoxin
The rabbit pyrogen test method is the currently required method for testing
biological products for pyrogenic substances (21 CFR 610.13).  Although the
pyrogenicity test is required, there may be specific cases where this test method can
not be performed for release due to properties of the final product (i.e., short product
shelf life, toxicity of product in rabbits).  Under these circumstances, a test method
such as the Limulus Amebocyte Lysate test method (LAL) may be used as an
alternative method, but prior to licensure must be shown to provide equal or greater
assurances of safety, purity, and potency (see 21 CFR 610.9).  The 1987 FDA
Guideline on Validation of the Limulus Amebocyte Lysate (LAL) Test as End-
Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological
Products, and Medical Devices (Ref. 15) sets forth acceptable conditions for use of
LAL.
For any parenteral drug, except those administered intrathecally, we recommend that
the upper limit of acceptance criterion for endotoxin be 5 EU/kg body weight/hour.
For intrathecally-administered drugs, we recommend an upper limit of acceptance
criterion of 0.2 EU/kg body weight/hour.  You should describe in your IND the
pyrogenicity/endotoxin testing you conduct, and your acceptance criterion for
release.
Note to FDA Reviewers:  Document in your review the specification for
pyrogenicity/endotoxin testing and verify that testing is on the final product and
that results are available prior to lot release.
D.
Potency
You should describe and justify all assays you will use to measure potency.  We
recommend that these assays be quantitative, but in addition, they may include a
qualitative biological assay.  For a Phase 1/Phase 2 study, we recommend that the assay
quantify the expression of a gene therapy vector product.  For a Phase 3 study, we
recommend that the potency assay consist of in vivo or in vitro tests that measure an
appropriate biological activity.  If development of a quantitative biological assay is not
possible, then a quantitative physical assay which correlates with and is used in
conjunction with a qualitative biological assay can be used.  Note that potency assays are
part of lot release and should be validated prior to licensure.
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Contains Nonbinding Recommendations
E.
Other
1.
General Safety Testing
Testing for general safety is required for licensure of all gene therapy vector products,
unless the product is exempt under 21 CFR 610.11(g).  General safety testing is
performed on biological products intended for administration to humans and specific
tests are described in 21 CFR 610.11.  You should inform FDA whether general safety
testing is being performed during product development.
Note to FDA Reviewers:  Advise the sponsor regarding the applicability of the general
safety test for the product under review.  (Revisions to the General Safety Requirements
for Biological Products (68 FR 10157; March 4, 2003) (Ref. 16)).
2. Viability
You should establish minimum release criteria for viability.  For genetically
modified cellular therapies, the minimum acceptable viability specification is
generally set at 70 percent.  If this level cannot be achieved, we recommend that you
submit data in support of a lower viability specification, demonstrating, for example,
that dead cells and cell debris do not affect the safe administration of the product
and/or the therapeutic effect.
3. Cell Number/Dose
If your final product is a genetically modified cell therapy, you should develop
specifications for the minimum number of viable and functional cells as part of
product testing and release.  We recommend that you inform FDA whether a
maximum number/dose of cells to be administered has been established, and the
basis for that level.  For administration of a gene vector, you should describe your
dose as the concentration of plasmid DNA, viral particle number, or titer.
V.
FINAL PRODUCT RELEASE CRITERIA TESTING
The final product is the final formulated product used for administration to human subjects.
Final product release criteria testing should be performed on each lot of product manufactured.
In some situations, each dose could be considered a single lot, depending on the manufacturing
process.  The results from final product release criteria testing should be available prior to
administration to a human subject.  If results from final product testing will not be available prior
to release, we recommend that you clearly indicate this in the IND, together with your
specifications, and include a description of the reporting notification process if the acceptance
criteria are not met.  We recommend that you provide, in table format, all of your proposed
specifications (tests for safety, purity, potency, and identity as described in Section IV, test
methods, and acceptance criteria), including test sensitivity and specificity, where appropriate,
24

Contains Nonbinding Recommendations
for the final product.  (Note that, before the product may be licensed, these parameters must be
validated (21 CFR 211.165(e)).
VI.
PRODUCT STABILITY
Stability testing must be performed during early phases of the clinical trial to establish that the
product is sufficiently stable for the time period required by the study (21 CFR 312.23(a)(7)(ii)).
Data supporting a final formulation and dating period will be necessary for licensure.  You
should describe the stability measures you will use to support your studies.  For further
information, refer to ICH Q5C:  “Quality of Biotechnological Products:  Stability Testing of
Biotechnological/Biological Products,” (Ref. 17), ICH Guideline Q1A(R):  “Stability Testing of
New Drugs and Products” (revised guideline) (Ref. 18), ICH Guideline Q1E:  “Evaluation of
Stability Data” (Ref. 19).
Note to FDA Reviewers:  Assess the product development plan in the IND review to determine
how much stability data are needed for the current phase of investigation and whether sufficient
data are included in the submission.  If submitted, include preliminary data in your review.
Assess whether proposed expiration dating is appropriate.
Stability Testing
Under 21 CFR 312.23(a)(7)(ii), you must conduct stability testing in all phases of the
IND, to demonstrate that the product is within acceptable chemical and physical limits
for the planned duration of the proposed clinical investigation.  If a very short term
clinical investigation is proposed, the stability data submitted may be correspondingly
limited.  You should submit a stability protocol and data for both in-process material and
the final gene product.  A proposed stability protocol should include a measure of
product sterility, identity, purity, quality, and potency.  For each test conducted, you
should describe the test method, sampling time points (there should be a zero-time point),
testing temperature, and other appropriate information, including your justification of the
assays used to indicate product stability, measuring those parameters for the duration of
storage required by the clinical protocol.  We recommend sterility testing be performed at
zero-time, end of stability study, and at an intermediate point during the study.
Note to FDA Reviewers:  If the sponsor plans to use the product past the duration of the
clinical trial (e.g., for a separate trial being conducted after the initial trial), verify that
testing establishes stability throughout the relevant time period.
1)  In-Process Stability Testing
You should describe the stability protocol that will be used to ensure that the final
product is stable during the period of cryopreservation, measuring the parameters
described in Section VI.A, as appropriate.  A comparison is often made of
analyses carried out pre-freeze and post-thaw.  You should describe any stability
25

Contains Nonbinding Recommendations
testing performed on the product during the holding steps, such as
cryopreservation of final product and storage of bulk product.
2)  Final Product Stability Testing
You should include any data that demonstrate that the product is stable between
the time of product formulation and infusion to subjects to aid in establishing an
expiration-dating period.  We recommend that you conduct the testing at the
appropriate temperatures and at time points consistent with predicted storage
times.  If the product is shipped from the manufacturing site to the clinical site,
describe the time and shipping conditions (e.g., packaging, temperature).  Your
stability protocol should be adequate to demonstrate that product integrity,
sterility, and potency are maintained under the proposed shipping conditions.  We
further recommend that validation studies using conditions that stress the system
be initiated by Phase 3 and completed prior to submission of a BLA.
VII.
OTHER ISSUES
A.
Product Tracking
For all products, you should establish a product tracking and segregation system.  An
adequate system should allow identification of the therapeutic product from collection to
administration of the product and should include procedures to ensure that the product is
segregated from other products in incubators, hoods, and cryopreservation units.  You
must establish and maintain a system of tracking that enables the tracking of all products
from the donor to the consignee or final disposition, and from consignee or final
disposition to donor (21 CFR 1271.290(b)).
B.
Labeling
You should describe the product labeling used throughout the manufacturing process and
on the final product container.  As described in 21 CFR 312.6(a), the label for an
investigational product must contain the following statement:  “Caution:  New Drug –
Limited by Federal law to Investigational Use.”  To minimize the potential for mix-ups
for products manufactured in multi-use facilities and for patient-specific products, we
recommend that the product label contain the date of product manufacture, storage
conditions, expiration date and time (if appropriate), product name, and two non-personal
patient identifiers.  As HCT/Ps, labeling for ex vivo genetically modified cell therapy
products must meet the requirements in 21 CFR 1271.250.  For autologous donors and
other situations described in 21 CFR 1271.90(a) for which a donor eligibility
determination is not required, you must include the applicable required labeling in 21
CFR 1271.90(b).  For example, for autologous cells intended for autologous use you
must label the product “FOR AUTOLOGOUS USE ONLY” (21 CFR 1271.90(b)(1)),
and “NOT EVALUATED FOR INFECTIOUS SUBSTANCES” if donor testing and
26

Contains Nonbinding Recommendations
screening is not performed (21 CFR 1271.90(b)(2)).  For more information, refer to Ref.
3.
C.
Container/Closure
You should describe the types of container and closure used, and that you have
determined that the containers and closures are compatible with the product.  For more
information, refer to “Guidance for Industry:  Container Closure Systems for Packaging
Human Drugs and Biologics; Chemistry, Manufacturing, and Controls Documentation”


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Pesticides

Regulating Pesticides

Restricted & Canceled Uses

Acetochlor

Immunoassay Methods of Acetochlor Detection

Immunoassay Methods of Acetochlor Detection

Review of Existing Immunoassay Kits for Screening of Acetochlor and Other  Acetanilides in Water

Acetochlor Registration Partnership

c/o ZENECA Ag Products

1800 Concord Pike

Wilmington, Delaware 19897

March 1995

TABLE OF CONTENTS

SUMMARY

INTRODUCTION

FACTORS USED IN COMPARISON OF IMMUNOASSAYS

Dose-Response Curves

Analytes of Interest

Cross-Reactants

Threshold Screening Levels

COMPARISON OF COMMERCIALLY AVAILABLE IMMUNOASSAYS FOR ACETANILIDES

Metolachlor (Ohmicron)

Metolachlor 1.0(Idetek/Quantix)

Alachlor 1.0 (Idetek/Quantix)

Alachlor (Millipore)

Alachlor (Ohmicron)

Acetanilide (Millipore)

Summary

EVALUATION OF ENVIROGARDTM ACETANILIDE PLATE KIT

Background

Principles of the Assay

Reproducibility of the Standard Curve

Cross-Reactivity

Accuracy and Precision

ENVIRONMENTAL HEALTH LABORATORIES (EHL) ACETANILIDE ASSAY

Background

Principles of the Assay

Reproducibility of Standard Curve

Cross-Reactivity

Accuracy and Precision

PERFORMANCE TESTING OF ACETANILIDE IMMUNOASSAYS

Purpose

Experimental Design

Results and Discussion

CONCLUSIONS

TABLES

Tables

REFERENCES

Top of page

SUMMARY

As part of the conditional registration of acetochlor in March 1994, the Acetochlor Registration Partnership (ARP) agreed to provide immunoassay methods for cost effective  state monitoring of ground and surface water samples for the presence of acetanilide  herbicides.  This report is submitted to the Environmental Protection Agency in  fulfillment of that agreement.

At the time of registration of acetochlor, only specific acetanilide assays, directed toward the detection of a single analyte had been reported.  Whilst engaged in an assessment of the potential utility of these specific immunoassays, the ARP became aware  of a class-specific assay in pre-commercial development by Millipore Corporation.  This  acetanilide immunoassay will be available in a microtiter plate format for use in  experienced laboratories with appropriate equipment and an automated format available as  an analytical service from the Environmental Health Laboratories (EHL) in South Bend,  Indiana.

A summary of the performance and characteristics of the existing analyte specific assays has been compiled and included within this report.  Also experimental data are presented for an ARP sponsored independent evaluation of the Millipore assay at the Water  Quality Laboratory (WQL) of Heidelberg College in Tiffin, Ohio and the results of the  analysis of blind fortified surface and ground water samples at the both the WQL and the  EHL.

Five commercially available analyte specific acetanilide immunoassays were evaluated for cross-reactivity with acetochlor.  Two of the assays were designed for metolachlor and three were directed toward alachlor.  Three of the assays were reported to have some  cross-reactivity towards acetochlor but the sensitivities as measured by I50 (concentration of analyte resulting in a 50% inhibition of control response) were from 6.55 to 70 ppb acetochlor.   Based on the typical characteristics of immunoassay
dose-response curves, these levels of sensitivity were considered inadequate to provide sufficient precision of measurement at lower concentrations of interest.

The pre-commercial acetanilide assay of Millipore was reported to have I50's for metolachlor, acetochlor and alachlor of 0.26 ppb, 1.7 ppb and 4.9 ppb, respectively, suggesting that it offers the best prospects for an acetanilide screen including acetochlor.  The independent WQL evaluation of this kit confirmed the sensitivity of the  assay for the reported acetanilides as well as demonstrating that dimethenamid is also  detected with an I50 of 0.5 ppb.  Atrazine and the common soil metabolites of alachlor  and acetochlor were not detected by the assay at concentrations up to 500 ppb.   Calibration of the assay using acetochlor calibrators provided by Millipore was reproducible and an acceptable level of accuracy for acetochlor in reagent water was  demonstrated at 0.1 and 1.0 ppb. The within-assay and between-assay precision varied with  the level of measurement but were typical of this type of immunoassay (% CV 8.3 to  35.2%).  The automated assay available from the EHL gave similar results except with  slightly better precision as would be anticipated for instrumented operation.

The analysis of blind surface and ground water samples fortified with acetochlor at 0.1 or 1.0 ppb provided a direct comparison of the manual operation of the acetanilide  assay at WQL with the automated version available at the EHL using samples with a typical  matrix composition.  In addition, since these samples were analyzed before and after  fortification by GC/MS for corn herbicides, it was possible to establish potential  interferences due to the presence of other acetanilides.  The results of the blind  analyses by the WQL and the EHL were in general agreement but differed significantly from  the GC/MS in their estimation of acetochlor level for the unfortified and the 0.1 ppb  fortifications.  These discrepancies were shown to be due to the presence of an average  of 0.091 ppb metolachlor in the surface water samples.  Improved agreement among all  assays was obtained for the 1.0 ppb fortifications.

The results described in this report indicate that the acetanilide immunoassay in
pre-commercial development by Millipore when used as a plate kit or in an automated format has sufficient accuracy and precision to serve as a screen for acetochlor in water  at 1.0 ppb.   Use of the assay as a screen at 0.1 ppb was considered to be unadvisable  due to low precision at this level and the fact that the lowest calibrator provided in  the kit was 0.1 ppb acetochlor.  The assay was shown to have improved precision at 0.2  ppb and to be within the linear range of the standard curve at this level.  These  observations suggest that 0.2 ppb would be a reasonable low level threshold for  application of the assay.

The acetanilide immunoassay of Millipore is not spectific and does not differentiate among alachlor, acetochlor, metolachlor and dimethenamid but rather detects their presence in varying degrees.  Therefore, it is imperative that any positive detects obtained using this immunoassay as a plate kit or in an automated format be confirmed  with another analytical method, such as GC/MS, to establish the presence and level of  acetochlor in water.

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INTRODUCTION

As part of the conditional registration of acetochlor in March 1994, the EPA requested  that the Acetochlor Registration Partnership provide immunoassay methods for cost  effective state monitoring of ground and surface water samples for the presence of  acetanilide herbicides.

The ARP has adopted a structured approach towards fulfilling this requirement. In the first instance, this has involved discussions with commercial immunoassay kit manufacturers, in order to gain a clear understanding of the potential issues associated with the development, validation, and use of appropriate methods. Subsequent discussions  with possible end-users have served to define additional criteria for the acceptability  of such methods in routine use.

On the basis of these initial findings, the ARP has reviewed the potential  applicability of currently available immunoassays against the criteria listed below:

Only commercially available immunoassays are suitable for the proposed  	application.  This reflects the need to ensure a guaranteed, quality-assured supply  	to the end-user, with an appropriate level of after-sales support.

The chosen assay must be sufficiently rugged to operate reliably in the 	hands of end-users who may not have extensive experience in the use of this 	technology.

The assay must offer the prospect of a rapid, high-throughput screen for  	samples prior to confirmatory analysis of positive detects.

The assay should perform with a suitably high degree of reliability at the  	requisite levels.  The incidence of "false negatives" (samples  	incorrectly identified as not containing acetanilides above a specified threshold)  	must be suitably low.  The permissible incidence of "false positives" may  	be relatively higher, but should not be so high that the potential benefits are  	negated by unwarranted repeat analysis.

The assay should be able to detect those acetanilides currently used as corn  	herbicides, namely alachlor, acetochlor, dimethenamid and metolachlor.

The possible inadvertent detection of interfering compounds
	("cross-reactants") must be defined. The incidence of such detects must  	not prevent the application of the assay for detection of the specified analytes.

At the time of registration of acetochlor, only specific acetanilide assays, directed towards the detection of a single analyte, had been reported:  this encompassed both commercially available assays and those developed by agrochemical companies1,2.  Consequently, the ARP concentrated attention on commercially available assays (see Section IV) designed for specific acetanilides; the reported levels of cross-reactivity for related compounds in these assays were used to provide an initial assessment of their  potential utility for class-specific determination of acetanilides.  Additionally, whilst  engaged in this process, the ARP became aware of a potential class-specific assay in
pre-commercial development.  The basic performance characteristics of all these assays  were subsequently compared as a means of prioritizing them for further evaluation.

The factors taken into account in comparing the assays are discussed in Section III.  The assays are described and compared in Section IV.

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FACTORS USED IN COMPARISON OF IMMUNOASSAYS

Dose-Response Curves

Immunoassay dose-response curves are usually defined in terms of the inhibition  	of binding of a labelled ligand to an antibody as a function of concentration of  	the analyte in a test sample.  The concentration of the analyte is related to the  	absorbance (color) produced by the bound, labelled ligand.  As the concentration of  	the test analyte increases, the color produced by the labelled ligand decreases.   	The resulting absorbance versus log [concentration] curves are sigmoidal and  	express the inverse relationship between concentration of the test analyte and  	intensity of color.

Two pieces of information are often quoted to define these curves:  I50 and I10  	or I20.  The I50 concentration defines the limiting analyte concentration producing  	50% inhibition and is the mid-point of the dose-response curve.  The I10 or I20 	concentration defines the limiting analyte concentration producing a 10% or 20% 	inhibition of color formation, respectively.  A sigmoidal dose-response curve is 	necessarily steepest at the I50 and shallowest at its upper and lower (I10)  	extremes.  Consequently, the relative precision is higher at the I50 and lower at  	concentrations represented by the lower inhibition values3.

Analytes of Interest

The analytes of interest are acetochlor, alachlor, metolachlor and dimethenamid.   	A suitable class-specific assay must be capable of identifying any sample in which  	any of these analytes occur at or above their respective threshold levels.  It is,  	however, unlikely that any assay will be capable of measuring all four analytes  	with the same degree of sensitivity.  Under these circumstances, it is important to  	evaluate the candidate assays in terms of their performance for all four potential  	analytes.

Cross-Reactants

An important criterion of immunoassay suitability is the selectivity or  	specificity of the assay for the analytes of interest.  Interference caused by  	materials of related structure can lead to a high level of "false 	positives" and severely limit the usefulness of the assay for measuring  	acetochlor, alachlor, metolachlor and dimethenamid.  Previous studies with an  	alachlor immunoassay have shown the potential of metabolites such as alachlor  	ethane sulfonic acid to cause false positive results4,5.

The state ground and surface water programs outlined in the acetochlor  	registration agreement do not include degradates of acetochlor since these  	materials are not of toxicological concern. Therefore, an assessment of the
	cross- reactivity of degradates or metabolites of the analytes is a key factor in  	determining the suitability of an assay.

Threshold Screening Levels

The use of immunoassays to screen for the presence of analytes in environmental 	samples has been successfully tested for a number of analytes including alachlor6  	and atrazine7.  The need for standardized validation of screening procedures has  	been recognized and a number of groups including the Association of Official  	Analytical Chemists (AOAC), Analytical Environmental Immunochemical Consortium  	(AEIC) and International Union of Pure and Applied Chemistry (IUPAC) are reported  	to be developing guidelines for the  evaluation of immunoassay kits8.  The need to  	verify immunoassay results at a specified threshold point by an alternative  	technique such as GC/MS is generally recognized as a requirement of a suitable  	validation program.

The acetochlor registration agreement has specified the detection of acetochlor  	at 0.10 ppb in ground water in the state monitoring program as a 	"trigger" for  further regulatory action.  In other monitoring programs  	outside of the Prospective Ground Water (PGW) program or the State Monitoring  	Programs, detection of acetochlor at 0.20 ppb requires follow up action.   	Regulatory action for the State Surface Water Monitoring Program is based on  	exceeding an annual time-weighted mean concentration of 2.0 ppb for acetochlor or a  	single peak concentration of 8.0 ppb for this analyte.

In determining the suitability of an immunoassay for use as a screen for  	threshold levels of acetochlor and other corn herbicides two action levels are  	worthy of consideration:

0.1 - 0.2 ppb and

1.0 - 2.0 ppb.

Sufficient sensitivity at the lower level would provide an assay capable of  	satisfying both thresholds since the more concentrated samples could be easily  	diluted.

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COMPARISON OF COMMERCIALLY AVAILABLE IMMUNOASSAYS FOR ACETANILIDES

Acetanilide immunoassays either currently commercially available, or in
pre-commercial development, are listed below:

Metolachlor (Ohmicron)

Metolachlor 1.0 (Idetek/Quantix)

Alachlor 1.0 (Idetek/Quantix)

Alachlor (Millipore)

Alachlor (Ohmicron)

Acetanilide (Millipore)

The first five of these are already commercially available, and are promoted as specific assays for the title compound.  The class-specific acetanilide kit is currently in evaluation/development by Millipore.  The properties of these assays are described below.

Metolachlor (Ohmicron)

The Metolachlor Ohmicron RaPID Assay Kit9 is intended for the detection of 	metolachlor in water and soil.  The key assay characteristics are outlined in Table  	1.  The assay shows high sensitivity towards metolachlor (I10 = 0.05 ppb; I50 =  	0.85 ppb), and some cross-reactivity towards acetochlor (I10 = 0.06 ppb; I50 = 6.55 	ppb).  The level of cross-reactivity towards alachlor is, however, comparatively  	low (I10 = 1.30 ppb; I50 = 84.0 ppb), making the assay of limited use for detection  	of the latter.

Notably, metalaxyl, N-(2,6-dimethyl-phenyl)-N-(methloxyacetyl)alanine methyl  	ester, also shows strong cross-reactivity, comparable to that of acetochlor, and  	might therefore constitute a significant source of false positive detects for  	chloroacetanilides were this kit to be used.

Metolachlor 1.0 (Idetek/Quantix)

This assay10 shows high specificity towards metolachlor (I50 = 0.75 ppb). The  	levels of cross-reactivity to both alachlor and acetochlor are very low (I50 = 100  	ppb and 70 ppb, respectively).  I10/20 values are not quoted in the technical data  	sheet, but the I50 values indicate that this assay would be of no use for the  	detection of either alachlor or acetochlor.

Alachlor 1.0 (Idetek/Quantix)

This assay11 is specific for alachlor (I50 = 0.48 ppb). The levels of
	cross-reactivity towards acetochlor (I50 = 23 ppb) and metolachlor (I50 = 80 ppb)  	indicate that the assay would not be of use for the detection of the compounds.   	Limited cross-reactivity was demonstrated for alachlor metabolites as indicated:   	alachlor sodium oxanilate (I50 = 50 ppb),   alachlor sulfonic acid (I50 = 10 ppb)  	and alachlor sulfinyl acetic acid (I50 = 7 ppb).  I10/20 were not reported.

Alachlor (Millipore)

The EnviroGard Alachlor Plate Kit12 has a least detectable dose for alachlor of  	0.046 ppb and an I50 of 0.6 ppb.  The least detectable dose of metolachlor is 0.6  	ppb, with an I50 of 40 ppb, again indicating that the assay would be of little  	utility for metolachlor detection.  No data describing the cross-reactivity of  	acetochlor in this assay were reported.

Alachlor (Ohmicron)

The characteristics of the alachlor Ohmicron RaPID Assay Kit13 are summarized in 	Table 2.  The assay does not have sufficient cross-reactivity towards metolachlor 	(I10/20) to form the basis of a useful screen for acetanilides.  In addition, there  	is significant inadvertent cross-reactivity to the ethane sulfonic acid metabolite  	of alachlor.

Acetanilide (Millipore)

The EnviroGard Acetanilide Plate Kit14 is the only one of the assays described  	which is primarily intended as a class-specific screen for acetanilides including  	acetochlor.  The assay characteristics are summarized in Table 3.  The assay shows  	the highest sensitivity towards metolachlor (I10 = 0.02 ppb; I50 = 0.25 ppb), but  	also has strong cross-reactivity towards acetochlor (I10 = 0.02 ppb; I10 = 1.7 ppb)  	and alachlor (I10 = 0.55 ppb; I50 = 4.9 ppb).

Summary

Table 4 shows a direct comparison of the six assays with respect to their  	capabilities to detect metolachlor, acetochlor and alachlor.  Published data for  	dimethenamid were not available for any kit.  The metolachlor (Idetek and Ohmicron)  	and alachlor (Idetek, Millipore and Ohmicron) assays are all clearly deficient in  	their capabilities to detect at least one of the three analytes.  This is most  	readily demonstrated where I10/20 values, corresponding to the least detectable  	doses, are in excess of the concentrations of interest.  In those cases where  	I10/20 values are not quoted, large I50 values indicate that the sigmoidal
	dose-response curves must be very shallow, and therefore imprecise, if indeed even  	measurable, at the low concentrations of interest.

The Millipore Acetanilide assay offers the best prospects for a acetanilide  	screen including acetochlor.  The least detectable doses for alachlor, acetochlor  	and metolachlor are relatively low, and the ranges of the respective dose-response  	curves appear sufficiently narrow to enable reasonably precise measurements to be  	made.

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EVALUATION OF ENVIROGARDTM ACETANILIDE PLATE KIT

Background

The pre-commercial development of an acetanilide immunoassay by Millipore 	Corporation was confirmed on December 15, 1994 with Brian A. Skoczenski, Vice 	President of Research and Development of Immunosystems Inc., a division of 	Millipore. Based on this information it was clear that the EnviroGardTM  	Acetanilide Plate Kit offered the best prospects for an acetanilide screen and the  	ARP was able to obtain, by special order, twelve kits for independent evaluation.

The ARP selected the Water Quality Laboratory at Heidelberg College in Tiffin,  	Ohio to conduct an evaluation of the acetanilide kit because of their extensive  	experience in water analysis using a variety of methods and their prior work with  	Millipore immunoassays4.  A protocol was written which specified the design of the  	study and was finalized by the signature of the study director on January 4, 1995.   	The study was conducted with clear guidelines for record keeping and documentation  	but not under the specific dictates of Good Laboratory Practices (GLP) as written  	in 40 CFR Part 160.

The objective of the evaluation was to determine how the kit performed for the 	analysis of acetochlor and other corn herbicides when used by experienced 	investigators.  Included within this evaluation was an analysis of blind coded  	surface and ground water samples that had been fortified with acetochlor at 0.10  	and 1.0 ppb levels.

Principles of the Assay

The Millipore assay is a competitive ELISA method that uses a microtiter plate 	format (12 strips of 8 antibody-coated wells each, in a strip holder) supplied by  	the manufacturer with wells precoated using the acetanilide antibody.  Water  	samples of calibration standards are added to individual wells of the microtiter  	plates, followed by an acetochlor-enzyme conjugate for binding to the limiting  	concentration of antibody during a fixed incubation period.  Following a wash step,  	which serves to remove any unbound acetochlor-enzyme conjugate, an enzyme substrate  	is added.  This results in the formation of a blue color in the presence of the  	bound acetochlor-enzyme conjugate.  The intensity of the color is inversely related  	to the concentration of acetanilide in the sample.  The actual concentration is  	measured by reference to a standard curve of log[concentration] versus absorbance  	which is generated concurrently during the analysis using standards of known  	concentration.  Since acetochlor calibrators are used, the concentration is  	expressed as acetochlor equivalents.  However, since the assay has broad  	specificity for a number of acetanilides including alachlor, acetochlor and  	metolachlor, a positive response in the assay does not mean that acetochlor is  	present in the sample; the assay will only detect unspecified cross-reacting  	acetanilides.

The method performance has been evaluated  in terms of the following assay  	characteristics:

Reproducibility of the standard curve

Cross-reactivity towards related analytes

Precision and accuracy of measurement of replicate analyses of 			fortified samples in reagent water

The results of these evaluations are reported and discussed below.

Reproducibility of the Standard Curve

The acetanilide kit relies on a three point calibration curve using the  	following acetochlor calibrators in reagent water:  0.0 ppb (negative control or  	blank), 0.1 ppb, 0.5 ppb and 5.0 ppb.  The standard curve is expressed as %Bo  	[(average absorbance of calibrator)/(average absorbance of negative control) x  	100%] versus log [acetochlor].  The reproducibility of the standard curve was  	evaluated by analyzing in triplicate a series of acetochlor standard solutions on  	ten separate days.    The data indicates acceptable reproducibility (%CV of 5.7 to  	14.5%).

Cross-Reactivity

The ARP provided a certified sample of acetochlor as well as the following  	reference materials for testing:

Alachlor

Metolachlor

Dimethenamid

Atrazine

Alachlor Sulfonic Acid

Alachlor Oxanilic Acid

Alachlor Sulfinyl Acetic Acid

Acetochlor Oxanilic Acid

Aceotchlor Sulfonic Acid

Acetochlor Sulfinyl Acetic Acid

Compounds 5-10 represent the major soil metabolites of alachlor and acetochlor.   	A stock solution of each standard was prepared in methanol and dilutions in water  	gave a series of solutions between 500 ppb and 0.1 ppb.  Each of the aqueous  	solutions were tested using the acetanilide kit, sigmoidal plots of %Bo versus log 	[concentration] were prepared and the values of I10 (10% inhibition) and I50 (50% 	inhibition) were determined from the graphs. The results are shown in Table 5.

The sensitivity of the assay for acetochlor, alachlor and metolachlor was  	confirmed as demonstrated by the I50 values (Table 5).  The acetanilide data sheet  	from Millipore for this assay lists a lower I50 for metolachlor than acetochlor  	contrary to the results of the Heidelberg College determination.  The reason for  	this discrepency is not known but one possibility could be the use of linear plots  	of %Bo versus log [concentration] by Millipore.  Since Heidelberg College used  	sigmoidal rather than linear fit of the data different estimates would be obtained.  	Dimethenamid was shown to be a sensitive analyte in the assay with a lower I50 than  	acetochlor.  Atrazine and the soil metabolites of alachlor and acetochlor were  	unreactive even at 500 ppb demonstrating that these materials have a very low  	probability of giving false positives at typical environmental concentrations.

Accuracy and Precision

The acetanilide assay was used to measure samples, generally in triplicate, on  	ten different days.  At total of twenty nine individual measurements were made at  	each fortification.

At 0.1 ppb, individual concentration measurements ranged from 0.09 - 0.29 ppb.  	Mean concentrations from daily measurements ranged from 0.10 - 0.26 ppb and the 	overall mean was 0.16 ppb.

At 1.0 ppb, individual concentrations ranged from 0.72 - 1.90 ppb.  Mean daily 	concentrations ranged from 0.83 - 1.48 ppb, while the overall mean was 1.06 ppb.

The within-assay precision of measurement of samples fortified at 0.1 ppb ranged 	between 10.0 - 23.2% (CV).  The between-assay precision was 32.5% (CV).

The within-assay precision of measurement of samples fortified at 1.0 ppb ranged 	between 8.3 - 35.2% (CV). The between -assay precision was 17.6%.

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ENVIROMENTAL HEALTH LABORATORIES (EHL) ACETANILIDE ASSAY

Background

A draft Standard Operating Procedure for an automated acetanilide immunoassay  	was received by the Partnership on December 7, 1994 from Jerry Thoma, President of 	Environmental Health Laboratories (EHL) located in South Bend, Indiana.  The EHL 	was founded in 1986 with the goal of providing drinking water analysis to both the 	public and private water suppliers as well as complex analytical testing for the  	commercial sector and state and federal contracting agencies.  The laboratory is 	certified in 33 states and EPA Regions to perform laboratory analyses for regulated 	parameters monitored by Public Water Supplies for compliance with the Safe Drinking  	Water Act.

The EHL, in an exclusive agreement with Millipore, adapted the EnviroGard 	Acetanilide Plate Kit for an automated assay format.  The goal of the program was  	to provide an automated acetanilide immunoassay service to public and private water 	suppliers.  The first circular advertising the service was distributed January 12,  	1995 and more information is available by contacting the Client Services Department  	of the EHL at<PHONE_NUMBER>.

Principle of the Assay

EHL assay relies on reagents supplied by Millipore Corporation and has been  	fully automated using a Bio-Tek Els1000 integrated microplate system.  In all other 	respects the assay resembles the typical competitive ELISA method that is the basis 	for the EnviroGard Acetanilide Plate Kit.  The Standard Operation Procedure for the 	EHL method 1294A contains information on the performance on the assay which will be  	summarized below.

Reproducibility of Standard Curve

The EHL method relies on a four-point standard curve using the following  	acetochlor calibrators in reagent water: 0.0 ppb (negative control or blank), 0.1  	ppb, 0.25 ppb, 1.0 ppb and 5.0 ppb.  The standard curve is determined in the same  	way as the acetanilide plate kit by expressing %Bo versus log [acetochlor].

Ten observations of the standard curve, each in duplicate, were made on separate 	occasions between October 27, 1994 and December 1, 1994.  The reproducibility of 	the automated EHL method is acceptable and somewhat better than that observed for 	the manual plate kit assay (see Table 5.).

Cross-Reactivity

The cross-reactivity results obtained with the EHL method are shown in Table 6  	and are in general agreement with the results reported in the acetanilide plate kit  	data insert.  The EHL analysis indicates that metolachlor is a more sensitive  	analyte than acetochlor with I50's of 0.25 and 0.71 ppb, respectively.

Accuracy and Precision

The EHL assay was used to measure samples in triplicate on each of seventeen  	days, giving a total of fifty one individual determinations at each concentration.

At 0.1 ppb individual concentration measurements ranged from 0.081 - 0.126 ppb.  	Mean concentration measurements from daily determinations ranged from 0.088 - 0.119  	ppb while the overall mean was 0.103 ppb.

At 1.0 ppb, individual concentrations ranged from 0.845 - 1.309 ppb. Mean 	concentrations from daily measurements ranged from 0.934 - 1.228 ppb, and the 	overall mean was 1.100 ppb.

The within-assay precision of measurement of samples fortified at 0.1 ppb ranged 	between 0.9 - 13.1% (CV).  The mean between-assay precision was 7.9% (CV, n=17).

The within-assay precision of measurement for samples fortified at 1.0 ppb  	ranged from 1.7 - 17.0% (CV).  The mean between-assay precision was 7.0% (CV,
	n=17).

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PERFORMANCE TESTING OF ACETANILIDE IMMUNOASSAYS

Purpose

This performance test was conducted as part of the evaluation of the
	EnviroGardTM Acetanilide Plate Kit and the automated acetanilide  	immunoassay test.

Experimental Design

Water samples were collected from 14 finished surface water sites in the greater 	metropolitan area surrounding St. Louis, Missouri and from one raw surface water 	site on the Missouri River near Defiance, Missouri.  In addition ground water from 	two Missouri wells, one well in Illinois and two wells in Wisconsin were also 	collected.  These samples were analyzed by GC/MS for the presence of corn 	herbicides and then fortified with acetochlor at either 0.1 or 1.0 ppb.  The  	samples were reanalyzed by GC/MS to confirm the fortification level and identical  	coded sets were sent to the Water Quality Laboratory (WQL) at Heidelberg College in  	Tiffin, Ohio and to the Environmental Health Laboratories (EHL) in South Bend,  	Indiana for analysis using an acetanilide immunoassay.

Results and Discussion

No acetochlor or dimethenamid was detected in the surface and ground water  	samples analyzed by GC/MS. Low levels of atrazine and metolachlor which averaged  	0.225 ppb and 0.091 ppb, respectively, were found primarily in the surface water.   	Alachlor was detected at trace levels that were <0.05 ppb in all cases.

Analysis of the unfortified water samples by immunoassay using the
	EnviroGardTM Acetanilide Plate Kit (WQL, Heidelberg College) and an  	automated acetanilide assay (EHL) gave results which were in general agreement but  	differed significantly from the acetochlor analysis by GC/MS.  No acetochlor was  	present in these samples and yet the WQL analysis indicated an average of 0.19 ppb  	acetanilide with five samples <0.10 ppb and the EHL results gave an average  	level of 0.32 ppb acetanilide with 9 samples <0.10 ppb.  The discrepancy was due  	primarily to the presence of metolachlor  which has been shown to be a strong  	cross-reactant in this immunoassay.

Following fortification at 0.10 ppb with acetochlor and reanalysis by GC/MS, the 	water samples were shown to contain 0.091 ppb acetochlor in good agreement with the  	theoretical concentration.  Immunoassay at WQL and EHL gave acetanilide results 	that averaged 0.26 and 0.37 ppb, respectively, once again reflecting the presence  	of other chloroacetanilides in addition to acetochlor.  Fortification at 1.0 ppb  	with acetochlor gave immunoassay values that were more precise and accurate and in 	better agreement with the GC/MS.  WQL and EHL measured an average of 1.20 ppb 	acetanilide compared with 1.09 ppb acetochlor obtained by GC/MS.  This agreement 	between ELISA and GC/MS at 1.0 ppb acetochlor was due to the fact that interfering 	acetanilides were minor compared with acetochlor and the values were within the 	range of the immunoassay where good precision for this analyte was obtained (I50).

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CONCLUSIONS

The sensitive and specific ELISA immunoassays developed for alachlor and metolachlor  have been shown in some cases to cross-react with acetochlor. However, the I50's  (concentration of acetochlor required to achieve 50% inhibition) in these assays were  between 6.55 and 70 ppb (see Table 4) making them unsuitable for measuring acetochlor in  water at 0.1 to 2 ppb.

A new kit,the EnviroGard Acetanilide Plate Kit, in pre-commercial development by Millipore Corporation has been shown to have greater sensitivity for acetochlor (I50 =  0.7 ppb) and broad cross-reactivity for other acetanilides including metolachlor and alachlor with I50's of 0.26 ppb and 4.9 ppb, respectively.  These characteristics of the  new Millipore kit give it the potential for use as a screen for the acetanilide corn herbicides including alachlor, metolachlor, acetochlor and dimethenamid in water.  The  Millipore acetanilide kit has also been adapted for use in an automated format by the  Enviromental Health Laboratories in South Bend, Indiana and offered as a commercial  service to public and private water suppliers by this analytical laboratory.

With the support of the Acetochlor Registration Partnership, an evaluation of the EnviroGard Acetanilide Plate Kit was conducted by the Water Quality Laboratory of Heidelberg College in Tiffin, Ohio.  A similar evaluation of the automated format for the  Millipore acetanilide kit was provided by Enviromental Health Laboratories and presented  in their Standard Operating Procedure for EHL Method 1294A.

For this report four criteria were used to assess the characteristics and performance  of the two assays:

Reproducibility of calibration curve

Cross-reactivity toward related analytes

Accuracy and precision of replicate analyses of acetochlor

fortified reagent water

Accuracy of blind fortified surface and ground water samples

Since both assays are based on the same antibody and reagents from the same company  (Millipore Corporation), similar results would be expected. Differences are likely to  reflect the precision afforded by instrumented transfer and reproducible plate mixing,  washing and incubation provided by the automated assay.

Both assays gave reproducible standard curves.  The acetanilide plate kit utilizes a three point calibration curve with acetochlor calibrators at 0.1 ppb, 0.5 ppb and 5.0 ppb.  The EHL method relies on a four point calibration that includes calibrators of 0.1  ppb, 0.25 ppb, 1.0 ppb and 5.0 ppb.  Somewhat better precision was observed for the  automated EHL assay.

Cross-reactivity results for both methods were consistent with those reported in the acetanilide kit technical data sheet from Millipore although differences in the absolute values for the I50's were observed.  Dimethenamid was found to be a sensitive analyte for  the assay with comparable levels of cross-reactivity to that observed for metolachlor.   The assay was shown to be non responsive to atrazine and the major soil metabolites of  alachlor and metolachlor at levels up to 500 ppb.

Investigation of the accuracy and precision using acetochlor fortified in reagent  water indicated improved accuracy and between-assay precision evident at 1.0 ppb.

A final aspect of the immunoassay evaluation was an analysis of representative blind surface and ground water samples fortified with acetochlor at 0.1 and 1.0 ppb.  The surface and ground water samples were analyzed by GC/MS prior to fortification to measure  the presence of indigenous acetanilides and after fortification to verify the level of  acetochlor.  The results of the two ELISA immunoassay systems were found to be relatively  inaccurate for measuring acetochlor at 0.1 ppb but satisfactory at 1.0 ppb acetochlor.   The inaccuracy at low levels of acetochlor appeared to be due to the presence metolachlor  in the surface water samples.

In summary, the EnviroGard Acetanilide Plate Kit and the EHL automated acetanilide assays have sufficient accuracy and precision to serve as a screen for acetochlor in water at 1.0 ppb.  Use of the assay as a screen at 0.1 ppb was considered to be unadvisable due to low precision at this level and the fact that the lowest calibrator provided in the kit was 0.1 ppb acetochlor.  At 0.2 ppb, values were found to range between 0.13 ppb and 0.34 ppb and improved precision was obtained.  In addition, 0.2 ppb  was shown to be within the linear range of the standard curve.  These observations  suggest that 0.2 ppb would be a reasonable low level threshold for application of the  assay.

The acetanilide immunoassay of Millipore is not specific and does not differentiate among alachlor, acetochlor, metolachlor and dimethenamid but rather detects their presence to varying degrees.  Therefore, it is imperative that any positive detects obtained using this immunoassay as a plate kit or in an automated format be confirmed  with another analytical method, such as GC/MS, to establish the presence and level of  acetochlor in water.

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TABLES/FIGURES

Table 1.

Cross-Reactivity of Metolachlor Ohmicron RaPID Assay Kit for Acetanilides

Compound
Least Detectable Dose
(I10 in ppb)
Dose-Response Mid-Point
(I50 in ppb)

Metolachlor
 0.05 	  0.85

Acetochlor
 0.06 	  6.55

Metalaxyl
  0.06 	  5.60

Butachlor
 0.26 	  52.0

Propachlor
 1.00 	  2500

Alachlor
 1.30 	  84.0

Table 2.

Cross-Reactivity of Alachlor Ohmicron RaPID Assay Kit for Acetanilides

Compound
Least Detectable Dose
(I10 in ppb)
Dose-Response Mid-Point
(I50 in ppb)

Alachlor
 0.05 	  ca. 1.0

Acetochlor
 n/aa 	  n/a

Butachlor
 6.0 	  ca. 100

Metolachlor
 5.6 	  ca. 80

Propachlor
 6000 	  n/a

Alachlor sulfonic acid
 0.03 	  n/a

a n/a = not available

Table 3.

Cross-Reactivity of EnviroGard Acetanilide Plate Kit for Acetanilides

Compound
Least Detectable Dose
(I10 in ppb)
Dose-Response Mid-Point
(I50 in ppb)

Metolachlor
 0.02 	  0.25

Acetochlor
 0.02 	  1.7

Metalaxyl
 0.02 	  0.24

Butachlor
 0.13 	  4.9

Propachlor
 0.18 	  9.2

Alachlor
 0.55 	  4.9

Table 4.

Comparison of  Cross-Reactivity of Immunoassays for Corn Herbicides

Assay
(Company)
Metolachlor
Acetochlor
Alachlor

I10 (ppb)	I50 (ppb)
I10 (ppb)	I50 (ppb)
I10 (ppb)	I50 (ppb)

Metolachlor
(Ohmicron)
0.05	0.85	0.06	6.55	1.30	84.0

Metolachlor
(Idetek)
n/aa	1.3	n/a	70	n/a	100

Alachlor
(Idetek)
n/a	80	n/a	23	n/a	0.6

Alachlor
(Millipore)
0.6	40	n/a	n/a	0.15	5.0

Alachlor
(Ohmicron)
5.6	ca.80	n/a	n/a	0.05	1.0

Acetanilide
(Millipore)
0.02	0.26	0.02	1.7	0.55	4.9

a n/a = not available

Table 5.

Cross-Reactivity of EnviroGard Acetanilide Plate Kit (WQL)

Compound
Least Detectable Dose
(I10 in ppb)
Dose-Response Mid-Point
(I50 in ppb)

Acetochlor	 ca. 0.1 	  1.0

Alachlor	 2.0 	  5.0

Metolachlor	 0.2 	  1.5

Dimethenamid	 0.1 	  0.5

Atrazine	 n/da 	  > 500

Alachlor Sulfonic Acid	 n/d 	  > 500

Alachlor Oxanilic Acid	 n/d 	  > 500

Alachlor Sulfinyl Acetic Acid	n/d	> 500

Acetochlor Oxanilic Acid	 n/d 	  > 500

Acetochlor Sulfonic Acid	 n/d 	  > 500

Acetochlor Sulfinyl Acetic Acid	n/d	> 500

a n/d = not determined

Table 6.
Cross-Reactivity of Automated Acetanilide Assay (EHL)

Compound
Least Detectable Dose
(I10 in ppb)
Dose-Response Mid-Point
(I50 in ppb)

Metolachlor	 0.04 	  0.25

Acetochlor	 0.08 	  0.71

Metalaxyl	 0.02 	  0.13

Butachlor	 0.81 	  8.9

Alachlor	 0.52 	  3.9

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REFERENCES

 Feng, P. C. C.; Wratten, S. J.; Logusch, E. W.; Horton, S. R.; Sharp, C. 	R., "Immunoassay Detection Methods for Alachlor In Immunochemical Methods for 	Environmental Analysis", Van Emon, J. M. and Mumma, R. O. Eds. American 	Chemical Society Symposium Series 442, Washington, D. C. 1990, pp 180-192.

 Schlaeppi, J. M.; Moser, H.; Ramsteiner, K., "Determination of  	Metolachlor by Competitive Enzyme Immunoassay Using a Specific Monoclonal  	Antibody", J. Agric. Food Chem. 1991, 39, 1533-1536.

 Harrison, R. O.; Braun, A. L.; Gee, S. J.; O'Brien, D. J.; Hammock, B. D.,  	"Evaluation of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Direct 	Analysis of Molinate (OrdamTM) in Rice Field Water", Food and  	Agric. Immunology 1989, 1, 37-51.

 Baker, D. B.; Bushway, R. J.; Adams, S. A.; Macomber, C., "Immunoassay 	Screens for Alachlor in Rural Wells:  False Positives and an Alachlor Soil 	Metabolite", Environ. Sci. Technol. 1993, 27, 562-564.

 Aga, D. S.; Thurman, E. M.; Pomes, M. L., "Determination of Alachlor  	and Its Sulfonic Acid Metabolite in Water by Solid-Phase Extraction and
	Enzyme-Linked Immunosorbent Assay", Anal. Chem. 1994, 66, 1495-1499.

 Feng, P. C. C.; Wratten, S. J.; Horton, S. R.; Sharp, C. R.; Logusch, E. 	W., "Development of an Enzyme-Linked Immunosorbent Assay for Alachlor and Its 	Application to the Analysis of Environmental Water Samples", J. Agric. Food  	Chem. 1990, 38, 159-163.

 Thurman, E. M.; Meyer, M.; Pomes, M.; Perry, C. A.; Schwab, A. P.,  	"Enzyme-Linked Immunosorbent Assay Compared with Gas Chromatography/Mass 	Spectrometry for the Determination of Triazine Herbicides in Water", Anal.  	Chem. 1990, 62, 2043-2048.

 Sherry, J. P., "Environmental Chemistry:  The Immunoassay Option. In  	CRC Critical Reviews in Analytical Chemistry"; Coleman, D. M.; Coleman, P. B.,  	Eds.; CRC Press, Inc., Boca Raton, FL; 1992; Vol. 23, p. 276.

 Lawruk, T. S.; Lachman, C. E.; Jourdan, S. W.; Fleeker, J. R.; Herzog, D. 	P.; Rubio, R. M., "Determination of Metolachlor in Water and Soil by a Rapid 	Magnetic Particle-Based ELISA", J. Agric. Food Chem. 1993, 41, 1426-1431.

 Metolachlor 1.0 Immunoassay Kit, Specification Sheet, Idetek/Quantix 	Systems, 1995, and Supporting Data from Idetek.

 Alachlor 1.0 Immunoassay Kit, Specification Sheet, Idetek/Quantix Systems, 	1995, and Supporting Data from Idetek.

 EnviroGard Alachlor Plate Kit (Catalogue No. ENVR P00 04), Technical Data 	Kit Insert, Millipore Corporation, 1994.

 Lawruk, T. S.; Hottenstein, C. S.; Herzog, D. P.; Rubio, F. M.; 	"Quantification of Alachlor in Water by a Novel Magnetic Particle-Based  	ELISA", Bull. Environ. Contam. Toxicol. 1992, 48, 643-650.

 EnviroGard Acetanilide Plate Kit (Catalogue No. SD3P228S4), Technical Data 	Kit Insert, Millipore Corporation, 1995 (See Appendix A).

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[Federal Register: May 16, 2007 (Volume 72, Number 94)]
[Notices]
[Page 27547-27548]
[DOCID:fr16my07-51]

-----------------------------------------------------------------------

DEPARTMENT OF ENERGY

Federal Energy Regulatory Commission

[Docket No. CP05-15-006]

Caledonia Energy Partners, L.L.C.; Notice of Compliance Filing

May 9, 2007.
    Take notice that on May 2, 2007, Caledonia Energy Partners, L.L.C.

(Caledonia) submitted a filing to comply with the April 27, 2007 order
of the Director, Division of Tariffs and Market Development--East of
the Federal Energy Regulatory Commission in Docket No. CP05-15-003.
    Caledonia states that copies of the filing were served on parties
on the official service list in the above-captioned proceeding.
    Any person desiring to protest this filing must file in accordance
with Rule 211 of the Commission's Rules of Practice and Procedure (18
CFR 385.211). Protests to this filing will be considered by the
Commission in determining the appropriate action to be taken, but will
not serve to make protestants parties to the proceeding. Such protests
must be filed on or before the date as indicated below. Anyone filing a
protest must serve a copy of that document on all the parties to the
proceeding.
    The Commission encourages electronic submission of protests in lieu
of paper using the ``eFiling'' link at http://www.ferc.gov. Persons

unable to file electronically should submit an original and 14 copies
of the protest to the Federal Energy Regulatory Commission, 888 First
Street, NE., Washington, DC 20426.
    This filing is accessible on-line at http://www.ferc.gov, using the

``eLibrary'' link and is available for review in the Commission's
Public Reference Room in Washington, DC. There is an ``eSubscription''
link on the Web site that enables subscribers to receive e-mail
notification when a document is added to a subscribed docket(s). For
assistance with any FERC Online service, please e-mail
<EMAIL_ADDRESS>or call<PHONE_NUMBER> (toll free). For

TTY, call<PHONE_NUMBER>.
    Protest Date: 5 p.m. Eastern Time on May 23, 2007.

Kimberly D. Bose,
Secretary.
[FR Doc. E7-9362 Filed 5-15-07; 8:45 am]

BILLING CODE 6717-01-P


MAR 23 2005 Dr. Demetra Vlachos Senior Manager, Regulatory Affairs Syngenta Seeds, Inc. P. O. Box 12257 Research Triangle Park, NC 27709 2257 Dear Dr. Vlachos:

Subject: Your 12/ 15/ 2003 Application and Revised Section G Dated 2/ 4/ 2005 Modified Cry3A Corn EPA Experimental Use Permit No. 67979 EUP 4 Effective Dates: Plantings Through February 28, 2006 and Associated Field Operations Through October 15, 2006 Quantity Authorized: 17,250,000 mCry3A Corn Plants ( 15.53 Grams Active Ingredient)

On the basis of the information furnished by the applicant and the annexed program, this experimental use permit ( EUP) under § 5 of the Federal Insecticide, Fungicide, and Rodenticide Act is issued to test modified Cry3A Bacillus thuringiensis protein and the genetic material necessary for its production ( via elements of pZM26) in Event MIR604 corn ( SYN IR6

4 5). Shipment and/ or use under this Permit is subject to the provisions of 40 CFR Part 172. Tolerance exemptions currently under 40 CFR 180.1252 and being issued under 40 CFR Part 174.456 apply to the plant incorporated protectant in corn to be tested under this permit.

Prior to shipment and/ or use of the subject corn, you must consult with the pesticide regulatory officials and, if applicable, the biotechnology regulatory officials of the State( s) in which your experimental program will be conducted, and obtain a State permit or license if such is required. ( Note Hawaii's reporting requirement for genetically modified organisms under section 321 11.6 of the Hawaii revised statutes.) Issuance of this Federal permit does not negate the need for permission from individual States. Prior to initiating this experimental program in any State, you are to notify the lead Agency of the State( s) in which your experimental program will be conducted of the specific testing program ( when, where, how much, etc.). Failure to do so may result in revocation or modification of this EUP. You must comply with all requirements of the USDA/ APHIS as they apply to the proposed testing.

You must provide notice to the State pesticide regulatory Agency at least 72 hours before you begin to apply the experimental use product. You must also provide a copy of this authorization letter, approved label, and experimental program section G to all cooperators, participants, and users prior to the initial pesticide application made in accordance with this EUP. The Agency is also notifying the relevant EPA Region( s), by sending them copies of this EUP. 2 Based upon the submitted experimental program, the subject corn seed may be shipped for use under this permit to the following State( s) and be planted on the following acreage, provided that the that the agronomically justified acreage as listed below is not exceeded and that States agree to the trials and all other requirements of the USDA/ APHIS have been met as they apply to the testing.

States Acres of Plants Derived From mCry3A Corn Acres of Plants from Non mCry3A Genotypes that are Part of an EUP Field Trial Protocol, Including: Control/ Check Plants and any Plants in Border Rows, and Other NonmCry3A Genotypes Planted Adjacent to the EUP Plot CO 3 41 HI 46 62 IA 244 401 IL 48 762 IN 23 259 KS 4 52 KY 3 21 MD 10 20 MI 13 127 MN 39 327 MO 18 207 MS 1 7 NE 32 415 OH 18 206 PR 41 127 SD 10 91 TX 2 14 WI 20 196 Totals 575 3335 Protocol Type EUP Acreage* mCry3A Acreage Non mCry3A Acreage Total EUP Acreage Breeding and observation 117 279 396 Efficacy field trials 94 1,320 1,414 Agronomic observation 26 284 310 Inbred and hybrid production 298 1,342 1,640 Regulatory field trials ( e. g. IRM and non target insect field trials, etc.) 40 110 150 Total 575 3,335 3,910 * EUP acreage is the sum of the acreage found within the perimeter of all associated experimental blocks. This includes mCry3A plants, non mCry3A plants, and border rows included within the blocks. 3 Conditions under which the EUP is being issued are as follows.

1. The total amount of corn seed to be used will not exceed the quantity authorized by the EUP and its planting acres.

2. The permittee must immediately notify the Environmental Protection Agency of any findings from the experimental uses that have a bearing on safety ( i. e., reporting to the Agency of any adverse effects from the use of, or exposure to, the pesticide is required). The permittee will also keep records of production, distribution and performance, and, on request, make the records available to any authorized officer or employee of the Environmental Protection Agency.

3. You must provide a final report within180 days of the expiration of the permit. The final report must include all of the items set forth in 40 CFR § 172.8( b).

4. Per 40 CFR § 172.4( b)( v), you must provide the name and street address of all cooperators as soon as available and prior to planting.

5. The labeling submitted in connection with your application is acceptable and must be used for all shipments of modified Cry3A seed subsequent to receiving this approval and be in possession of the user under this EUP.

6. This experimental use permit is not a limited seed increase registration under § 3 of FIFRA and plantings solely for commercial seed increase are prohibited Sincerely,

/ s/

Janet L. Andersen, Ph. D., Director Biopesticides and Pollution Prevention Division ( 7511C) Enclosure