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II. Salmonella/Mammalian Microsome Reverse Mutation
Assay
1) Section II.C.6.a
The title Negative and Solvent Controls is
revised to Negative Controls (untreated and/or solvent)
2) Section II.C.6.b. Positive control:
The second and third sentences were revised as follows: Sodium
azide (without S9) will be the positive control for TA1535 and
9-aminoacridine (without S9) for TA1537. The positive controls
for TA98 and TA100 will be 2-aminofluorene (with S9).
II. Reasons for Revision
1) This revision is to clarify the language.
2) The original protocol was submitted as a general protocol
for costing purposes without detailed review. The protocol was
revised at the start of the study to make it consistent with the
Standard Operating Procedures actually followed in the
laboratory. The revision was to show that 2-aminofluorene with S9
would be used as a positive control for TA100 instead of sodium
azide (without S9). Sodium azide was not
used as the positive control for TA100 without S9 because it was
already used as the positive control for TA1535. Since
TA100 is derived from TA1535 and the fact that both detect
base-pair mutagens, using sodium azide as the positive control
for both TA100 and 1535 would only yield redundant data. In
addition, the positive controls that were used adequately showed
the sensitivity of the assay, because the positive response with
2-aminofluorene adequately showed that the S9 system was capable
of activating mutagens, and also demonstrated the sensitivity of
each strain to detect mutagens.The sentence regarding the
positive control for TA98 and TA1537 was only for clarifying the
ambiguity in the original protocol.
We did not obtain approval for the revised changes in the
protocol from the study sponsors because of the time constraints
associated with this study.
IV. In Vivo
Mouse Bone Marrow Micronucleus Test
1. Section IV.C.2. Test agent:
The last part of the first sentence, single
intraperitoneal (ip) injection, is changed to gavage
for three consecutive days.
2. Section IV.C.4. Dosing and sampling:
a. The one-dose, one-sampling
in the first sentence is changed to multiple dosing (one
dose per day for 3 days).
b. Deleted the following part from the second sentence:
or appropriate solvents and administered by single
intraperitoneal (i.p.) injection
Revised to:
and administered by gavage dosing.
3. Section IV.C.7. Micronuclei observation:
The last part of the first sentence was modified. The
frequency of micronucleated cells is observed in 1000
polychromatic erythrocytes (PCES) per animal by using
blind-coded slides.
Reasons for Revision
1. This revision reflects the change in the dosing route of
ammonium perchlorate (see the rationale in #2a).
2. a. Oral gavage is a preferred route of administration
(Brusick, 1994), since ammonium perchlorate is soluble in water.
Intraperitoneal (i.p.) injection is used only if the test
chemical's characteristics preclude its use in gavage dosing (for
example, the test chemical is soluble only in DMSO, etc.).
Another reason to opt for gavage dosing is based on the
assumption that since AP is being evaluated to determine its
potential toxicity in humans, one of the possible exposure routes
is probably by the oral route. This is indicated in the report of
a serious environmental AP contamination in drinking water in the
Lake Mead area in Nevada (McKinnon, 1998). Multiple dosing of AP
is preferred to make sure that a potential mutagenic effect is
not missed from a single dosing, as cell cycle delays can affect
the outcome of the experiment.
b. This revision reflects the change in the dosing route of
ammonium perchlorate (see the rationale in #2a).
3. The original protocol omitted to note this procedure. The
practice of blind-coding slides is the recommended procedure for
eliminating technician bias.
Because of time constraints for this study, we did not attempt
to obtain an approval from the study sponsors for the revised
protocol. But these revisions have significantly improved the
methods cited in the original protocol.
VIII. Reports and Deliverables (See attached)
This section is replaced by the following section:
Technical progress will be communicated informally to
the Project Officer prior to completion of genotoxicity assays;
because of additional tests run, the completion date is revised
to May 22, 1998. The draft final report will be submitted on May
26, 1998. TERA will provide written comments by June 12, 1998, or
as soon as possible thereafter. A final report will be submitted
by June 26, 1998, or 14 days after receipt of comments from TERA,
whichever is later. If extensive changes are needed to address
the comments received, an additional price will be negotiated.
The final draft report constitutes the study's deliverable, and
ManTech considers acceptance of the deliverable to occur when it
is received by TERA.
VIII. Reasons for Revision
A revision was made at the request of TERA to include a draft
final report. The approved revised schedule for reports and
deliverables is included in the signed protocol revision. (See
Appendix D.)
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