Memorandum - Konjac Foods · 2013. 6. 7. · starch, konjac flour and guar gum, all within 24 h. By 48 h significant amounts of gas were produced in cultures supplemented with D-glucose,

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DEPARTMENTOF HEALTH& HUMAN SERVICES Public Health Servicez:-:“+‘+

>%,,a Memorandum ‘-

Date●March 29, 1999

From Team Leader, Chemistry and Environmental Review Team (CERT)Scientific Support Branch, Devision of Product Policy (HFS-205)

Subject GRASP 6G0418 – Konjac Flour for use as an FMC CorporationIngredient in Human Food – Environmental Review c/o Keller and Heckrnan

Washington. DC 20001Tci Division of Petition Control (HFS-2 15)

Attention: Andrew Laumbach

The petitioner submitted a claim of categorical exclusion under 21 CFR 25.32(r). Thisexclusion is for approval of a food additive, color additive, or GRAS affkrnation petition fora substance that occurs naturally in the environment. The petitioner stated the proposedaction and the section of the CFR that the GRAS petition proposed to amend. The petitionercited $ 25.32(r) as the section of the CFR under which the categorical exclusion is claimed,

and said that the exclusion criteria are met and that, to the knowledge of the petitioner, noextraordinary circumstances exist that require submission of an EA.

We reviewed the submission and concluded that the petitioner’s claim of categorical

exclusion is warranted.

cc:HFS-200 TarantinoHFS-207 Batarseh

HFS-246 RF/GRASP 6G0418

--

LAW OFFICES

KELLER AND HECKMAN LLP1001 G STREET. N.W.SUITE 500 WEST

WASIEIFTOTON.D.C. Z?OOOITELEPHONE (202) 434-41OO

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TELEPHONE 32(2) 541 OS 70

FACSIMILE 32(2) 541 05 00

—WWW.ICHLAW.COM

March 25, 1999

EAND DELIVERED

JOSEPH Z KULER [1907JEROME H HECUMANWILLIAM H BORQHESANI,MALCOLM O MAcARTHURWAYNE v 8LAcXTcnRE.NCE D dONESMARTIN W. BERCOVIC!JOHN S ELDRCORICHARD d LEl OHTONALFRED S RCGNERYDOUGLAS d BEHRRAYMOND A KOWALSK1,MICHAEL F MORRONEJOHN S RICHARDSJEAN 5AVIGNY.0JQHN B DUBECI(PETER L os LA CRUZMELVIN S DROZEMLAWRENCE P HALPRINRALPH A SIMMONSRICHARD F MANN

Andrew D. Laumbac~ Ph.D.Division of Petition Control (I-IFS-215)Office of Premarket ApprovalCenter for Food Safety and Applied NutritionFood and Drug Administration1110 Vermont Avenue, 12th FloorWashington D.C. 20204

.19941

JR.

C DOUGLAS JARRCTTSHEILA A. MILLARGCORGC G. M15K0PATRICK J HURD

CLIZAE?ETH N HARRISONJOw a RODGERSJOAN C SYLVAINOMARTHA E 14AR RAPESENtCOLE S DONATH

DEBORAH ROSEN WHITEDAVID R .lOYFREDERICK A STEARNSTODD A. HARRISON.JOHN F FOLEY10NYE RuSSCLL EPPS7H0MAs c breamRACHIDA SEMAIL*OJOHN 00 BINSONOJOMN F C, LUCDKEKOM AL J “CR SliBCR@PAuLA DCZAJOHN S OLOUOHLIN JRJENNIFER A 130L0STE8NWDEVON W“ HILL*DANIEL OUINTART*OTASHIR J LECAMY E FORTCNBET?R”*ANN M BOECKMAN*

●NOT ADMITTED IN DC,ORESIOWT SRUSSELS

Sclunw-lc STAFF

DANIEL S. DIXLER, Pn. 0.CHARI.ES v. sRcDca, w, 0.

ROSERT A. MAfl+EWS. P“. 0., D. A.0,1.JOHN P. MODDERMAN, P“. D.

11s44-19s8)HOLLY HUTMIRE FOLEYJAN ICTTE HOUK, Pn. 0.

LESTER 80 R001NsKY. P“. o.THOMAS C. BROW

MICHAEL T FL~D, Pn. D.ANDREW P XWANOV!Cli PH. D

ANNA GEROELY, P“, D.0STEFANIC M COROITT

JUSTIN .I FRED ERICO. P“. D.RACHEL F JOYWER

ELIZABETH A HEQCR

TELECOwNICATIONS

ENGINEERRANDAU O. YoUNO

WRITSI?’S DIRECT ACCESS

(202) [email protected]

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Re: FMC Corporation - GRAS Aflhmation Petition No. 6G0418 forKonjac Flour

Dear Dr. Laumbach:

The purpose of this letter is to provide you with a revised statement of categoricalexclusion from the need to submit an Environmental Assessment for the above-reference GRASMlirmation Petition. This responds to your recent request for the same to the Petitioner, FMCCorporation. As we understand it, the Food and Drug Administration (FDA) has determined thatthe basis for the previous claim of categorical exclusion under21 C.F,R. $ 25.42(b)(7), i.e., thatthe petition requests GRAS affirmation for the same uses for which Konjac flour is currentlymarketed, is not applicable because the product is not now employed in all of the applicationsenvisioned in the petition.

For these reasons, and based on conversations with Dr. Layla Batarseh of theEnvironmental Impact Section, we are hereby submitting a claim for categorical exclusion inaccordance with 21 C.F.R. $ 25.32(r). This exclusion pertains to GRAS Afl%-rnationPetitions forsubstances that occur naturally in the environment, when the action does not alter significantly theconcentration or distribution of the substance, its metabolizes, or degradation products in the

Andrew D. Laumbach, Ph.D. KEUER AND HECKMAN LLP

March 25, 1999Page 3

The fermentation study examined the change in pH and gas volume of bacterial mediacontaining the various test compounds over a period of 96 hours. Significant reductions in pH(i.e., increases in acid content) were observed within 24 hours for soluble starch, pectin, and guargum, and within 48 hours for Konjac flour and xanthan gum. Similarly, gas production wassignificantly elevated in samples containing soluble starch, Konjac flour, and guar gum within 24hours, and with pectin within 48 hours. No significant fermentation was observed withcarboxymethyl cellulose or sodium alginate.

The results of this testing indicate that konjac flour is fermented by human fecal florasimilarly to other common food ingredients. At each time interval, the pH and gas productionobserved with konjac flour was within the ranges seen with these “control” food ingredients.Thus, it maybe concluded that the use of konjac flour as a food ingredient will not significantlyalter the concentrations and distributions of these metabolizes (i.e., fermentation products)released to the environment.

The Petitioner hereby certifies that no extraordinary circumstances are expected thatwould disquali~ the Petition for categorical exclusion.

For the foregoing reasons, we respectfi.dly submit that an Environmental Assessment forthe action requested in the Petition is not required in accordance with21 C.F.R. $ 25.32(r).Should you have any t%rther questions regarding this matter, please do not hesitate to let usknow.

Very truly yours,

‘ John B. Dubeck

Enclosure

cc: Ms. Eunice M. CuirleAndrea Holtzman Drucker, Esq.

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I . . . .-

-.

‘) EXHIBIT 20

,-*

FINAL REPORT

SRonsor:

FMC Corporation,

Princeton, New Jersey

Studv Title:

h vitro Fermentation of Konjac Flour FW-KON by Human Fecal Flora

FMC Study: 194-1976

Protocol CR 01 O-RJC-001

Date”-

9 May, 1995

Performing Laboratory:

Tech Lab, Inc.VPI Research Park

1861 Pratt DriveBlacksburg, Virginia 24060

1

,

FMC study: 194-1976CR 01 O-RJC-001

TechLab Quality Assurance Statement

/n vitro Fermentation of Konjac Flour FW-KON by Human Fecal Flora


The report for this study has been reviewed by the Quality Assurance Officer of

Techlab, Inc. in accordance with Good Laboratory Practice Regulations as set forth

in 21 CFR 58.35 (b) (6) (7). The report identifies and/or describes the authorizedmethods and Standard Operating Procedures followed in the conduct of this study. The

Quality Assurance Officer has also conducted the following inspections of the facilities

used in the conduct of this study and has submitted a written report of theseinspections to the Study Director. Written status reports of inspections and findings

are submitted to the management according to Tech Lab Standard Operating

Procedures. .

Date of Inspection Type of Inspection Date Issued to Study Director

02/23/95 Protocol Review 02/23/95

03/20/95 Test Nlatsrial Preparation 03/20/95

03/29/95 Data Review 03/29/95

03/30/95 Draft Report Review

05/09/95 Report Review

03/30/95

05/09/95

5/9/5f“

Date

Tech Lab Quality Assurance Officer

2

l., }

F;IIC Corporation .

A .vAb .kuuy. im-lyi~

CR O1ORJC-OOI

QUAL~Y ASSURANCE STATEMENT

FMC STUDY NUMBER194-1976

This report and raw data were reviewed for accuracy and compliance with the studyprotocol, FDA Good Laboratory Practice Regulations, EPA Good LaboratoryPractice Standards and OECD Principles of Good Lalxxatory Practice by the I?MCToxicology Department’s QuaIity Assurance Unit. Revisions were made wherenecessary.

‘jv-L31&i2kWilLiam D. BartaQuality Assurance Supem-isor

.

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t

FMC study: 194-1976

CR 01 O-RJC-001

STUDY IDENTIFICATION

FMC Study: 194-1976


h vitro Fermentation of Konjac Flour by Human Fecal Flora,1

Test Material Konjac Flour FW-KON

Study Monitor Lois A. Kotkoskie, PhD, DABT,

Senior Research Toxicologist,FMC Corporation,

Chemical Research and Development Center,Box 8,

Princeton, New Jersey 08543

Phone: (609) 951 3724

Fax: (609) 951-3837

●

Study Director

Study Location

Study TimetableStart DateEnd Date

Robert J. Carman, PhD,Director of Research,Tech Lab, Inc.,1861 Pratt Drive,

Blacksburg, Virginia 24060Phone: (703) 231-3943

Fax: (703) 231-3942

TechLab, Inc.VPI Research Park

1861 Pratt Drive

Blacksburg, Virginia 24060

March 1, 1995March 28, 1995

4

*

Robert J. Carman, PhD.

Donna T. Evans, BS.!

Tracy D. Wilkins, PhD.

FMC study: 194-1976

CR 01 O-RJC-001

Key Personnel

Study Director

, Quality Assurance Officer

Scientific Consultant

*

Title page

TechLab Quality Assurance Statement

FMC Quality Assurance Statement

FMC study: 194-1976

CR 01 O-RJC-001

Contents

Study Identification

Key Personnel

Contents “

Summary

Objective

Regulatory Compliance

Test Material

Safety

Experimental Design

Data Analysis

Results

Conclusions\

Table 1. Mean PI+ and gas production results ofcontrol and test compounds incubated with pooledand diluted human feces 1.A. Time = 24 h

Table 1. Mean pH and gas production results ofcontrol and test compounds incubated with pooledand diluted human feces 1 .B. Time = 48 h

..%,..

6

E19s

1

2

3

4

5

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8

9

9

9

9

9

10

10

11

12

13

FMC study: 194-1976

CR 01 O-RJC-001

Table 1. Mean pH and gas production results of

control and test compounds incubated with pooled

and diluted human feces 1.C. Time = 96 h 14

Appendix A - TechLab Standard Operating Procedures 15

Appendix B - Protocol CR 01 O-RJC-001 37

Appendix C - Protocol Amendment 46

Protocol Deviations 50

Final Report Approval 51

b

7

*

Summary

FMC study: 194-1976

CR O1O-RJC-OOI

Control and test compounds were incubated in a bacteriological mediuminoculated with a dilute suspension of bacteria prepared from pooled human feces.

At each time interval, four tu,bes were prepared, consisting of two sets of two. Eachpair was inoculated from a suspension prepared from two different aliquots of a single

lot of pooled feces. The pH of the cultures were recorded at 24, 48 and 96 h. At thesame time, the volume of gas produced during incubation was measured. The meanpH and gas volumes were compared to a control (basal medium) using an unpaired t-

test. While none of the compounds were fermented sufficiently to observe a drop of

1 pH unit greater than the drop seen in basal medium, statistical differences were

observed. Within 24 h D-glucose, D-galactose, soluble starch, pectin and guar gumhad significantly lower pHs than the basal medium control. Within 48 h significantly

more acid was. produced by fermentation of the following additional compounds:

konjac flour and xanthan gum. Only L-glucose, carboxymethyl cellulose and sodium

alginate remained unfermented. Compared to the basal medium controls, there was

significantly elevated gas production in the tubes containing, D-gal actose, solublestarch, konjac flour and guar gum, all within 24 h. By 48 h significant amounts of gaswere produced in cultures supplemented with D-glucose, D-galactose, soluble starch,

konjac flour, pectin and guar gum. By 96 h only L-glucose, carboxymethyl cellulose

and sodium alginate showed no significant gas production compared to the control,

unsupplemented basal medium. In conclusion, at each time interval, the mean pH and

volume of gas produced by incubating test compound (Konjac flour FW-KON) withfecal suspensions were approximately between the upper and lower levels seen whenfour control food grade compounds were incubated with the same suspensions.

8


Objective

The purpose of this study

fermentation of test material

was to determine the potential for, and extent of.

by fecal bacteria in vitro;

Regulatory Compliance

The study was done according to the Food and Drug Administration’sLaboratory Practice Regulations for Nonclinical Laboratory Studies, 21 CFR 58.

GoodThe

protocol, study conduct and final reports were monitored by the Quality

Unit in compliance with FDA regulation 21 CFR 58.35.

Test Material

Assurance

Konjac Flour FW-KON Lot No. R94026. The composition and 30-day stability ofKonjac Flour FW-KON were determined in FMC Study Number P94-0085. Resultsindicated the material was stable at room temperature for 30 days and the test

material was composed of 83.4% soluble dietary fiber (data supplied by Sponsor).

Safety

The safety precautions used throughout this study were those required by TechLab’s

normal work practices, supplemented by those indicated in MSDS supplied by theSponsor.

Experimental design

Test and control compounds were incubated with fecal suspensions according toTechLab SOPS (see Appendix A)

Feces were collected, pooled and processed according to TechLab SOPS (seeAppendix A). Feces used to prepare the pool were provided by two adult males and

two adult females. None had received antibiotics within the preceding 4 weeks.

Biographical . data (age, sex, dietary habits, general information and recent

antimicrobial history) is stored at Tech Lab. The identity of the donors will remain

confidential. No diagnoses has been or will be made using data collected from thisstudy. Collection of the samples was voluntary and involved no risk to the donors.

. The donors have each signed a release form attesting to the facts listed above.

Inocula were prepared according to SOPS (see Appendix A) and two different aliquotsof the same lot of pooled feces.

9


Inoculated broth aliquots were dispensed aseptically and anaerobically into sterile

tubes. The tubes were stoppered anaerobically (see Appendix A for SOPs).

Inoculated broths were incubated in a press to prevent gas produced during incubation

from expelling the stoppers. However, three tubes did blow their stoppers after

removal from the incubator but before the gas production could be recorded. This has

no effect, of which Tech Lab is aware, on the final pH of the culture. In theseinstances the mean volume produced was an average of less than 4 datum points.

Tubes loose their stoppers primarily because of the abundance of gas produced, rather

than their stoppers are ill-fitting. Incubations were for O, 24, 48 and 96 h, under

anaerobic gasses and at 37 ‘C.

Acid production was analyzed using a pH meter and an electrode standardized at pH4.0 and 7.0 at ~oom temperature. Tubes were brought to room temperature before

being assayed. Gas production was assessed by fluid displacement. The SOPS are

included in Appendix A.

The following control compounds were tested: D-glucose, D-galactose, L-glucose and

soluble starch. The following extra control compounds were tested by request of theSponsor: food grade guar gum, food grade xanthan gum, food grade sodium alginate,

food grade carboxymethyl celiulose and food grade pectin. Food grade materials met

the current specifications for purity and identity as stipulated by FAO/WHO, the ECand Food Chemical Codex (data available from Sponsor).

The anaerobic transport device (SOP #5000) was used whenever appropriate toprovide a “bench top” anaerobic atmosphere.

Data Analysis

To analyze data the approach taken was that described in the Protocol Amendment

(see Appendix C). Briefly, each measured pH value was converted to its hydrogen ion

concentration and the means were calculated and then compared using an unpairedt-test. The analysis was done using computer software (SigmaPlot).

Results

.The results are shown in Table 1. While none of the compounds were fermented

- sufficiently to observe a drop of 1 pH unit greater than the drop seen in basal medium,statistical differences were observed. Within 24 h D-glucose, D-galactose, solublestarch, pectin and guar gum had significantly lower pHs than the basal mediumcontrol. Within 48 h significantly more acid was produced by fermentation of the

10

FMC study: 194-1976

CR 010-RJC-001

following additional compounds: konjac flour and xanthan gum. Only L-glucose,

carboxymethyl cellulose and sodium alginate remained unfermented. Compared to thebasal medium controls, there was significantly elevated gas production in the tubes

containing, D-galactose, soluble starch, konjac flour and guar gum, all within 24 h.

By 48 h significant amounts of gas were produced in cultures supplemented with D-glucose, D-galactose, so[uble starch, konjac flour, pectin and guar gum. By 96 h only

L-glucose, carboxymethyl cellulose and sodium alginate showed no significant gasproduction compared to the control, unsupplemented basal medium.

Conclusions

At each time interval, the mean PH and volume of gas produced by incubating testcompound (Konjac flour FW-KON) with fecal suspensions were approximately

between the upper and lower levels seen when four control food grade compounds

were incubated’with the same suspensions.

11

. . .. .

FMC study: 194-1976

CR 01 O-RJC-001

Table 1. Mean pH and gas production results of control and test compounds

incubated with pooled and diluted human feces

1.A. Time = 24 h

Z!Erl :~’nRange of pH Gas production (ml)& SD2Basal medium 6.31 6.28-6.36 6.0 & ().9 (n=3)

D-glucose 5.45’ 5.42-5.48 13,9 & 6.2 (n=2)

D-galactose 5.54” 5.49-5.58 12.9 & 0.5”

L-glucose 6.37* 6.36-6.39 5.8 & 0.1

Soluble starch 5.89” 5.80-5.94 12.5 & 1.1”

FG konjac flour 6.21 6.13-6.29 8.8 & 0.7” I

FG xanthan 6.25 6.19-6.31 5.2 ~ 0.7

FG pectin 6.06” 5.99-6.11 7.0 * ().3

FG guar gum 6.08” 6.05-6.11 9.4 * 0.7”— .—

FG carboxymethyl- 6.33 6.29-6.35 5.5 Y 0.6cellulose

FG sodium alginate 6.34 6.31 -6.37 4.9 * 0.2”

ln = 4; means of pH calculated using hydrogen ion concentrations (see Appendix C)2 n = 4 unless indicated

FG = Food grade“ Statistically different (unpaired t-test, p = 0.05) from basal medium

12

*.

FMC study: 194-1976

CR 01 O-RJC-001

Table 1. Mean pH and gas production results of control and test compounds

incubated with pooled and diluted human feces

1.B. Time = 48 h

Y :~’n‘angeOfpHGas production(ml) & SD2Basal medium 6.37 6.34-6.39 7.7 * 0.5

D-glucose 5.67* 5.50-5.99 18.8 k 2.2*

D-galactose 5.86” 5.81 -5.91 14.7 ~ 0.6’

L-glucose . 6.35 6.31 -6.40 7.8 & 0.4

Soluble starch 6.04” 6.00-6.07 14.5 * 0.5”

FG konjac flour 6.12- 6.03-6.29 12.1 * 0.4”

FG xanthan 6.21” 6.15-6.33 7.3 * 1.9

FG pectin 5.81” 5.69-5.99 11.2 * 0.9”

FG guar gum 5.74” 5.69-5.77 13.9 * 0.7”

FG carboxymethyl- 6.38 6.35-6.41 7.8 k 0.4

cellulose

FG sodium alginate 6.35 6.31 -6.41 7.1 * 0.2

ln = 4; means of pH calculated using hydrogen ion concentrations (see Appendix C)

2 n = 4 unless indicated

FG = Food grade“ Statistically different (unpaired t-test, p = 0.05) from basal medium

.

13

,.. .


*

I8

Table 1.

incubated

Mean pH and gas production results of control and test compounds

with pooled and diluted human feces

I.C. Time = 96 h

Zz3 :~’n‘angeOfpH(ml)*sD2Gas productionBasal medium 6.46 6.42-6.53 11.2 A0.6

D-glucose 5.90” 5.76-6.12 21.2 t 0,6”

D-galactose 6.00” 5.96-6.08 16.7 ~ 0.8”

L-glucose . 6.49 6.40-6.60 10.3 * 0.4”

Soluble starch 5.89” 5.79-6.00 17.0 * 1.2”

FG konjac flour 6.09 5.80-6.32 15.9 ~ 2.9*(n=3)

FG xanthan 6.03’ 5.90-6.11 12.1 * 0.2”

FG pectin 5.94” 5.84-6.08 14.2 ~ 1.1”

FG guar gum 5.94” 5.87-6.00 17.3 * 0.5”

FG carboxymethyI- 6.48 6.46-6.51 10.8 & 0.3cellulose

? 6-26” 6“’’4-6-37 (“:3)

112 A0.3

1 n = 4; means of pH calculated using hydrogen ion concentrations (see Appendix C) ..

2 n = 4 unless indicatedFG = Food grade“ Statistically different (unpaired t-test, p = 0.05) from basal medium

.

14

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.


Appendix A - TechLab Standard Operating Procedures

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FMC study: 194-1976

CR 01 O-RJC-001

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TITLE

PPI Aaaerobic Culttie Systemsfkdvc SE? J J Q7 ~=

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4.2 Light the bunsen burner and turn on the anaerobic gas tank.‘flamecannulas. Be sure that there is gas coming out of thecannulas.

4.3 If you are using culture tubes, flame the stoppered culturetubes and the bent hemostat. Remove the stopper (with a~rocking~ motion) and immediately insert the flame-sterilizedcannula. Whether you are inoculating Zrom a culture tube ora culture plate, flame the stoppered tubes to be inoculatedand insert the flame-sterilized Cannula.

4.4 Inoculating tubes from tubes:

4.4.1 It is better to use the pipettor and pipet tipswhen it is known that there is not a lot of growthin the tube. Transfer 200 pl into t!!efresh brothmedia. Discard tip.

4.4.2 If there is adequats growth, one can use a sterilePasteur pipet or an inoculating loop. If using asterile pipet, open the canister and flame the topsof the pipets. Remove one pipet and attach therubber bulb to the end. Fl?uaethe entire pipet.Do not let it melt. Place the pipet halfway Lntothe culture tube (do not touch media) and a squeezethe bulb a few tines to suck in the anaerobic gasand to cool the tip. Place the pipet into theculture izedia. Squeeze the zedia into the pipet afew times (to mix). Suck a small amourrtof theculture media and draw the pipet straight up andout the tube. Place the pipet halfway into thefresh broth media tube (be careful aot to touchanything with the pipet) and squeeze a fev dropsinto the broth. Discard the pipet. If using aninoculating loop, flame the entire loop. Place ithalfway into the culture tube and allow it to coolslightly. Place the loop into the culture mediaand obtain.a loopful of culture. Draw theinoculating loop straight out of the tube and placeit into the fresh media. Swirl it around andremove the Loop. FLame the loop.

I1

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Dismouuon I... .!

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FMC study: 194-1976

CR 01 O-RJC401

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FMC study: 194-1976CR 01 O-I?JC-001

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FMC study: 194-1976CR O1O-RJC-OOI

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TITLE Euoxi”e‘J~~ : “ ‘s= ‘=

Pooling Euman Fecal Ssmplas T>y : : !>

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20


IiIL&Sffccdvc:J.% pa=

Dispensing Pooled!Kuman ..Fecal Samples

ksud ..~1 ‘of 2

1.

2.

●

3.

Pun Osez

To describe procedure to prepare frozen stocks of pooledhuman fecal samples to be used in in vitro metabolismstudies.

paterials 6 Ecruiument

2.1 Culture tubes 25 X 12510BI2.2 Beaker2.3 Mettler scale (SOP #5001)2.4 Anaerobic transfer device (SOP #5000)2.5 Stopper2.6 Figure B (appendix)

zocedu7e

3.1 ?reoaze feces for dispensing (SOP #6001)

3.2 Place a 25 x 123 mm culture tube into a glass beaker andplace the beaker onto a balance and tare.

3.3 Squeeze the pooled, nixed, feces through the tube,c!ispensing5 gram (+/- O.lg) into tube (see diagra”m,parta).

3.4 Ixoediately place the tube containing the Pooled Human Fecesonto :he anaerobic transfer device; flush and stopper tube.uncleranaerobic gas.

3.5 Date and label t~e tube. Store the tubed aliquot of pooledhuman feces at -LO “C untiL used to prepare the human fecalinoculum.

Aliquots are good for one year.

21

,.

.

FMC study: 194-1976

CR 01 O-RJC-001

/ispensinq Pooled HWSan -..!. ‘ W2Issued~~’~‘-“ Zofzecal samples /

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FMC study: 194-1976

CR 01 O-RJC-001

‘---

Preparing Euman )

~

IFecal Inoculum LM ; :1‘= lofz~

1.

2*

*

3.

To describe vrocedure used to ureuare a human fecal inoculum forstudies on ~a in vitro metaboiis~ of foreign compounds.

Materials G Mu imnent

2.1 5 “g pooled human fecal samples (SOP #6001)2.2 Anaerobic transfer device (SOP #5000)2.3 TY broth, 40 mlftube2.4 Stoppers2.s Vortex2.6 Pipette (5 ml)2.7 Figure 1.0 (appendix)

Procedure

3.1

3.2

3.3

3.4

3.5

3.6

3.7

Thaw a 5 g tube of pooled humanwater. Place the tube onto theanaerobic gas.

feces by placing the tube in coldanaerobic transfer device under

Pouring from the TY tube, add 40 ml of TY broth to the cube offeces and stoppez the tube anaerobically.

Vortex the tube vigorously for 2-3 ainutes until completelymixed.

Return the tube to the anaerobic transfer device and allow thediluted pooled human feces to settle undisturbed for 5 ainutes.

From the center region of the tube of diluted human feces,anaerobically transfer 2 ml into a fresh 40 ml tube of TY broth.(See Figure 1.0).

Stopper the tube and vortex for 1 minute.

Label the tube “human fecal inoculum”.

THIS Is THZHUMAN FECAL INOCtm.m.

Rm-d By Dme I Approvcday . IDisoibucion

\ . . ‘-“-,,. .. . . .. . . . .!,, .. . ...”.RobenJ.Cuman 1/lQ192 Donna T. Evans QA, R&D

23

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FMC study: 194-1976CR O1O-RJC-OO1

. .

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\

.mdi&Diluted,

‘re~cd 9y i%e ~ppmvcd By Disoibuaaa..- ---

-. .,”.

Roben J. Ca.marI 1/[0/92 D, ,I,fla T. Evans QA, R&D

24

— ,=-fastkg .PZ4-:C:. U3

_ .Cant.ralHomcqRnous”3aC3ndaa$lon


*

1. PWOOSQ

To describe procedures foron in Vitro fermentations.

2. Hateria19 and Euuiu alent

supplementing

2.12.22.32.42.s2.62.72.82.92.102.112.12

1 M NaOH0.1 M NaOHTY broth tubes, 40 iul/tube10 % Phenol Red solution (SOP #6021)AnaeWbic transfer device (SOP #5000)pH MeterVortexweighing paperSpatulasMettler scalesPasteur pipettesTest substrate

3. Procedure:

3.1

3.2

3.3

3.4

3.5

3.6

3.7

3.8

?repara a Phenol Red control brothdrop of a phenol red solution fromfresh 40 ml TY broth tube. pH the7.5 using 1 H andjor 0.1 X NaOH.

Weigh out 0.2 g of substrate (test

Remove the stopper ot the 40 ml TYon the anaerobic transfer device.

TY broths for studies

by aseptically adding 1a Pasteur pipette to atube. Adjust to a pH of

material) .

broth tube and place tube

Add the substrate to the tube, then add 1 drop of 10% PhenolRed solution to the TY broth tube and stopper the tubetightly.

Vortex the tube vigorously for 30-60 seconds.VisuallycomPare the color with the CONTROL phenol red - see #3.1.

Place the tube back onto anaerobic setup and add either 0.1or 1 H NaOH until the color of the broth matches the phenolred control.

Tightly stopper the tubes under anaerobic gas.

THESE ARE NOw ‘lSUPD L~E7VTF~ M TY BROTHS.cp-q ay Dmc Approwda~ , .~.-~-#.,.X.’)“2:’.1m..,. Distribution. -.”:., -.j -,,.’ --.’. , - ’- J. Cm= 1212819C CUma T. Evans 1/14/91 QA, RLD

J

I

I1

25

.


Efrcdw25.Y

IPi>:z

‘ej suQerSakS. -------,....,.,.. .. ....-..- ., A ML I

InoculatiZ@ ~t ad TV 9rctm I

1.

2.

3.

Pumos e

To ciescribeprocedures for inoculating supplemented TY broths withhuman fecal bacteria for in vitro fe-mencation studies.

y’atsrials and Eou Liumen

2.1 iIumanfecal inoculum (SOP #6003)2.2 Suppl=menc=d TY broth, 40ml/cube (SOP %6016)2.3 Anaerobic cr3nsfsr device (SOP *5000)2.4 E culzc=e cukes, L3 r.%diameter, with rei~forced Ii?

(sterils)2.5 5 ML pipeccss2.5 1 ml pi~e2c2s2.7 ?asceu= ?~~ezce with bulb2.8 “ A

. .

FMC study: 194-1976

CR 01 O-RJC-001

. . . . . .. .“~;;’y:$j~w.*@=y

RccdJreX2.6017

~~*>&&~;’-””.. .. .RobercJ.Cmnan “v-loS/’)5 Donna T. EVWM QA,?d,R&D

\ /

27

.

FMC study: 194-1976CR 010-RJC-001

1.

2.

b

3.

yJ rpose

To describehuman fecal

procedures for incubating “inoculated” TY broths withbacteria for in vitro fermentation studies.

~terial~ and SOUiument

2.1 Inoculated TY broths2.2 Rack .~ithrubber mat2.3 ?ress & xbbez pad2.4 37-C :r)cabator2.5 Yreezez2.6 l-l.:/501

3.1

3.2

3.3

3.4

3.5

(SOP #6017)bottom

TY Broths into a test tube rack with ar“~>zezxat beneath tube bottoms and place rack into a press.Place a rdbber pad between the stoppers of the tubes and the.Dresscop. Tighten the press evenly and firmly. (See Figure~.o).

~,-.c.

, .**


....6. .,$w.’”*..*~**::: x.?.-?.*%:%M *f&A* ~y.-+ti w.:,


3.1.4 Whefithe liquid in the graduated tube stopslowering, mark with the spot ‘#ithan O ring.Record the zeading irnnetilacelyon TLF $-according to SOP #6018.

3.1.5 Write the reading direccly on the tube .with aniadelible ink pen.

3.1.5 ~emave the needle of the “gas meter” from the t~e.

.

Prepared3y Oau Appluvui3y.,,.....Diso’ibudo!l

‘ :.”-..., .,,. ... ... - -..

. .. ;>, , .- -----\;.$ _--:. .:-.

RoberC2. C&m’an 12/28/90 &mna T. Evans 1/14/91 QA, RfJ J

30

r,’,,-

FMC study: 194-1976CR 010-RJC-001

criteriaforFe~en[atiOnofsubsrraq

1“ I %gupmluchr II* [P=ialPsTc=Iumim(Durham.&~’% fuilotsa.s) I

.M.xinnl w pmdwxion (Durtum tube fuU of gq

.*. .

.YD Gar producdonmatdcremincd (bred! WM CM ;chdnous w viscow)

[Ii Insoluble suiwme. psrdculxe SUspmsinn fomed

I X I [nsolubl e $u&%[e. $ubmxe rmukd in c!ump or (aye I

I $ I C’mplm!ySoluole$lbmarc I

[ +lmdiulll [ 6J ,- (0.,) I 6.6,- (m) I 6.6,- (02) I

Susch pm (43) I 5.3 / - (8.S) I 5.3 / -- (10.6) /

Smd! I53 /* (4,1) 5.2/- {9.4) A5.7I+++ (12.1)

TLRVZQ369WI?U

Prcpaml By Dxc ApprovedBy . Disuibution,, . . .

- ...r -.. .!.. ,- .. . .: -.. “-. . . ...” .

RoW J. Cman 12/28/90 &nna T. Evans 1/14/91 CA, ~~D

31


-:~umw 60L,

TITLE

Plea surina~=uvc..: ~ “ ,.. ~w:

Gas Production .-ln TY Brot hs kswd--””-”.. .’ 4of4

\

VolumetricGasMeasuringDevice

b

L.Gu!d A

RilcterTubing

x“RubkrGp

n

21:.Vedc

4

?cpamlBy Due ,Approted 3Y.- i- .:-, .,.. ..; ... . .. . .. . .-! . J’,,..RobertJ. Carman 12/28/5o

- ‘ rConna T. .Wans

,

32

.


.

1.

2.

b

3.

mrw Ose:

To measure acid production in supplemented TY broths incubatedwith Fecal Bacteria.

Materials and Eouiument

2.1 ‘7ncubatedH T’ibroths2.2 TLP form #6012.3 pH meter (SOP #5001)2.4 Gloves2.5 Deionized Water2.6 Tissues2.7 Wash Bottle2.8 Beaker 500ml

m ocedure

[~ Xeasuring the gas, jneasure tSe pli using a combinationpencil eleczrada (Fisher Electrode ciec?icatedto pH deteninationof fernentaticn broths) standardized against pH 7.0 Q@ pH 4.0ixlffer.

3.1

3.2

3.3

3.4

3.5

3.6

3.7

Remove the stopper fzorothe mixed fecal culture and~lace the electrode into the bottom of the tube ~ep. tQ thelnvezted glass tube.

Gently swirl the tube (do not vortex) several time to mix thetube and equilibrate the electrode.

Read the PH.

Gently swirl the tube again and read the pii.

If the pH is stable, not changed, record the pH - if the pHis not stable (has changed > 0.1 unit) swirl the tube againand rezead the pH. Continue until the pH has stabilized.Record data according to SOP $6018 on TLF #601.

Remove the electrode from the tube and wash it off thoroughlywith distilled water, rinse bottle into beaker with washbottle, BLOT (do not wioe!) th- zlectrode with a tissue.Discard the tissue into a discard pan to be autoclave.

Autoclave discard Dan and wash water.

Pqa-cdBy Dme AootovcdBy : Disuihtion. ,.. ........._ ~ .+-- , .“ .. . . . . !: .L:,”.1: . . . .. . . .. . . . .

Ro”m J. Carmen 12/28/90 ~Md T. ~Ve”S 1/14/91 QA. 5!LD

33


TITLE Srfccd= ‘“’ “’” ~~

Preparation of Phenol ---- loflRed Solution

-,:. ::~-,.

,

1. yurmose:

To describe procedures used to prepare Phenol Red stock solutionfor carbohydrate fermentation studies.

2. )faterials E Equ ipment:

2.1 Phenol Red (Sigma). 2.2 PRAS dilution fluid (Anaerobe Lab Manual)

2.3 Vortex2.4 Weiqhing paper2.5 Anaerobic transfer device (SOP #5000)2.6 Spatulas2.7 Hettler scales2.3 Stapper

3. Procedure:

3.1 Weigh out I g Phenol Red.

3.2 Using E>.SAnaerobic transfer device, add the 1 g Phenol Redto 10 ai of the PRAS (Pre-Xeduced &Taerobically Sterilized)dilution fluid.

3.3 Stopper the tube. Vortex for 30 secacds.

-PqmcdBY Dw ApprovdBy Oisc~ouUonl-. .I- “+..,. . . ,. . . .. “... “. ,. : .-. .,.

F@xrt J. C.==ran 12/28/90 coma ‘r. Evans 1/14/91 QA, ?.&3

34

FMC study: 194-1976

CR 01 O-RJC-001

e

~ggztiGgti’’’~&E$$~’‘-’’””N”’022,,‘ ~: z;.,,..,,1 ,.. < ~,. ,+..: .::..:.. ,. ‘“”’””*

-, ..-’$’=:Y-+;:.:;2,;;,.:;;’”asket and fold the opening edges of the bag into aCuff (like a shirt). See Figure 1.0.

3.2 Raise t>.e sea: of the toilet bowl, and place the ‘*basket/bagdSSenbly” LZES the bowl Sussended f~on the basket a~s over the

bowl ria.

3.3” Immediately a:tzr the sample is “donated” unfold the cuff andexclude =Gs= of the air and seal the bag closed.

3.4 Seal the hag tightly, flattening the fecal saaple and pressingOUt as much of the gas as possible, and freeze at -IO”C.

3.5 ‘Whena wire collection basket is not available, ‘de c?onorcan dowithout by squatting over the open bag. It is wise to urinatebefore a:tezpting this.

F’rcpandBy; Dac Approval’5y Disc..iwtion,.’~“n~...r ./:L-u.:!.,.l,..-.c.-

-_, ;


--. ..—. . ..●

,

36

* .

Appendix B - Protocol CR 01 O-RJC-001

FMC study: 194-1976

CR 01 O-RJC-001

ORIGINAL

PROTOCOL

Soonsor

FMC Corpomcion.PrinceIon. New Jersey

●

Smdv Tide:

In vitroFermentation of Konjac Flour by Human Fecal Flora

FMC Study: 194-1976P:omcol CR O1O-RJC-OO1

k“

17 February, 1995

~CffOITlliXl!?Lib Or.tow:

Tectib, Inc.WI Research Park

1861 pram DriveBlacksburg, Virginia 24060

1

37

I

i

,,

,.1*


FMC study: 1941976CR O1O-RJC-OO1

STUDY IDEiiC/iTION

In vim Fermentation of Konjac FJour by Human

Test LMa[eriaJ Konjac Flour FW-KON

Study Director

Study ~Monitor Lois A. Kockoskie, PhD, DABT,Senior Reseu-ch Toxicologist,

b FMC Corporation.Chemical Research and Development Center,BOX 8,Princeton, New Jersey 08543

Phone: (609) 9513724Fax: (609) 951-3837

Robefi J. Carman, PhD,Direc[or of Researeh,TeehL.ab, Inc.,1S61 Pi-an Drive,Blacksburg, Viiginia 24060

Phone: (703)231-3943Fax: (703) 231-3942

Study Location Tectib, Inc.YTI Research Park1861 Pram DriveBlacksburg, Virginia 24060

Proposed Study T-hnerableStarr Date March 1, 1995End Date ,March 28, 1995

.

38

1. study: .

2. %pose:

3. Justification.

4. Regulatory Compliance:

5. Quality Assurance:b

6. Test Material:

A. [dentifico,tion

B. Storage-stabiliry

C. Chamc:etistics

D. Safery

E. Retention

F. Stem.ge

7. E~perirnenral design:

A. Ourline



(n vim Ferrnenration of Konjac Flour by Human Fecal Flora

To determine rhe por.ential for, and ewenc of, fermentation oftest marerial by fecal bacteria invh.

To help collect&u for rhe Sponsor’s possible submission toregu[a~o~ authorities.

The srudy wilI be done according to rhe Food and Drug.Adminisrracion (FDA) Good Labomtory Practice Regulations forNonchicd bbora;ory Studies.

The protrxo[. srudy conduct and fircal reporu will be monitoredby tie Quality Msurance Unit in compliance wirh FDAreguhclon 21 CFR 58.35.

KW3C ~OUK FW-KON LOCNO. W4026

To 5C te~ned under FMC .5kudyNo. P94-0085

Inforr.ac:on on che production. puriry, stability and orherkzrures r.?3[define tie restmaterial will be determined urderFMC Swdy No. P94-0085.

,%srcquxd by TechLab’s normal work practices, unlessSpocsor indicues additional requirements. The sponsor willprov!dc a MSD sheet for r-hetest compound.

.by urmsec! test material wil[ be returned to tie Sponsor afterissumct of the Fd Report or disposed of by TechLab.

Room ternpenrure

Test and conrrol compounds will be incubated with fecalsuspensions. Wherher or not die compounds are fermented wilfbe judged on the basis of pH shifts and gas production.

.

39

-=..””

B. Procedures

C. Reagenrs

D. ?vfedia

E. Feces

F. kIOCUhlriS

G. Inoculation

H. Incubation

FMC study: 194-~976CR 010-RJC-001

FMC srudy: 194-1976CR O10-WC-001

AH experimerual procedures will be conducred in accordancew-kh Tectib’s SOPS. i%ny SOP revisions resulting from oroccurring during rhis srudy will & documented and included inTecblab’s historical SOP file,

Prepared media, media componenr.s and conrrol subsuatcs willbe purchased. They will include: nypticase, yeast exuact,scdium chloride, herein. viramin K. Phenol red, L-cyweine, D-/glucose, so[ub[e srmh, !ylaccose. L-glucose, w =Ym,/armgccrran. This is not a comp[ere list. Equivalent suppliesmay be used when deemed necessary by the Srudy director.

Trm(:clse-yeast exmct broth (TY broth) will be mpplememed,

3ciording [0 SOPS, wirh test or control compounds (0.5%wrf,+01fir~ concen~cion).

31c$npn:cal dau (age,sex,dietary habiss, general infomsationL-d -:cem mirnicrobial history - see Appendix A) will bewcu::d ~mm all donors. The identity of rhe donors will remainccr~”.der.cal. No diagnoses will be made using data collectedf::m L’.S sr~dy. No donor will have received antibiotics withinJ zL.-A.zwr of’ tie prec:ding 4 weeks. Co Uection of tie samples~:il ‘:c :n[lreiy voiun~ and will involve no risk (O rhe donors,who ,wIII SIgI 3 release form arlestirg to this and tie ocher facts!k.ttd ibOVC.

Feces wdl be collec[ed, pooled and processed according toTechLab SOP which will be inc[uded in che F-ml Reporr.

Tle e,x~erm.erm.l inoculum will be prepared according to SOPS(to be included in the F“mal Reporr) and the same 10CofiEccdlum will be used tioughout rhe srudy.

From inoculated broths aliquors will be dispensed asepticallyand amerobicdlyinros~erilerubes.Therubeswillbes~opperaiaraerobicdly:eachsetofrubeswillcomprisetie‘testaridconrrolincubadonsbroths”fortieshorrterminw“moincubationsruc!ies.

fich ser of brotis will be incubated in a press to prevent gassesproduced during incubation from expelling rAe stoppers.

,

4

40

II

*


FMC 5mdY: 194-[ 976CR 010-WC-COl

t lncubacions wifl be ForO, 24.48 and 96 h periods.underanaerobicgassesandu 37 “C.

I. .%rsa[ysis Each rest and conrrol compound will be analyzed in duplicareoneachofrwoseparatecccasions.

i. Acid Acid production will be analyzed using a pH meter and anekcrrode srandarrfized at pH 4.0 and 7.0.

ii. Gas Gas production will be assessed by fluid disp[acemenc. TheSOP will be included in tie Final Reporr.

. The fo[lowing conrrol compounds will be purchased byii. ControlsTechhb: D-glucose, L-gIucow, soluble starch and D+@ac[ose,~e fo[lowirrg extra concro[ compounds will be provided by theSponsor: focal giade guar gum, food grade xanrhan gum, foodgrade sodium algirwe, focal grade carboxymetiylcellu[osc andfood grade pectin. Dau on the storage and srabiliry of controlrcagerms provided by tic Sponsor will be ktpc on ri[e at rheSponsor’s. The Sponsor will provide MSD sheets for theseadditional conrro[s.

iv. Dara AppendL. B lists rhe cnd point dctinhioru of fermemafion.

J. .Anaerobiosis Tbe anaerobic transporr device will be used wheneverappropriate to provide a bench Iopmanaerobic atmosphere,

8. Repom A final repom icc!uding, but not linked [o, rhose items !iscedbelow will be submirted. Onc copy of rhc drarl reporr and wocopies of the final report will be provided. A copy of tie rawdara will be provided ;O tie Sponsor wish rhe Draft FmlRepolZ

Rcsuks will be reporrcd in rabular form “Mcadmg (for eachdup[icme substme and COIXXO[rube at each drne irxervai) Chep~and rhe volume (ml) of liquid displacement (see Appendix B).

9. Data Maintenance Original Dara, or copies thereof, will be rnainrained at Tech.hbto facilicace auditing during i~ progress and before acceptance OfChe final reporr. When he fml report is completed. all originaldara, compurer data and the original final reporr will be rerained.

5

i

Ii

. . .

41

,....



in r.he m-chives of TechLab.

I... .

t

1

I

42

.,,,

},?


FMC mudy: [W~976CR O1O-RJC-OOI

.\ppendLx .A - Donor Questionnaire Form

●

-1

.

43

.,,. .

FMC study: 194-1976CR O1O-RJC-CO1

.4ppendLx B - Deftition of Fermentation on the Basis of pH Change

FMC study: 194-1976CR 010-RJC-001

;

1. Fcrmesmuion defined by pH change

7-. A ksc macerid will be deemed [o be fcrmenrable if, when incubamd withhuman colonic micmflora.thereisadmp inpH by 1 pH unitor greatercompa- LOanyfallinhe conaolincubation.

3. “Fast” fermentation is defined as a drop of 1 pH unit within 24h; ‘slow”fermentarioa k a drop of 1 pH unit over more than 48h. A fail in 1 or morepH units between 24=18h W besaid(obc “medium”.

b

t

I

I

. .

44

.

PROTOCOL .WPROVAL


FMC study: 194-1976CR OIC-RfC-001

2.~L-t,-

Lofi A. Kotostie, PhD, DABT, Date

Study Monitor,‘MC Coqlorarion

Srudy Dkector,TechLab. Inc.

Qualiry Assurance Off~cer.Tccb.Lab, Inc.

TechJab, Inc.

9

/,

.

r,.{,

,..

Appendix C - Protocol Amendment

.—

,! ,/ORIGINAL


I

PROTOCOL AMENDM&NT

SL?QIMK

FMC Corporation,Princeton. New Jersey

b

$rudv Title:

In ti~o Fermentation of Konjac Flour FW-KON by Human Fecal Flora

FMC Study: 194-1976Procccoi CR 010-WC-001

Q?&

26 Apd, 1995

TechLab, Inc.WI Research Park

1S61 Prart DriveBlacksburg, Viginia 24060

1

I

46

FMC study: 194-1976

CR 01 O-RJC-001

FMC srudy: 194-1976CR O1O-RJC4OI

STUDY IDENTIFICATION

in vifro Ferrnentdion of Konjac Flour FW-KON by Human Fecal Flora

Test Material

Study Monitor .

Study Direccor

Study Lccation

Konjac Flour FW-KON

Lois A. Kodcoskie, PhD, DABT,Senior [email protected],ChemicalResearchandDevelopmentCenter,Box8.Pnncecon.New Jersey08543

Phone:(609)9513724Fax: (609)951-38S7

Robcn J.Cm-man. PbD.Direc:or of Research.Tec.tib. klC..[S61 Prarr Drive,Bhcksburg, Virginia 24060

Phone: (703) 231-3943Fu: (703)23L-3942

TechLab, Inc.VPI Resemis Park1S61 PmtI DriveBlacksburg, Wgirda 24060

. --

.

“47

FMC study: 194-1976

CR 01 O-RJC-001

i

.FMC srudy: [9A [976CR O1O-FLTC-OO1

,,1. Purpose of zsnendmem

To describe the sdsucal arraIysis of fermentation dara.

M. pH data analysis: The following is a sample caIcularion of the mean of obsemed pHvalues. The data used for this sample is from the pH dar.a collected at 24 bouts for the Basalmedium. Thk approach Wa used throughout COgenerate means for all pHs.

Sample TabIe Basal Medium

Tiie = 24 h,,

Sample n~mber I pH(

7

Hydrogen IOISConcentration

1 6 ;6 4.37 x 10’

2 531 4.90 x IfY’

II3 623 ] 5.?5 x 10-7 I4 631 4.s0 x 10J I

,Meiut ~i

Observed pH dara were convcfled :0 hydrogenion concencrarions (ie. -log,O of the observedpH). To calculate the recur PH. ~hesum of the hydrogen ion concenrmcions was divided byrhe sample size and the mmn hydrogen :on coacenf.rarionwasconverted back co pH units.

In r-he worked example r.bis was:

pH = -[og(4. S6 .x [0-7) = 6.31

Conversion[0andlogarkhms andback again were doneusingS@naP1occompurer sohvare.

Comparisons of pH dara by unpaired t-ccst were done on the hydrogen ion concentration (ie.the negative antigorichrn of the obscmd pH values). The level of significmce was p =0.05.

lC. Gas production data amdysis: The mean and standard deviations of rhe volumes ofgas produced, in MI. were generated using compucer sofrware (Sigmaplot).

!

i

I

“.. . .

3

48

,/

FMC study: 194-1976

CR 01 O-RJC-001

FMC study: 194-1976CR O1O-FUC-OO1

PROTOCOL .&%lENDMENT APPROVAL

4Q5i4-Lois A. &XkOSki% PhD, DM3T,Study Monitor.FMC Corporation

h.u’l’-LL.-b (?2W,.CURoberI J. Ca.rman. PhD,Study DUCCKOC.TechLab, hlC.

!

Doma ‘Evans. BS,

‘f--27. ffDate

Quaky Asurance Offlc:r,Tectib, InC.

K’

Prcs{den~Techbb, Inc.

.

!

. .

49

? f‘.+.,&


Protocol Deviations

There were nodeviations from the Protocol and Protocol Amendments.

50

. . :a–.-


Final Report Approval

‘-$ b.% s- w-k‘ Lois A. Kotkoskie, PhD, DABT, Date

Study Monitor,FMC Corporation

WA &.J-k%Robert J. Carman, PhD,

Study Director, ●TechLab, Inc.

.

Jb’mdd/h+’#Officer,

,/ ;/’

5-/t/qs-Date

DateDonna Evans, BS,Quality AssuranceTechLab, Inc.

& ~+~;ly’-..<Pfiracy D ‘

/ilkins, ‘Ph D,

Scientific Consultant,Tech Lab, Inc.

51

. ..

.:-,...-

.

—–,:. :. ~+.,.J:=&.:4: f.* ... ., ~.:7._=., .Z,.. -..:=

Dear

This

Mr. Dubeck:.—.-~. T.---L. -,,.1. ,,..-

.7.- ;@

letter acknowledges receipt on March 26, 1999, of your amendment dated March 25. 1~.--”

for the subject petition.

,.

We have determined that this information does not constitute a substantive amendment.

tiorrnation has been incorporated into the petition jacket.

Sincerely yours,

Sylvia DodsonOffice of Premarket Approval, HFS-200Center for Food Safety

and Applied Nutrition..

..&

CC:HFS-200 HFS-215 HFS-225 HFS-246 “E=~...“F/T:sDD:3/31/99

....-,;

Documents

Memorandum - Konjac Foods · 2013. 6. 7. · starch, konjac flour and guar gum, all within 24 h. By 48 h significant amounts of gas were produced in cultures supplemented with D-glucose,